UNIVERSIDADE DE LISBOArepositorio.ul.pt/bitstream/10451/2476/1/ulsd059472_Td_Ana_Vaz.pdf · À...

UNIVERSIDADE DE LISBOA

FACULDADE DE FARMÁCIA

IDENTIFICATION OF CELLULAR TARGETS FOR SPECIFIC

THERAPIES IN NEURODEVELOPMENTAL DISORDERS

Ana Rita Mendonça Vaz

Doutoramento em Farmácia

(Biologia Celular e Molecular)

2010

UNIVERSIDADE DE LISBOA

FACULDADE DE FARMÁCIA




Research advisor: Dora Maria Tuna de Oliveira Brites, PhD.

Co-advisor: Maria Alexandra de Oliveira Silva Braga Pedreira de Brito, PhD.

Doutoramento em Farmácia

(Biologia Celular e Molecular)

2010



IDENTIFICAÇÃO DE ALVOS TERAPÊUTICOS ESPECÍFICOS PARA O TRATAMENTO DE DOENÇAS DO

NEURODESENVOLVIMENTO

Dissertação apresentada à faculdade de Farmácia da Universidade de Lisboa para

obtenção do grau de Doutor em Farmácia (Biologia Celular e Molecular)


2010

Para a elaboração da presente tese de doutoramento foram usados integralmente

como capítulos, artigos científicos publicados, ou submetidos para publicação, em

revistas científicas internacionais indexadas. Estes trabalhos foram realizados em

colaboração com os seguintes autores: Sandra L. Silva, Maria Delgado-Esteban,

Andreia Barateiro, Adelaide Fernandes, Ana Sofia Falcão, Juan P. Bolaños, Angeles

Almeida, Maria Alexandra Brito e Dora Brites.

De acordo com o disposto no ponto 1 do artigo nº41 do Regulamento de Estudos Pós-

Graduados da Universidade de Lisboa, deliberação nº 93/2006, publicada em Diário

da República – II Série nº 153 – 5 de Julho de 2003, o Autor desta dissertação declara

que participou na concepção e execução do trabalho experimental, interpretação dos

resultados obtidos e redacção dos manuscritos.

Os estudos apresentados nesta dissertação foram realizados no grupo de

investigação “Neuron Glia Biology in Health & Disease”, Research Institute for

Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farmácia da

Universidade de Lisboa. Parte do trabalho foi também realizado no Departamento

de Bioquímica e Biologia Molecular da Universidade de Salamanca, Espanha, sob

a supervisão dos Professores Doutores Juan P. Bolaños e Angeles Almeida.

O trabalho foi subsidiado pelos projectos FCT-POCTI/SAU/MMO/55955/2004,

FCT-PTDC/SAU-NEU/64385/2006 concedidos à Professora Doutora Dora Brites

pela Fundação para a Ciência e Tecnologia (FCT), sendo que a Autora usufruiu de

uma bolsa de Doutoramento (SFRH/BD/30292/2006) concedida pela FCT, Lisboa,

Portugal,.

Agradecimentos __________________________________________________________________________

Agradecimentos

As minhas primeiras palavras de agradecimento vão para a Professsora Doutora Dora

Brites, orientadora deste trabalho. Agradeço-lhe por me ter recebido ainda enquanto

estudante de Licenciatura e me ter dado a a conhecer o mundo da investigação. Agradeço-

lhe também a oportunidade e o incentivo de fazer este Doutoramento, assim como todos os

conhecimentos que me transmitiu ao longo destes anos. Estou certa que o elevado nível de

exigência e rigor que estiveram sempre presentes na orientação científica deste trabalho

contribuíram de uma forma muito positiva para a elevada qualidade do mesmo. Do ponto de

vista científico, a sua capacidade de criação e raciocínio é bastante inspiradora, mesmo a

partir de ideias que ainda estejam a nascer nas nossas mentes, o que me faz sempre

acreditar no sucesso de novos projectos em que a Professora esteja envolvida. De um ponto

de vista mais pessoal, agradeço ainda a inteira disponibilidade para a orientação desta

Tese, assim como por me fazer acreditar que, se dermos o nosso melhor, podemos alcançar

o nível de excelência naquilo que fazemos, e assim contribuirmos activamente para nos

tornarmos pessoas especiais.

Agradeço também à Professora Doutora Alexandra Brito, minha co-orientadora. A

orientação científica que sempre me disponibilizou foi muito importante para a progressão

deste trabalho. Agradeço-lhe a disponiblidade para a orientação desta Tese, assim como a

constante motivação para fazer mais e melhor. Consigo aprendi que o “só mais um

esforçozinho” compensa quando queremos ser bem sucedidos. E quando alcançarmos esse

tão desejado sucesso, podemos acreditar que as coisas só têm tendência a melhorar!

Pessoalmente, agradeço-lhe toda a simpatia e preocupação que demonstrou comigo, assim

como o carinho que sempre me deu.

Me gustaría también agradecer a los Profesores Ángeles Almeida y Juan Bolaños del

Departamento de Bioquímica y Biología molecular de la Universidade de Salamanca por la

oportunidad que me ofrecieron de pasar parte significativa de mi doctorado en su

laboratorio. Durante esos periodos, me fue dada la oportunidad de aprender varias y muy

útiles metodologias experimentales. Nuestra colaboración se ha revelado muy provechosa

para el desarrollo de la presente tesis, una vez que los resultados ahí obtenidos constituyen

uno de los puntos clave aquí presentados.

À minha colega e mais que tudo, amiga, Sandra Guedes, deixo um agradecimento muito

especial. A tua amizade foi um dos achados mais preciosos desde que enveredei pelo

caminho da Ciência, quase que dava para a incluir no capítulo das conclusões! Sabes que

sempre contei com a tua força e sentido prático das coisas, tão importantes nas fases mais

Agradecimentos __________________________________________________________________________

difíceis. Esta fase final foi trabalhosa e houve momentos em que parecia que o caminho

estava continuamente a ser acrescentado mas ambas conseguimos lá chegar, e por isso

estamos de Parabéns! Ver-te concluir esta etapa ao mesmo tempo que eu vai ser uma fonte

adicional de satisfação. Para o futuro, desejo-te toda a felicidade, quer a nível profissional

como a nível pessoal. Não tenho dúvidas que serás bem sucedida nas duas pois para além

de inteligente, és muito justa e sensível e ainda por cima é fácil trabalhar e aprender contigo.

Quero deixar um OBRIGADA às minhas queridas colegas e amigas Adelaide Fernandes e

Sofia Falcão. Durante estes anos, vocês foram sempre os primeiros alvos das minhas

questões e dramas existenciais, tão característicos de quem procura o que ainda mais

ninguém encontrou. Adelaide, o teu conhecimento científico é uma fonte de inspiração, já

para não falar na tua capacidade de organização para teres sempre tempo para toda a

gente que te pede orientações sobre os próprios projectos e a quem tu nunca negas ajuda.

Sofia, a tua boa disposição e simpatia são contagiantes e as tuas orientações ao meu

trabalho foram sempre um grande contributo. Tem sido muito gratificante trabalhar e trocar

ideias convosco.

Quiero agradecer también a María Delgado-Esteban, que fue la responsable de

acompañarme en el laboratorio de la Universidad de Salamanca. Gracias por tu paciencia

com una recién estudiante de doctorado, acabada de llegar, siempre llena de preguntas y a

veces com menos respuestas. Contigo aprendí las bases de las regulaciones enzimáticas y

descubrí que se puede estudiar todo un mundo alrededor del metabolismo energético. De

esta colaboración nació también nuestra amistad. Creo que no podría haber tenido más

suerte con la persona con la que me tocó trabajar. Gracias por recibirme tan bien, incluso

fuera del laboratorio, y por hacerme sentir como si estuviera en casa en una ciudad

extranjera.

Me gustaría agradecer a todos los elementos del grupo de la Universidad de Salamanca, en

especial a Julia, Ángel y Mónica por la simpatia y amistad com que me acogieron y por el

esfuerzo constante en entender mi pseudo-español.

Agradeço também de uma forma muito carinhosa aos restantes elementos do grupo Neuron

Glia Biology in Health & Disease. Ao Professor Doutor Rui Silva agradeço a boa disposição

e a ajuda sempre presentes quando a ele recorri; consigo aprendi que às vezes o mais

importante é procurar qual é a pergunta certa! À Andreia um muito obrigada por seres

companheira de todas as horas; mesmo longe sei que posso contar sempre contigo. À Ema

e à Inês, minhas “pupilas” do coração, agradeço a amizade constante e os bons conselhos

que às vezes são tão precisos. À Filipa agradeço a espontaneidade que tantas vezes me fez

Agradecimentos __________________________________________________________________________

rir…Meninas, a todas vocês eu desejo as maiores felicidades para a continuação do vosso

Doutoramento e para a vossa vida posterior. Agradeço também à Cibelle e à Eduarda, que

mesmo tendo estado menos tempo connosco, contribuíram para as memórias felizes e

inesquecíveis que eu guardo destes anos.

A todos os colegas do Centro de Patogénese Molecular, obrigada pela vossa simpatia e

também partilharem das nossas venturas e aventuras do dia-a-dia.

Agradeço também a todos os amigos e familiares que me apoiaram na decisão de fazer o

Doutoramento, e que sempre me ouviram com entusiasmo e atenção a falar dos “meus

ratinhos”, ainda que por vezes não entendessem muito bem daquilo que eu falava…

Um especial agradecimento à minha Tia Nanda e ao meu Tio Tiago, por terem sempre

incentivado as minhas escolhas, provavelmente inspiradas neles, que foram as primeiras

pessoas mais próximas de cientistas que eu conheci. Eu sei que posso sempre contar com

vocês e vocês sabem que podem sempre contar comigo. A ti, Tiaguinho, agradeço o dom

que tens de me deixar sempre contente, mesmo nos momentos em ando mais desanimada.

E obrigada por me dizeres que também queres ser cientista como eu quando cresceres, faz-

me acreditar ainda mais naquilo que eu faço!

Aos meus Pais, Lídia e Francisco, deixo um agradecimento do tamanho do Mundo!

Obrigada pelo incentivo constante em querer estudar mais e procurar um futuro melhor,

especialmente porque neste momento isso ainda depende muito do vosso contributo. Bem

sei que às vezes não estive presente nem cheguei a horas mas nem por isso vocês

deixaram de me apoiar. E essa estabilidade em que me mantiveram a todos os níveis

contribuiu de forma decisiva para me tornar naquilo que eu sou, penso ou faço. Por isso,

tenho todo o prazer de partilhar esta Tese convosco, porque em verdade ela também é

vossa…

Ao Hugo deixo o meu agradecimento final. Aliás, a nós os dois. Porque a nossa vida estará

sempre ligada à Ciência e porque o gosto pela Ciência proporcionou que nos

conhecêssemos. Porque este ano foi atribulado e cheio de decisões difíceis e tu estiveste

sempre presente e deste-me força para continar. Porque todos os dias me fazes acreditar

que o que é necessário para estar contigo vale a pena…

Contents ________________________________________________________________________

xiii

Contents

I. Abbreviations ................................................................................................................ xix

Abstract ....................................................................................................................... xxiii

Resumo ........................................................................................................................ xxv

I. General Introduction ................................................................................................... 1 1. Redox status and cellular bioenergetics in central nervous system: regulation and

dysfunction ...................................................................................................................... 3

1.1. Free radicals, reactive species and antioxidants ............................................... 3

1.2. Pathways of glucose utilization .......................................................................... 7

1.3. Mitochondria: the powerhouse of the cell and the major source of ROS/RNS 10

1.4. Dysfunctional mitochondria ............................................................................. 12

2. Neuronal-glia actions and interplay in the brain ....................................................... 13

2.1. Glutathione shuttle ................................................................................................ 14

2.2. Glutamate shuttle ............................................................................................ 15

2.3. Lactate shuttle ................................................................................................. 17

2.4. Neuronal susceptibility to oxidative stress ....................................................... 18

2.4.1. Increased oxidant capacity in the brain ................................................. 18

2.4.2. Antioxidant capacity in the brain ............................................................ 20

2.5.Neuronal susceptibility bioenergetic crisis ........................................................ 21

3. Inflammation and cell death in central nervous system ........................................... 22

3.1. Cells involved in inflammation and CNS injury ................................................ 22

3.2. Inflammatory mediators and signalling pathways ............................................ 22

3.3. Neuronal susceptibility to inflammation ........................................................... 24

3.4. Death signalling pathways ............................................................................... 25

4. Bilirubin induced neurological damage and risk factors involved ............................ 27

4.1. Neonatal hyperbilirubinemia ............................................................................ 27

Contents ________________________________________________________________________

xiv

4.2. Prematurity as a risk factor of neonatal hyperbilirubinemia ............................. 28

4.3. Sepsis-associated neonatal hyperbilirubinemia .............................................. 29

4.4. Differential neuronal vulnerability among brain regions ................................... 31

4.5. Mechanisms underlying bilirubin-induced neurotoxicity .................................. 32

5. Promising molecules for modulation in hyperbilirubinemia ...................................... 34

5.1. Glycoursodeoxycholic acid (GUDCA) .............................................................. 34

5.2. N-ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) ............................. 35

5.3. N-acetylcysteine (NAC) ................................................................................... 35

6. Global aims of the thesis ......................................................................................... 37

7. References .............................................................................................................. 38

II. Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in immature cortical neurons. Assessment of the protective effects of glycoursodeoxycholic acid ................................................................................................. 59

Abstract ....................................................................................................................... 61

1. Introduction .............................................................................................................. 62

2. Materials and Methods ............................................................................................ 63

2.1. Chemicals ........................................................................................................ 63

2.2. Neurons in primary culture .............................................................................. 64

2.3. Treatment of neurons ...................................................................................... 64

2.4. Determination of the mitochondrial respiratory chain complex activities and

citrate synthase ............................................................................................................. 64

2.5. Detection of superoxide anion radical (O2.-) ..................................................... 65

2.6. Determination of oxygen consumption ............................................................ 65

2.7. Ѱm measurements ........................................................................................... 65

2.8. Metabolite determinations ................................................................................ 66

2.9. Assessment of apoptotic cell death by flow citometry ..................................... 67

2.10. Analysis of apoptotic cell death by 4'-6-diamidino-2-phenylindole (DAPI)

nuclear staining ............................................................................................................. 67

Contents ________________________________________________________________________

xv

2.11. Caspase-3 and -9 activity assays .................................................................. 67

2.12. Statistical analysis ......................................................................................... 68

3. Results ..................................................................................................................... 68

3.1. UCB selectively impairs cytochrome c oxidase activity in immature neurons,

which is prevented by GUDCA...................................................................................... 68

3.2. UCB produces oxidative stress in immature neurons, which is prevented by

GUDCA ......................................................................................................................... 69

3.3. UCB impairs cellular oxygen consumption and collapses ΔѰm in immature

neurons and GUDCA exerts a preventive effect ........................................................... 70

3.4. UCB increases extracellular ATP content, glycolysis and F2,6P2 levels in

immature neurons, which are counteracted by GUDCA ............................................... 71

3.5. UCB triggers apoptotic cell death in immature neurons, which is prevented by

GUDCA ......................................................................................................................... 72

4. Discussion ............................................................................................................... 73

5. References .............................................................................................................. 78

III. Pro-inflammatory cytokines intensify the activation of .NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin ................................................................................................................................. 83

Abstract ....................................................................................................................... 85

1. Introduction .............................................................................................................. 86

2. Materials and Methods ............................................................................................ 88

2.1. Chemicals ........................................................................................................ 88

2.2. Neurons in primary culture ............................................................................... 88

2.3. Treatment of neurons ...................................................................................... 89

2.4. Quantification of nitrite levels ........................................................................... 89

2.5. Western blot assay .......................................................................................... 89

2.6. Caspase activity determination ........................................................................ 90

2.7. MTT reduction ................................................................................................. 90

2.8. Densitometry and statistical analysis ............................................................... 90

Contents ________________________________________________________________________

xvi

3. Results ..................................................................................................................... 91

3.1. UCB, alone or in combination with TNF-α+IL-1β, induces nNOS expression

and .NO production in immature neurons, which are counteracted by l-NAME ............ 91

3.2. Inhibition of nNOS by l-NAME prevents the cascade of apoptosis induced by

UCB or UCB+TNF-α+ IL-1β in immature neurons ........................................................ 91

3.3. Inhibition of nNOS by l-NAME decreases P-JNK1/2 in immature neurons

treated with UCB or UCB+TNF-α+IL-1β ....................................................................... 94

3.4. Inhibition of P-JNK1/2 by SP600125 prevents the cascade of apoptosis

induced by UCB or UCB+TNF-α+IL-1β in immature neurons ....................................... 96

3.5. Loss of neuronal functionality in immature cells exposed to UCB is increased

by UCB+TNF-α+IL-1β and prevented by inhibition of nNOS and JNK1/2 activation .... 96

4. Discussion ............................................................................................................... 98

5. References ............................................................................................................ 103

IV. Selective vulnerability of rat brain regions to unconjugated bilirubin ............... 109 Abstract ..................................................................................................................... 111

1. Introduction ............................................................................................................ 112

2. Materials and Methods .......................................................................................... 114

2.1. Chemicals ...................................................................................................... 114

2.2. Neurons in primary culture ............................................................................ 114

2.3. Treatment of neurons .................................................................................... 115

2.4. Quantification of nitrite levels ......................................................................... 115

2.5. Western blot assay ........................................................................................ 115

2.6. Determination of cGMP concentration: .......................................................... 116

2.7. Glutathione measurement ............................................................................. 116

2.8. Assessment of ROS formation ...................................................................... 116

2.9. Evaluation of cell death ................................................................................. 117

2.10. Neurite Extension and Ramification ............................................................ 117

2.11. Densitometry and statistical analysis ........................................................... 117

3. Results ................................................................................................................... 118

Contents ________________________________________________________________________

xvii

3.1. UCB-induced nNOS expression and production of nitrites and cGMP is

enhanced in immature hippocampal neurons as compared to cerebellar or cortical

neurons ....................................................................................................................... 118

3.2. UCB-induced oxidative stress is highest in immature hippocampal neurons,

probably as a result of the lowest levels of total glutathione ....................................... 118

3.3. UCB-induced neuronal death is higher in immature cells from hippocampus

than in those from cortex or cerebellum ...................................................................... 121

3.4. UCB-induced neuronal oxidative stress and cell death in immature neurons is

prevented by NAC ....................................................................................................... 123

3.5. UCB regulates DJ-1 protein expression in immature neurons, mainly in those

from hippocampus, which is reverted by NAC ............................................................ 123

3.6. UCB-induced reduction of neurite outgrowth and branching mainly in

immature neurons from hippocampus, is closely followed by those from cerebellar

and cortical regions, and is prevented by NAC ........................................................... 125

4. Discussion ............................................................................................................. 126

5. References ............................................................................................................ 131

V. Final considerations ................................................................................................ 137 1. Concluding remarks and perspectives ................................................................... 139

2. References ............................................................................................................ 145

Abbreviations ________________________________________________________________________

xix

Abbreviations 7-AAD 7-amino-actinomycin

AGUDC Ácido glico-ursodesoxicólico

Ala Alanine

Apaf 1 Protease activating factor 1

ATP Adenosine triphosphate

BCAA Branched-chain amino acid

BIND Bilirubin-induced neurologic dysfunction

BNC Bilirrubina não conjugada

CAT Catalase

cGMP cyclic Guanosine monophosphate

CHAPS Cholamidopropyldimethylammonio-1-propanesulfonate

CNS Central nervous system

CO Carbon monoxide

CO2 Carbon dioxide

CuZnSOD Cooper/zinc superoxide dismutase

Cys Cysteine

Cyt c Cytochrome c

DHR 123 Dihydrorhodamine 123

DNIB Disfunção neurológica induzida pela bilirrubina

ERK 1/2 Extracellular signal-regulated kinases 1 and 2

F2,6P2 Fructose-2,6-bisphosphate

FADH2 Reduced flavin adenine dinucleotide

FasR Fas receptor

FBS Fetal bovine serum

FCCP Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone

G6P Glucose 6-phosphate

G6PD Glucose 6-phosphate dehydrogenase

Gln Glutamine

Glu Glutamate

Gly Glycine

GPx Glutathione peroxidase

GR Glutathione reductase

GSH Reduced glutathione

GSSG Oxidized glutathione

GST Glutathione S-transferase

GUDCA Glycoursodeoxycholic acid

H2O2 Hydrogen peroxide

HBSS Hanks’ balanced salt solution

Abbreviations ___________________________________________________________________

xx

HNE 4-Hydroxy-2-nonenal

HO Heme oxygenase

HSA Human serum albumin

IL-1 Interleukin-1

IL-1ra IL-1 receptor antagonist

IL-1β Interleukin-1β

ICE IL-1β-converting enzyme

IL-6 Interleukin-6

IM Inner mitochondrial membrane

IMS Intermembrane space

JNK 1/2 c-Jun N-terminal kinases 1 and 2

Leu Leucine

L-NAME N-ω-nitro-L-arginine methyl ester hydrochloride

L-NMMA N-ω-monomethyl-L-arginine

MAP-2 Microtubule-associated protein 2

MAPKs Mitogen-activated protein kinases

MEM Minimum essential medium

MnSOD Manganese superoxide dismutase

MPP+ 1-Methyl-4-phenylpyridinium ion

Mrp1 Multidrug resistance associated protein 1

NAC N-acetylcysteine

NAD Nicotinamide adenine dinucleotide

NADH Reduced nicotinamide adenine dinucleotide

NADPH Reduced nicotinamide adenine dinucleotide phosphate

NF-κB Nuclear factor κB

NH3 Ammonia

NMDA N-methyl-D-aspartate

NMDAR N-methyl-D-aspartate receptor .NO Nitric oxide

NOS Nitric oxide synthase

nNOS Neuronal isoform of NOS

mtNOS Mitochondrial isoform of NOS

iNOS Inducible isoform of NOS

eNOS Endothelial isoform of NOS

NOX NADPH oxidase enzymes

O2 Oxygen

O2.- Superoxide anion radical

O22- Peroxide anion

.OH Hydroxyl radical

Abbreviations ________________________________________________________________________

xxi

OM Outer mitochondrial membrane

ONOO- Peroxynitrite

PARP Poli (ADP-ribose) polymerase

PDH Pyruvate dehydrogenase

PFK1 6-phosphofructo-1-kinase

PFKFB 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Pi Inorganic phosphate

Pgp P-glycoprotein

pNA p-nitroaniline

PPP Pentose phosphate pathway

Pyr Pyruvate

R. Radicals

RNS Reactive nitrogen species

ROOH Peroxides

ROS Reactive oxygen species

SAPKs Stress-activated protein kinases

SOD Superoxide dismutase

TNF-α Tumor necrosis factor-α

TACE TNF-α converting enzyme

TNFR TNF-α receptor

tBid truncated Bid

TCA Tricarboxylic acid cycle

TMRE Tetramethylrhodamine

TUDCA Tauroursodeoxycholic acid

UCB Unconjugated bilirubin

UDCA Ursodeoxycholic acid

VEGF Vascular endothelial growth factor

α-KG α-ketoglutarate

γ-GluCys γ-L-glutamyl-L-cysteinylglycine

Abstract __________________________________________________________________________

xxiii

Abstract The present dissertation is focused in neonatal hyperbilirubinemia, a very common

condition in the neonatal period, characterized by increased concentrations of unconjugated

bilirubin (UCB). High levels of UCB may lead to bilirubin-induced neurologic dysfunction

(BIND), particularly in premature infants, which may be a starting point to the appearance of

long-term neurodevelopment disabilities. In cultures isolated from rat brain, toxicity induced

by UCB is more pronounced in neuronal cells that in those from glia, and immature cells are

more prone to this injury. Among the UCB-induced cytotoxic effects, are the extracellular

accumulation of glutamate and the up-regulation of inflammatory pathways (mainly in glial

cells), permeabilization of the mitochondrial membrane (in isolated mitochondria), impairment

of neuritic development (in immature cortical and hippocampal neurons) and oxidative stress

(in differentiated neurons). Firstly, we intended to better understand the mechanisms of

neurotoxicity by UCB, mimicking a condition of prematurity, regarding oxidative stress,

mitochondrial dysfunction associated with bioenergetic alterations and cell death, as well as

to study the role of known modulators of oxidant species production in the prevention of

UCB-induced neuronal injury. The obtained results showed that rat immature cortical

neurons exposed to UCB undergo oxidative stress, mitochondrial dysfunction associated

with respiration failure and cell death, effects that are prevented in the presence of

glycoursodeoxycholic acid, a compound with antioxidant and anti-inflammatory properties. In

addition, since prematurity is often associated with sepsis, these studies evaluated the

additional effects of inflammation on hyperbilirubinemia. We demonstrated that UCB induced

nitrosative stress, c-Jun N-terminal kinases 1 and 2 signalling and cell death and that these

effects are intensified by pro-inflammatory cytokines tumor necrosis factor-α and interleukin-

1β, through the same cascade of mediators. Finally, it was investigated whether there is a

dissimilar brain regional susceptibility to UCB-induced oxidative damage and neurite

outgrowth and branching disruption in immature neurons, which might determine the

preferential UCB deposition and brain damage in specific brain areas characteristic of

kernicterus, such as cerebellum and hippocampus, and also the mechanisms that are

involved in the modulation of UCB-induced neurotoxicity. Rat hippocampal neurons were the

most susceptible to UCB-induced oxidative and nitrosative stress, as well as to UCB-induced

neuritic impairment and cell death. N-acetylcysteine, a precursor of glutathione synthesis,

was able to counteract the UCB-induced neurotoxicity. Taken together, these studies will

substantiate target-driven approaches to the prevention and treatment of BIND, and provide

fruitful opportunities for future investigations.

Abstract __________________________________________________________________________

xxiv

Keywords: Bilirubin-induced neurological dysfunction (BIND); BIND-associated

inflammation; oxidative and nitrosative stress; antioxidants; mitochondrial dysfunction;

caspase activation; brain regional vulnerability.

Resumo __________________________________________________________________________

xxv

Resumo A presente dissertação é dirigida para o estudo da hiperbilirrubinémia neonatal, uma

situação clínica frequente durante a primeira semana de vida, resultante da elevação das

concentrações da bilirrubina não conjugada (BNC). A disfunção neurológica induzida pela

bilirrubina (DNIB) poderá ser o ponto de partida para algumas doenças do

neurodesenvolvimento, especialmente nos bebés prematuros. Utilizando modelos de

culturas celulares obtidas a partir de cérebro de rato, verificou-se que a toxicidade induzida

pela BNC é mais pronunciada em neurónios do que em astrócitos, sendo as células mais

jovens particularmente susceptíveis. Do vasto leque de mecanismos moleculares envolvidos

na toxicidade induzida pela BNC, destacam-se a acumulação de glutamato extracelular e a

resposta inflamatória (nas células gliais), a diminuição do desenvolvimento neurítico (em

neurónios do córtex e do hipocampo) e o stresse oxidativo (em neurónios diferenciados).

Numa primeira fase, este trabalho teve como objectivo compreender os mecanismos

associados à lesão pela BNC, no que respeita ao stresse oxidativo, disfunção mitocondrial e

morte celular, assim como avaliar o efeito protector do ácido glico-ursodesoxicólico

(AGUDC), um composto com propriedades anti-oxidantes, em condições que mimetizam

uma situação de prematuridade. Para tal foram utilizadas culturas de neurónios corticais

com 3 dias, obtidos de cérebros de rato. Neste modelo, a exposição à BNC conduziu ao

stresse oxidativo, disfunção da respiração mitocondrial, e consequente morte celular, efeitos

que foram prevenidos na presença do AGUDC. De seguida, avaliaram-se os efeitos

adicionais da incubação concomitante da BNC com as citocinas pro-inflamatórias,

mimetizando uma reacção inflamatória associada à hiperbilirrubinémia. Utilizando o mesmo

modelo, observou-se que tanto o stresse nitrosativo, como a morte celular surgem

aumentados após esta a incubação concomitante da BNC com as citocinas pro-

inflamatórias, estando envolvidos os mesmos mediadores e vias sinalizadoras. Por fim,

investigou-se de que forma o padrão de deposição específico da BNC encontrado na

patologia de kernicterus é determinado pela diferente vulnerabilidade regional à lesão

oxidativa e ao desenvolvimento neurítico pela BNC. Para tal, isolaram-se neurónios não só

do córtex mas também do hipocampo e do cerebelo de rato. Os neurónios do hipocampo

mostraram ser mais susceptíveis ao stresse oxidativo e nitrosativo induzidos pela BNC,

assim como à disfunção do desenvolvimento neurítico e à morte celular. A incubação com o

precursor da síntese da glutationa N-acetilcisteína preveniu os efeitos tóxicos induzidos pela

BNC. Em conclusão, estes resultados contribuem para o melhor conhecimento dos

mecanismos moleculares subjacentes à DNIB no período neonatal, tendo as moléculas com

capacidade anti-oxidante um efeito notório na prevenção desta disfunção.

Resumo __________________________________________________________________________

xxvi

Palavras-chave: disfunção neurológica induzida pela bilirrubina (DNIB); DNIB associada

à sépsis; stresse oxidativo e nitrosativo; anti-oxidantes; disfunção mitocondrial; activação

das caspases; vulnerabilidade regional do encéfalo.

Chapter I

I. General Introduction

General Introduction _________________________________________________________________________

3

1. Redox status and cellular bioenergetics in central nervous system: regulation and dysfunction

1.1. Free radicals, reactive species and antioxidants Oxidative stress is classically defined as an imbalance between the levels of oxidants

and antioxidants and has been implicated in the cell death pathways of several disorders in

the central nervous system (CNS). Under normal circumstances, cells can regulate the

production of oxidants and antioxidants, resulting in redox equilibrium. Oxidative stress

occurs when cells are subjected to excess levels of reactive oxygen/nitrogen species

(ROS/RNS), or as a result of depletion in antioxidant defences Figure I.1. ROS result from

the body’s homeostatic response to the presence of molecular oxygen. Since it contains two

unpaired electrons, molecular oxygen is considered to be a diradical, accordingly with the

definition of a free radical - any chemical species containing one or more unpaired electrons

occupying an atomic or molecular orbital and can generate highly reactive species (Poli et

al., 2000).

Figure I.1 - Oxidative stress results from imbalance between the levels of reactive oxygen and nitrogen species (ROS/RNS) and antioxidants. Under normal circumstances, cells are able to balance the production of ROS/RNS and antioxidants, resulting in redox equilibrium. Oxidative stress occurs when cells are subjected to excess levels of ROS/RNS, or as a result of depletion in antioxidant defences.

ROS/RNS were originally considered to be exclusively detrimental to the cells but

nowadays they are recognized as key modulators in cellular functions, such as regulation of

redox cell signalling, gene modulation, neuromodulation, activation of signalling cascades,

differentiation, apoptosis and necrosis (Circu and Aw, 2010, Finkel, 2000, Yoneyama et al.,

ROS/RNSAntioxidants

ROS/RNS

Antioxidants

Equilibrium

Oxidative stress(Depleted Antioxidants)

ROS/RNS

Antioxidants

Oxidative stress(Excess ROS/RNS)

Chapter I __________________________________________________________________________

4

2010). Therefore, the classical concept of oxidative stress as “an imbalance between the

production of oxidants and the occurrence of cell antioxidant defences”, proposed by Sies H.

(Sies, 1997), is now being redefined as “a disruption of redox signalling and control that

recognizes the occurrence of compartmentalized cellular redox circuits”, as reviewed by

Packer and Cadenas (2007).

Among ROS group, the most common species are: superoxide anion radical (O2.-),

hydrogen peroxide (H2O2), peroxide anion (O22-) and hydroxyl radical (.OH). Among RNS, the

most important species are peroxynitrite (ONOO-) and nitric oxide (.NO). Mitochondria are

the main source of ROS, since generation of O2.- occurs during oxidative phosphorylation.

O2.- is easily converted into H2O2 by the action of superoxide dismutase (SOD). H2O2 can

originate .OH in the presence of iron (Fe2+) or copper (Cu+), by Fenton’s reaction. .OH is a

very potent inducer of lipid peroxidation and, along with peroxidation products, such as 4-

hydroxy-2-nonenal (HNE), is capable of impairing protein and acid nucleic functions, as well

as destroying cell membranes (Brito et al., 2007). .NO is a free radical generated from L-arginine, which is converted to L-citrulline in the

presence of O2, reduced nicotinamide adenine dinucleotide phosphate (NADPH) and

tetrahydrobiopterin, by a reaction catalysed by nitric oxide synthase (NOS) (Knowles et al.,

1989). In post-synaptic neurons, .NO is generated subsequently to activation of glutamate

receptor, mainly of the N-methyl-D-aspartate (NMDA) subtype. After this activation, Ca2+ is

transiently increased in the cytosol and forms a complex with calmodulin that binds to and

activates constitutive neuronal NOS (nNOS). Glial cells (astrocytes, microglia and

oligodendrocytes) synthesize .NO after the transcriptional expression of a Ca2+-independent

inducible NOS (iNOS). There is a third isoform of NOS, endothelial NOS (eNOS), that is

Ca2+-dependent (as nNOS) and is able to generate and release .NO from the brain

microvessels (Knowles and Moncada, 1994, Merrill et al., 1997). Different isoforms of NOS

are involved in distinct processes: nNOS is mainly involved in neuronal signalling, iNOS is

generally induced after an inflammatory stimulus and eNOS is involved in vasodilation

(Moncada and Bolaños, 2006). More recently, it was described a fourth isoform of NOS,

mitochondrial NOS (mtNOS), in rat liver mitochondria (Ghafourifar and Richter, 1997). The

mtNOS was identified as the splice variant α of the nNOS with the post-translational

modifications of myristilation and phosphorylation (Elfering et al., 2002). Although the

presence of mtNOS have been confirmed in several tissues, organs and cells, the NOS

isozyme that accounts for the formation of mtNOS is still a matter of debate. However, as

reviewed by Ghafourifar and Cadenas (2005), there is a growing notion that mtNOS is an

enzyme associated with the matrix face of the mitochondrial inner membrane, which


5

generates .NO in a Ca2+-dependent manner. It is also believed that .NO produced by mtNOS

regulates mitochondrial respiration. .NO can interact with O2

.-, generating ONOO-, which is very unstable and also a potent

inducer of lipid peroxidation. In addition, ONOO- participates in the nitration of tyrosine and in

oxidation of glutathione, processes that can impair several cellular functions (Moncada and

Bolaños, 2006). Besides being essential for neurotransmission, .NO accumulation leads to

excitotoxicity caused by over-activation of NMDA receptors (Dawson et al., 1991). However,

it should be taken into account that .NO is an important intercellular neuronal modulator and

plays a fundamental role not only in neuronal death but also in neuronal survival pathways.

Being an intercellular messenger, the rate and concentration of .NO are critical for its

modulatory function in the brain, as reviewed by Laranjinha and Ledo (2007).

In order to fight against oxidative injury, cells possess mechanisms to destroy or to expel

reactive species; these are called antioxidant defences, which can be divided enzymatic and

non-enzymatic systems. The most relevant antioxidant enzymes are SOD, catalase (CAT),

glutathione peroxidase (GPx) and glutathione S-transferase (GST). SOD, CAT and GPx

mainly have a preventive action, since they avoid oxidative damage by destroying or

inactivating ROS. GST acts by a repair mechanism, eliminating ROS-derived molecules,

such as hydroperoxides. Non-enzymatic systems are constituted by low molecular weight

compounds that act against peroxyl radicals. Some examples are: (i) α-tocopherol, that

inhibits lipid peroxidation by scavenging peroxyl radicals at the expense of a poorly reactive

radical generation, α- tocopheryl; (ii) ascorbic acid, that is able to remove the radical α-

tocopheryl, generating ascorbyl, a much less reactive radical; (iii) glutathione (γ-glutamyl-L-

cysteinylglycine), a tripeptide that serves as subtract to GPx and GST and also reacts

directly with radicals in non-enzymatic reactions, as represented in Figure I.2 (Brito et al.,

2007).

Glutathione is the most abundant cellular thiol present in mammalian cells. This

molecule constitutes one of the primary antioxidant defences of the cells, as it reacts directly

with radicals in nonenzymatic reactions and is also a donor of electrons in the reduction of

peroxides catalized by GPx (Dringen, 2000). The thiol group (SH) of cysteine serves as a

proton donor and is responsible for the biological activity of glutathione. Provision of this

amino acid is the rate-limiting factor in glutathione synthesis by the cells. In addition,

glutathione is essential for cell proliferation (Cotgreave and Gerdes, 1998) and regulation of

apoptosis (Ghibelli et al., 1998, Lu, 2009). In vivo, glutathione is synthesized by the action of

two enzymes: (i) γ-glutamylcysteine synthetase, which uses L-glutamate and cysteine to form

γ-glutamylcysteine; (ii) glutathione synthetase, which adds glycine to γ-glutamylcysteine,

Chapter I __________________________________________________________________________

6

originating the tripeptide glutathione. Both reactions require energy in the form of adenosine

triphosphate (ATP), being the first one the rate-limiting step in glutathione synthesis (Dringen

et al., 2000). Glutathione antioxidant action is extremely important in brain injury. In fact,

glutathione levels are reported to be markedly decreased in case of ischemia-reperfusion

lesion and inhibition of the enzymes involved in glutathione synthesis results in amplification

of brain damage (Mizui et al., 1992). In addition, GPx activity is considered determinant in the

recovery of the immature mouse brain subjected to traumatic brain injury (Tsuru-Aoyagi et

al., 2009) and several in vitro and in vivo studies support the neuroprotective effect of N-

acetylcysteine (NAC), an important precursor of cellular glutathione (Dringen, 2000,

Zachwieja et al., 2005) in lipid peroxidation and in antioxidant enzyme activities deficiencies

of rats’ brain (Nehru and Kanwar, 2004), as well as in hypoxia-induced oxidative stress in rat

cultured hippocampal neurons (Jayalakshmi et al., 2005).

Figure I.2 – Schematic representation of glutathione protective role in oxidative stress. Glutathione reacts directly with radicals (R.) in non-enzimatic reactions and is also a donor of electrons in the reduction of peroxides (ROOH), a reaction catalyzed by glutathione peroxidase (GPx). The resulting oxidized glutathione (GSSG) is recycled through the action of glutathione reductase (GR), a reaction dependent of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Adapted from Brito et al. (2007).

Another compound that may have some antioxidant properties is bilirubin. The ability of

low nanomolar concentrations of bilirubin to overcome large amounts of oxidants by

efficiently scavenge peroxyl radicals was explained by a redox cycling mechanism, whereas

biliverdin reductase plays a key role. Through this catalytic cycle, and as schematically

represented in Figure I.3, bilirubin is oxidized to biliverdin by reactive species, neutralizing

Gly – Cys – Glu|

S|

Gly – Cys – Glu

NADPH

NADP

GR

ROOH

ROH+ H2O

2 R.

2 RH

GPx

2 GSH

GSSG

Gly - Cys - Glu


7

their toxicity, and then is regenerated by the action of biliverdin reductase, an enzyme

dependent of NADPH (Barañano et al., 2002, Stocker et al., 1987, Brito et al., 2006).

Figure I.3 – Amplification of the antioxidant properties of bilirubin by a redox cycling mechanism. Large amounts of oxidant species (R•) can be neutralized (RH) through bilirubin oxidation to biliverdin, which is rapidly reduced back to bilirubin by biliverdin reductase, a reaction dependent of nicotinamide adenine dinucleotide phosphate (NADPH). Adapted from Brito et al. (2006).

1.2. Pathways of glucose utilization

Maintenance of cellular activity within CNS requires large amounts of energy. Catabolic

pathways, in which organic nutrient molecules are converted into smaller and simpler end

products such as lactic acid, carbon dixode (CO2) and ammonia (NH3), release energy, some

of which is conserved in the formation of ATP and reduced electron carriers [reduced

nicotinamide adenine dinucleotide (NADH), NADPH, and reduced flavin adenine

dinucleotide (FADH2)]; the rest is lost as heat. In spite of fatty acids and aminoacids can be

bioenergetic precursors, glucose constitutes the main source of energy for most cells, being

the only one in the brain. Glucose is stored as high molecular weight polymers, such as

glycogen. However, glycogen stores are very limited in the brain, thus a permanent glucose

supply via the blood stream is necessary in order to maintain brain function. In the resting

brain, oxygen is mainly used for the oxidation of glucose. In fact, although brain represents

only ~2% of the total body weight, it contributes to more that 20% of the total consumption of

both oxygen and glucose. When energy demands increase, glucose is released from

glycogen and used to produce ATP either aerobically or anaerobically. In the first step of

glycolysis, glucose is activated for subsequent reactions by its phosphorylation to yield

glucose 6-phosphate (G6P), with ATP as the phosphoryl donor, in an irreversible reaction

catalyzed by hexokinase. G6P is then degraded during the sequential reactions of glycolysis,

NADPH

NADP2 R.

2 RH

Bilirubin

Biliverdin

Biliverdinreductase

Chapter I __________________________________________________________________________

8

where some of the free energy is conserved in the form of ATP and NADH. This process

occurs in the cytosol. The end product of glycolysis is pyruvate, which may have three

distinct metabolic fates, depending on tissue and environmental conditions (Nelson and Cox,

2005). Under normoxic conditions, pyruvate is converted into acetyl-coenzyme A by pyruvate

dehydrogenase (PDH) complex, a cluster of enzymes located in the mitochondria of

eukaryotic cells. The acetyl group is then oxidized to CO2 in the tricarboxylic acid cycle

(TCA), a process where energy of oxidation is temporarily held in the electron carriers FADH2

and NADH. The electrons resulting from these oxidations are passed to O2 through a chain

of carriers in the mitochondria (mitochondrial respiratory chain), in a process called oxidative

phosphorylation. The energy released by the flow of electrons through the mitochondrial

respiratory chain complexes is used to pump protons out of the inner mitochondria

membrane through complexes I (NADH dehydrogenase), II (succinate dehydrogenase), III

(ubiquinone: cytochrome c oxidoreductase) and IV (cytochrome c oxidase), coupling NADH

oxidation and passage of protons between mitochondrial matrix and intermembrane space.

This passage generates an electrochemical gradient across the inner mitochondrial

membrane, called proton-motive force, which drives protons back into the matrix, providing

the energy necessary for ATP synthesis, by the phosphorylation of ADP into ATP by F0F1-

ATPase (complex V, ATP synthase), a process denominated chemiosmotic theory. O2

serves as the ultimate electron acceptor and is reduced to water (Nicholls and Ferguson,

2002, Bolaños et al., 2010). Pathways of glucose utilization are schematically represented in

Figure I.4.

Under conditions of hypoxia or anoxia, or in case of impairment of the components of the

mitochondrial respiratory chain, NADH cannot be re-oxidized to nicotinamide adenine

dinucleotide (NAD), in spite of its requirement as an electron acceptor for the further

oxidation of pyruvate. Under these conditions, glycolytic rate increases and pyruvate is

reduced to lactate, accepting electrons from NADH and thereby regenerating the NAD

necessary for glycolysis to continue. Although this process is less efficient from

bioenergetics’ point of view, it may occur in a necessary level to provide the energetic needs

of the cells. This alternative is made at the expense of an increase rate in glucose

consumption (Nicholls and Ferguson, 2002).


9

Figure I.4 - Schematic representation of glucose utilization pathways. Glucose catabolism can be divided into three stages: (i) glycolysis, where glucose is metabolized in enzimatic sequential reactions. In aerobic conditions, the end product is pyruvate. During glycolysis, a small portion of free energy is conserved in the form of adenosine triphosphate (ATP) and the electron carrier reduced nicotinamide adenine dinucleotide (NADH); (ii) tricarboxylic acid cycle (TCA), a process where energy of oxidation is temporarily held in the electron carriers reduced flavin adenine dinucleotide (FADH2) and NADH and also conserved in the form of ATP; (iii) oxidative phosphorylation, where the electrons carried by NADH and FADH2 passes through a chain of carriers in the mitochondria, that constitute the respiratory chain. This passage generates an electrochemical gradient across the mitochondrial inner membrane, providing the energy necessary for ATP synthesis. An alternative pathway for glucose utilization is the pentose phosphate pathway, necessary for the maintenance of redox capacity of the cell, with the formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Adapted from “Cellular respiration” from Department of Biology, University of Miami (2007).

In addition, G6P (the branching point of glucose metabolism) can be metabolized in the

pentose-phosphate pathway (PPP), an oxidative pathway where G6P is decarboxylated to

form ribose-5-phosphate, being NADPH the electron carrier that conserves the redox

potential. More important than participating in a bioenergetic metabolic route, NADPH is the

cofactor necessary for many reducing reactions, mainly those involved in fatty acids

biosynthesis and regeneration of reduced glutathione. The rate-limiting step of PPP is the

conversion of G6P into 6-phosphogluconate, catalyzed by glucose-6-phosphate

dehydrogenase (G6PD), an enzyme that is activated by oxidized glutathione (Eggleston and

Krebs, 1974) and in conditions of oxidative stress, in order to provide cytoprotection (Kletzien

et al., 1994). In addition, G6PD activation exerts its neuroprotective effects against .NO-

mediated apoptosis and glutathione depletion through up-regulation of PPP, which will

increase NADPH regeneration (García-Nogales et al., 2003, García-Nogales et al., 1999).

Therefore, NADPH plays a key role for the regeneration of reduced glutathione (GSH) from

GlycogenPentosephosphatepathway

NADPH

Acetyl-CoA

Lactate

TCAPyruvateGlucose

ATP ATP ATP

Mitochondrial respiratory chain

and oxidative phosphorylation

Glycolysis

Cytosol Mitochondria

Electronscarried via

NADH

Electrons carriedvia NADH and

FADH2

Chapter I __________________________________________________________________________

10

its oxidized form (GSSG), demonstrating that PPP is tightly connected with the maintenance

of cellular redox status (Figure I.5).

Figure I.5 - Branching point of glucose utilization. Glucose-6-phosphate (G6P), the metabolite resulting from glucose catalyzed by hexokinase, is metabolized either in glycolysis [first reaction catalyzed by glucose-6-phosphate isomerase (G6PI)] or in pentose phosphate pathway [first reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD)]. The main goal of glycolysis and subsequent metabolic pathways is energy production, whereas the main goal of pentose phosphate pathway is the maintenance of redox capacity of the cell, mainly due to production of reduced nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is an essential cofactor for glutathione reductase activity, which is responsible for regeneration of reduced glutathione (GSH) from oxidized form (GSSG).

1.3. Mitochondria: the powerhouse of the cell and the major source of ROS/RNS Mitochondria is the site where the oxidative phosphorylation machinery occurs.

However, during oxidative phosphorylation in mitochondria, electrons can directly react with

O2, generating ROS. As mentioned in section 1.2, O2 is the ultimate acceptor of electrons

that flow through mitochondrial respiratory chain complexes. However, electron leak to

oxygen through complexes I and III can generate O2.- (Figure I.6).

hexo

kina

se

ATP

Pyruvate, Lactate

Glutathione regeneration

Glucose

G6P

Energy production

Glycolysis

NADP+Glutathionereductase

GSH

GSSG

NADPH + H+

NADP+

Pentose phosphate pathwayG6PDG6PI


11

Figure I.6 - Respiratory chain is the major source of reactive species. The mitochondrial respiratory chain is embedded in the inner mitochondrial membrane (IM) and consists of complexes I–IV, coenzyme Q [ubiquinone (Q)] and ATP synthase (also denominated complex V). Cytochrome c is also a member of the chain, the only one present in the intermembrane space (IMS). These complexes are disposed in an electrochemical hierarchy based on their redox potentials. Electrons enter the chain through oxidation of either NADH at complex I or FADH2 at complex II and flow down the chain to complex IV to reduce O2 to H2O. However some O2 is reduced incompletely to superoxide anion (O2

-.) at the level of complexes I and III. In addition, mitochondria possess a NOS isoform (mtNOS), which is associated with the IM and generates nitric oxide (.NO) in a Ca2+-dependent manner. Mitochondrial .NO competes with O2 for binding to complex IV and regulates mitochondrial respiration. .NO produced by mtNOS reacts readily with O2

-. and produces the powerful oxidative species peroxynitrite (ONOO-). ONOO- produced inside mitochondria causes the release of cyto c and increases the peroxidation of mitochondrial membrane lipids. Outer mitochondrial membrane (OM). Adapted from Ghafourifar and Cadenas (2005).

The rate of O2

.- production is affected by mitochondrial metabolic state and increases

when the electron carriers harbor excess electrons, either from inhibition of oxidative

phosphorylation or from excessive calorie consumption (Nohl et al., 2005). In addition to O2.-

production, mitochondria also produces .NO, through the activity of mtNOS (Ghafourifar and

Cadenas, 2005, Ghafourifar and Richter, 1997). .NO is a physiological regulator of

mitochondrial respiration. In the arterioles, .NO promotes vasodilatation, increasing blood

flow and O2 delivery to the tissues (Clementi et al., 1999). However, .NO is capable of rapidly

and reversibly inhibit the mitochondrial respiratory chain by inhibition of complex IV, which

Chapter I __________________________________________________________________________

12

may be implicated in the cytotoxic effects in the CNS (Bolaños et al., 1994, Brown and

Cooper, 1994, Cleeter et al., 1994). When .NO is present at persistent higher concentrations,

it acts irreversibly at multiple sites, such as destruction of heme, compromising cellular

energy metabolism (Sharpe and Cooper, 1998). Additionally, inhibition of the mitochondrial

transport chain at the level of complex IV can further produce O2.- from O2 due to the

interruption of electron flow. O2.- can also react with .NO, generating the highly reactive

ONOO-. Damage to mitochondria by neurotoxins [such as 1-methyl-4-phenylpyridinium ion

(MPP+) and rotenone] generates more ROS from the electron transport chain and causes

oxidative damage that modifies proteins and other biomolecules (Szeto, 2006). Other

conditions can favor ROS production in the mitochondria, such as are high membrane

potentials, hyperoxia, excessive Ca2+ uptake and anoxia/reoxygenation (Kowaltowski, 2000).

1.4. Dysfunctional mitochondria Mitochondria plays a central position in the production of ATP and the decline of basal

metabolic rate and of physical performance in energy-requiring tasks is characteristic of

several neurological disorders. One example is mitocondrial dysfunction during the aging

process (Navarro and Boveris, 2007). An age-dependent impairment of mitochondrial

function includes: decreased electron transfer rates, increased permeability to H+ of the inner

membrane, and impairment of the driven ATP synthesis according to chemiosmotic theory.

As reviewed in Navarro & Boveris (2007), complexes I and IV activities are selectively

inhibited in isolated mitochondria from rat and mice liver, brain, heart, and kidney upon aging,

whereas complexes II and III are generally unaffected. Regarding enzyme activities of the

TCA cycle, only aconitase activity exhibited a significant decrease with age in isolated

mitochondria from kidneys of old mice and α-ketoglutarate dehydrogenase activity was

modestly decreased (Yarian et al., 2006). In the same study, the ratio of the

intramitochondrial redox indicator, NADPH/NADP+, was higher in young animals in

comparison to old ones, while the NADH/NAD+ ratio remained unchanged. Other metabolic

enzymes are reported to be selectively inhibited during the aging process, such as acyl

carnitine transferase, which catalyzes fatty acid transport to the mitochondrial matrix, thus

being essential for mitochondrial function (Liu et al., 2002). In addition, key glycolytic

enzymes activities, such as pyruvate kinase, α-enolase and triosephosphate isomerase, also

showed to be decreased in aging male monkey hearts (Yan et al., 2004).

Other neurological disorders present features of mitochondrial dysfunction, such as

hypoxia-ischemia. As reviewed by Vannucci et al. (2004), a cerebral hypoxic–ischemic event

rapidly depletes tissue energy reserves, promotes acidosis, glutamate excitotoxicity,

generation of ROS, with consequent inflammation and cell death (Vannucci and Hagberg,


13

2004). In addition, cultured neurons under conditions of hypoxia-ischemia demonstrated

specific loss of mitochondrial complex I activity, mitochondrial membrane collapse, ATP

depletion and consequent cell death (Almeida et al., 2002).

Mitochondrial dysfunction is also verified in sepsis. In fact, several studies have

implicated pro-inflammatory mediators in the impairment of metabolic function, namely at the

level of mitochondrial respiratory chain complexes and ATP production (Haden et al., 2007,

Suliman et al., 2004). Structural alterations of mitochondria were also found in intestinal

epithelial cells, hepatocytes and cardiomyocytes from septic animals, as reviewed by Wendel

and Heller (2010). Coupled with this less energetic efficiency, these neurological disorders

also present an increased production of free radicals, ROS and RNS in the mitochondria,

(Beckman and Ames, 1998, Sener et al., 2005, Vannucci and Hagberg, 2004, Wendel and

Heller, 2010). As a result, several mitochondrial proteins become nitrated, such as those

involved in TCA cycle, complex I, MnSOD, complex V, among others (Kanski et al., 2005),

which may cause inhibition of enzymatic activity. As a consequence, the mitochondrial

capacity to produce ATP is seriously compromised in this process.

2. Neuronal-glia actions and interplay in the brain Brain tissue encloses a complex network of different cells, each one with unique

structure and function. Neurons are the functioning unit of the CNS, with long processes

called dendrites and axons. Dendrites are multiple filaments that arise from the cell body,

often extending for hundreds of microns and branching multiple times, whereas axons are

single and usually ramified filaments that arise from the cell body. The interconnection of

these processes enables the reception, integration and transmission of information (Purves

et al., 2004). In contrast to neurons, glial cells do not fire action potentials, but instead

surround and enwrap neuronal cell bodies, axons and synapses throughout the CNS (Allen

and Barres, 2009). Astrocytes comprise about 85% of all glial cells, and contribute to the

maintenance of vascular, ionic, redox and metabolic homeostasis in the brain by providing

neurons with energy and substrates for neurotransmission, as well as glutathione precursors

(Allen and Barres, 2005, Dringen, 2000). Besides different brain cells have their own

particular functions and specialized machinery, bidirectional communication actually occurs

between neurons and astrocytes. This communication is essential in the maintenance of

several cellular processes, such as redox status regulation and metabolic pathways.

Chapter I __________________________________________________________________________

14

2.1. Glutathione shuttle

As mentioned in section 1.1, glutathione is synthesized by the action of two enzymes, at

the expense of ATP. Intracellular levels of glutathione are controlled by negative feedback of

γ-glutamylcysteine synthetase, thus, keeping glutathione homeostasis. These metabolic

steps occur in both neurons and astrocytes, however these two nerve cells use different

precursors for glutathione synthesis. In astrocytes, glutathione levels are limited by glutamate

content, and glutamine serves as a glutamate precursor when this aminoacid is not present.

In these cells, NAC and, most importantly, cystine serve as cysteine donors. However, since

neurons are not able to use cystine as cysteine donor, astrocytes supply the precursors

necessary for glutathione biosynthesis in neurons. Glutathione released by astrocytes is

hydrolyzed originating the dipeptide cysteine-glycine, which will be further hydrolyzed into

cysteine and glycine, taken up for neuronal usage (Dringen, 2000), as schematically

represented on Figure I.7.

Astrocytes also contain higher concentrations of glutathione, as well as greater activities

of enzymes involved in glutathione metabolism than neurons (Makar et al., 1994), indicating

that they are more resistant to ROS and that this ROS scavenging mechanism may function

to support neuronal survival. In fact, neurons co-cultured with astrocytes show increased

resistance to injury induced by .NO, H2O2 or O2.- than neurons cultured alone (Desagher et

al., 1996, Haskew-Layton et al., 2010, Lucius and Sievers, 1996) and differences in

glutathione content of neurons and astrocytes contribute to the increased susceptibility of

neurons to toxic agents that induce protein oxidation, such as unconjugated bilirubin (Brito et

al., 2008b).


15

Figure I.7 - Schematic view of interplay between astrocytes and neurons regarding glutathione metabolism. Neurons and astrocytes synthesize reduced glutathione (GSH) by the action of two enzymes: γ-L-glutamyl-L-cysteinylglycine (γGluCys) synthase, which uses glutamate (Glu) and cysteine (Cys) as substrates and GSH synthase, which combines γGluCys with Gly. While astrocytes are able to take up cystine through sodium independent channel and break it down to yield Cys. In contrast, neurons cannot use cystine as a Cys donor, therefore in these cells the rate limiting step of GSH synthesis is the usage of Cys and neurons rely on astrocytes for GSH synthesis. Reactive oxygen species (ROS) oxidize GSH to oxidized glutathione (GSSG), which is recycled back to GSH by the action of glutathione reductase (GR). Glutamine (Gln) and glycine (Gly). Adapted from Brito et al. (2007).

2.2. Glutamate shuttle

Glutamate toxicity plays an important role in neuronal cell death during brain injury (Yi

and Hazell, 2006). Therefore, control of extracellular glutamate levels is very important to the

prevention of neuronal excitotoxicity by excessive activation of glutamate receptors.

Astrocytes have an essential role in the maintenance of glutamate levels under the toxic

threshold, since they have Na+-dependent transporters which are responsible for the

clearance of glutamate from extracellular space, at the expense of ATP (Anderson and

Swanson, 2000). As schematically represented in Figure I.8, once taken up by astrocytes,

glutamate can be metabolized in different ways, of which glutamine formation and entry into

the TCA cycle are the most important. Glutamine formation is catalyzed by glutamine

synthetase, an enzyme present in astrocytes and in oligodendrocytes, but absent in neurons

(Suárez et al., 2002). Neuronal glutamate is also formed from α-ketoglutarate, a metabolite

produced in TCA cycle. Astrocytes take up leucine and transfer its amino group to

Neurons relie on astrocytes for GSH synthesis (Glu‐Cys‐Gly)

Chapter I __________________________________________________________________________

16

α-ketoglutarate by the action of branched-chain aminoacid transaminase, originating

α-ketoisocaproate. α-ketoisocaproate is then transferred to neurons and can originate

α-ketoglutarate by the reverse reaction (Daikhin and Yudkoff, 2000). Oxidative metabolism of

α-ketoglutarate produces more than 30 ATP, about 20-fold more than required for glutamate

uptake. In conditions of oxidative stress there is ATP depletion, which originates cessation of

glutamate uptake in astrocytes, together with its efflux. Accumulation of glutamate in the

synaptic cleft results in excitotoxicity phenomenon and neuronal death (Santos et al., 1996).

Therefore, astrocytes play an important role in the protection against oxidative stress-

induced excitotoxicity (de Arriba et al., 2006). In addition, astrocytes do use glutamate

released by neurons, for example in the synthesis of glutathione, as mentioned in 2.1, thus

removing excess of glutamate from the brain, which can be toxic when in elevated levels

(Dringen and Hamprecht, 1996).

Figure I.8 – Schematic view of interplay between astrocytes and neurons regarding glutamate metabolism. Astrocytes support neuronal glutamate metabolism. Glutamate (Glu) is released during neurotransmission and is taken up primarily by neighboring astrocytes through excitatory amino acid transporters. A portion of astrocytic Glu is converted to glutamine (Gln) by glutamine synthetase, which is abundant in astrocytes and absent in neurons. Gln is released from astrocytes and taken up by neurons through specific transporters. In neurons, Gln is deaminated into Glu by mitochondrial glutaminase. Neuronal Glu is also formed from α-ketoglutarate (α-KG). Astrocytes take up leucine (Leu), and the amino group of Leu is transferred to α-KG by branched-chain amino acid (BCAA) transaminase. Pyr, pyruvate; Ala, alanine; Pi, inorganic phosphate. Adapted from Chen and Swanson (2003).


17

2.3. Lactate shuttle

The coupling between synaptic activity and glucose utilization (neurometabolic coupling)

is a central physiological principle of brain function. Neurons and astrocytes are the two

major contributors for the massive consumption of oxygen and glucose in the brain. While

glycolysis occurs preferentially in astrocytes, most of the oxygen is consumed by neurons

(Jolivet et al., 2009). Under resting conditions, astrocytes metabolize ~85% of the glucose

consumed in lactate. As schematically represented in Figure I.9, glycogen, the main energy

store in the brain, is localized predominantly in astrocytes. Upon neuronal stimulation with

glutamate, both glucose uptake and lactate production are observed in surrounding

astrocytes (Pellerin and Magistretti, 1994). In addition to glucose, lactate (mainly provided by

astrocytes) can constitute a supplementary fuel for activated neurons. In fact, as reviewed by

Pellerin et al. (2007), a major glycolytic response in astrocytes upon activation, either by

direct application of glutamate or stimulation of glutamatergic pathways, represent an

important lactate source. Lactate accumulated in both extracellular and intracellular space in

astrocytes constitutes a pool readily available for neurons upon increased energy demands.

Upon neuronal activation, there is a rapid decrease in mitochondrial NADH in dendrites and

then an increase in TCA cycle activity, in order to replenish the mitochondrial NADH pool

(Kasischke et al., 2004). Moreover, there are additional reports that came to the conclusion

that lactate is the predominant oxidative substrate over glucose in cultured neurons (Itoh et

al., 2003, Bouzier-Sore et al., 2003).

In addition to glutamate/glutamine cycling between neurons and astrocytes referred in

2.2, neurons also rely on astrocytes for the supply of metabolic intermediates, particularly

oxaloacetate, formed by the condensation of pyruvate with CO2 (Haberg et al., 1998),

allowing the further synthesis of glutamate or γ-aminobutyric acid. Therefore, a transfer of

glucose-derived metabolites from glial cells to neurons is necessary for neuronal survival,

especially during severe hypoglycemia (Forsyth, 1996, Wender et al., 2000).

Chapter I __________________________________________________________________________

18

Figure I.9 - Neural activity triggers the release of the neurotransmitter glutamate (Glu) that is taken up into the astrocyte, and stimulates the breakdown of glycogen, the uptake of glucose, and glycolysis, to produce lactate in astrocytes. Astrocytic released glutamine (Gln) favors synaptic process, whereas astrocytic released lactate stimulates neuronal glucose uptake. Since neurons use more energy than they are able to produce by themselves, interplay with astrocytes constitutes an essential additional source of energy. Adapted from Magistretti (2006).

2.4. Neuronal susceptibility to oxidative stress

2.4.1. Increased oxidant capacity in the brain Mammalian brain cells are particularly susceptible to oxidative damage, since they

present higher oxidant capacities. The first reason is because large amounts of ATP are

required to maintain neuronal processes. As a consequence, in neuronal cells, a high O2 and

glucose consumption occurs, leading to a continuous production of ROS during oxidative

phosphorylation process. In fact, electrons leak to O2 through complexes I and III of the

respiratory chain, thus generating O2.-.

Brain cells are also more susceptible to oxidative stress because of the presence of

excitatory aminoacids. Oxidative stress damages neurons and induces the release of

glutamate. This aminoacid will bind to NMDA receptors on adjacent neurons, leading to an

increase in intracellular Ca2+ within them (Mailly et al., 1999). This increase in intracellular

Ca2+ concentrations can induce massive production of .NO, by activation of nNOS, a

Ca2+dependent enzyme, as mentioned in section 1.1. Rise in Ca2+ levels affects

mitochondrial function, contributing to the generation of O2.-. The excess of O2

.- may react

with .NO, generating ONOO-, which is responsible for inactivation of glutamine synthetase by

Glycolysis

Glycogen


19

tyrosine nitration (Görg et al., 2007). As a consequence of these events, it may occur an

increase in extracellular levels of glutamate, thus promoting excitotoxicity.

In addition, several neurotransmitters present in the brain, such as dopamine, serotonin

and norepinephrine are autoxidizable. By reacting with O2, they can generate O2.-, as well as

quinones/semiquinones that bind to thiol groups of reduced glutathione, causing its depletion

(Wrona and Dryhurst, 1998).

Another fact that accounts for brain increased susceptibility to oxidative stress is the

elevated concentrations of iron, mostly contained in ferritin in healthy brain (Burdo and

Connor, 2003). However, in damaged brain, iron accumulation is excessive relative to the

amount of ferritin and it will catalyze free radical reactions, namely Fenton’s reaction.

Neuronal membrane lipids are enriched in unsaturated fatty acids, which are thought to

be target molecules for free radical-induced peroxidation and neural cell damage, thus

playing a major role in the pathogenesis of many neurological diseases. HNE, one of the

main products of lipid peroxidation, especially induces neuronal cytotoxicity by increasing

Ca2+ levels, which will inactivate glutamate transporters and damage neurofilament proteins

(Mark et al., 1997). HNE also inactivates α-ketoglutarate dehydrogenase, a key enzyme in

TCA cycle (Sheu and Blass, 1999).

Brain metabolic pathways are also responsible for huge generation of H2O2, not only by

the action of SOD, as described in section 1.1, but also by other enzymes, being monoamine

oxidases A and B and flavoproteins located in the outer mitochondrial membranes of

neurons and glia particularly important for this process (Gal et al., 2005). Furthermore,

neuronal NADPH oxidase enzymes (NOX) become activated in response to oxidative stress

and may promote neuronal apoptosis. This process is extremely important during

development of the nervous system; however, if trophic support is lost in the developed

brain, NOX can become overactivated and leading to neuronal apoptosis (Sánchez-Carbente

et al., 2005, Tammariello et al., 2000).

Astrocytes and microglial cells can also contribute to oxidant environmental conditions,

when they become activated by inflammatory features, such as pro-inflammatory cytokines.

Activated astrocytes and, especially, activated microglia may produce O2.- and H2O2 and,

.NO, by activation of iNOS. Thus, activated glial cells are major players in oxidative stress

induced by inflammatory processes in the brain. Commonly, in studies with isolated cultures,

astrocytes appear less susceptible to ROS and RNS than neurons, since they have higher

glutathione levels and are more able to promote its synthesis under stress than neurons

(Halliwell, 2006).

Chapter I __________________________________________________________________________

20

2.4.2. Antioxidant capacity in the brain Efficiency of antioxidant defences of brain cells is low when compared to that of other

tissues. In fact, catalase levels are low in most brain regions, specifically located in

peroxisomes and are hardly able to counteract H2O2 produced in other cellular compartments

(Angermüller et al., 2009).

In order to battle against oxidative stress, all parts of the brain contain SODs with active-

site for manganese (MnSOD) in the mitochondrial matrix and for cooper/zinc (CuZnSOD) in

the mitochondrial intermembrane space and in the rest of the cell. Curiously, neurons

containing nNOS are reported to be relatively resistant to NMDA and .NO-mediated

neurotoxicity, by a mechanism involving MnSOD activation (Gonzalez-Zulueta et al., 1998).

Glutathione/GPx system is also present within all nervous cells. Since neuronal

concentrations of glutathione are lower than in glia, these cells might assist neurons by

supplying them with cysteinyl-glycine as a glutathione precursor. In fact, glutathione released

by astrocytes can be degraded by γ-glutamyl transpeptidase on their cell surface to produce

cysteinyl-glycine, which neurons then further cleave to release cysteine for uptake and use in

glutathione synthesis (Dringen et al., 2005).

In addition to glutathione, brain cells are enriched in low molecular compounds with

antioxidant activity, mainly ascorbate. In fact, neurons have specific transporters that

efficiently take up ascorbate and astrocytes take up dehydroascorbate and convert it to

ascorbate in intracellular space (Rice, 2000). However, in damaged brain, ascorbate can

stimulate the oxidation of Fe3+ and Cu2+ into Fe2+ and Cu+, respectively, thus potentiating

Fenton’s reaction and formation of .OH. Another low molecular compound that is present in

the brain is α-tocopherol, mostly derived from plasma high-density lipoprotein (Hayton and

Muller, 2004). Furthermore, brain contains elevated levels of histidine-containing dipeptides,

known for their antioxidant properties, namely, by chelating metal ions and binding cytotoxic

aldehydes produced during lipid peroxidation (Aruoma et al., 1989, De Marchis et al., 2000,

Decker et al., 2000).

Finally, bilirubin has antioxidant properties (Barañano et al., 2002, Stocker et al., 1987).

Heme oxygenase (HO) is a widespread enzyme in the brain, existing in both inducible

(HO-1) and constitutive (HO-2) isforms. HO catalyses the degradation of heme, with

generation of carbon monoxide (CO), which can act as neurotransmitter, and biliverdin that

will be further converted to bilirubin by biliverdin reductase. Although heme degradation

causes the release of Fe2+, which can potentiate .OH formation as abovementioned, HO is

involved in antioxidant mechanisms. HO-2 activation is able to prevent neuronal death in

cerebral ischemia (Doré et al., 2000, Doré et al., 1999) and HO-1 is rapidly upregulated by

oxidative and nitrosative stresses in some neurodegenerative diseases, as an attempt to


21

convert the highly damaging heme into the biliverdin and bilirubin (Calabrese et al., 2005).

However, bilirubin breakdown by ROS originates bilirubin oxidation products, which can

produce vasoconstricting compounds (Pyne-Geithman et al., 2005) and high levels of

bilirubin are neurotoxic, as it will be further discussed in section 4, due to its relevance for the

present thesis.

2.5. Neuronal susceptibility bioenergetic crisis

Astrocytes and neurons respond differently to .NO-induced inhibition of mitochondrial

respiration. In fact, whereas neurons suffer a rapid decline in ATP levels, a collapse in

mitochondrial membrane potential (ΔѰm) and apoptotic cell death, astrocytes utilize

glycolytically-generated ATP, thus maintaining their ΔѰm (Almeida et al., 2001). This

differential response in not exclusively to impaired mitochondrial respiration, since it also

occurs in case of over-activation of neuronal glutamate receptors, an event that inhibits

mitochondrial ATP synthesis or glucose uptake (Almeida and Bolaños, 2001, Porras et al.,

2004).

It was reported that one of the main reasons why neurons and astrocytes respond

differently to .NO-induced inhibition of respiration is the fact that they have very lower activity

levels of 6-phosphofructo-1-kinase (PFK1), a master regulator of glycolysis, in comparison to

astrocytes. The content of fructose-2,6-bisphosphate (F2,6P2), the powerful allosteric

activator of PFK1, is also lower in neurons. In addition, mitochondrial respiration inhibition

induced F2,6P2 in astrocytes, whereas has no effect on neuronal F2,6P2 (Almeida et al.,

2004). Recently, it was suggested that neurons are unable to increase glycolysis because

they almost does not possess 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

(PFKFB), which is the enzyme responsible to F2,6P2 formation (Herrero-Mendez et al.,

2009).

Glucose metabolism in neurons is directed mainly to the PPP, with the main goal of

reduced glutathione regeneration. In fact, the antioxidant function of the PPP in neurons was

demonstrated in response to pro-oxidant compounds exposure (García-Nogales et al., 2003,

Vaughn and Deshmukh, 2008) or glutamate receptor stimulation, mainly NMDA (Delgado-

Esteban et al., 2000). As described in section 1.1, over-activation of NMDA receptors favors .NO formation by activation of nNOS, a process implicated in the pathogenesis of several

CNS diseases (Suárez et al., 2002).

Chapter I __________________________________________________________________________

22

3. Inflammation and cell death in central nervous system

3.1. Cells involved in inflammation and CNS injury The inflammation in the CNS, also designated as neuroinflammation, represents an

essential response to peripheral inflammation and CNS injury or infection, necessary to the

maintenance of tissue survival, repair and recovery, and to conserve the energy of the

organism, by limiting the survival and proliferation of invading pathogens. The produced

damage may have different causes like infection, traumatism, ischemia, necrosis,

hemorrhage, among others. However, it is also recognized as a major contributor to acute

and chronic CNS disorders. As reviewed by Allan and Rothwell (2003), inflammatory

mediators, such as complement, adhesion molecules, cyclooxygenase enzymes and pro-

inflammatory cytokines, are increased in several neurological diseases, and intervention

studies in experimental animals suggest that several of these factors contribute directly to

neuronal injury.

In the past, studies on CNS injury have focused predominantly on neuronal death and

survival, since these cells largely determine CNS function and survival and cannot be

replaced once they are lost. However, the role of other cell types in CNS disease is

becoming increasingly apparent. Glial cells constitute the majority of the brain volume and

play an active role in normal physiology and pathology (Raivich et al., 1999). The primary

glial cells implicated in neuroinflammation are microglia. These are cells of the

monocyte/macrophage lineage, which are resident in the brain and are activated in response

to infection, inflammation and injury (Streit, 2002). They are important phagocytic cells and

release numerous inflammatory molecules, namely cytokines (Hanisch, 2002). Another glial

cell type important for neuroinflammation is astrocytes, which are the most abundant glial

cells, and play key physiological roles in supporting neurons, regulating ion and transmitter

concentrations and in electrical transmission, and are an important source of both

neuroprotective and inflammatory molecules (Allan and Rothwell, 2003). Oligodendrocytes,

the third type of glia, are crucial for myelination, but are also a source of specific

inflammatory molecules (Baumann and Pham-Dinh, 2001, Du and Dreyfus, 2002). Vascular

endothelial cells are also targets and sources of inflammatory mediators, being particularly

important in the adhesion of circulating cells of the immune system (Couraud, 1994).

3.2. Inflammatory mediators and signalling pathways Cytokines are among the major effectors of neuroinflammation. They can be involved in

either neuroprotection or neurodegeneration processes (Konsman et al., 2007). Specific pro-

inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) β,


23

have pleiotropic effects in the CNS, including their emerging role in neurodevelopment (Marx

et al., 2001) but are also described as mediators of neuronal apoptosis (Kajta et al., 2006).

TNF-α exerts its biological activity by binding to type 1 and type 2 receptors (TNFR1 and

TNFR2) and activating several signalling pathways. TNFR1 contains a common death

domain whereas TNFR2 does not. Thus, TNFR1 activation is involved in both cell survival

and cell death signalling, while TNFR2 mediates cell survival signals. However, it is

suggested that TNFR2 might potentiate death signal mediated by TNFR1 (Gupta, 2002).

Like TNF-α, IL-1β is a pro-inflammatory cytokine associated with several CNS

disorders. Activated microglial cells are the main source of this cytokine in the damaged

brain (Block and Hong, 2005). Released IL-1β directly affects neurons, astrocytes and

oligodendrocytes, promoting production of other cytokines and regulation of synaptic function

on hippocampal neurons (Bellinger et al., 1993). In the opposition of the dual role of TNF-α in

brain damage, IL-1β is mainly considered by its neurotoxic effects (Panegyres and Hughes,

1998, Yang et al., 1998).

The effects of cytokines depend on which cell type they act upon and whether it is a

direct or indirect effect (Allan and Rothwell, 2003). As reviewed by Allan and Rothwell (2003),

studies in vitro demonstrated that cytokines directly act on neurons, promoting changes in

Ca2+ entry, neurotransmitter release and synaptic plasticity, thus contributing to neuronal

viability in the injured brain. It is speculated that neuronal responses can be modified,

indirectly or directly, by cytokines. One example of this is in case of seizure activity, which is

enhanced by IL-1 administration (Vezzani et al., 1999). In addition, pro-inflammatory

cytokines such as IL-1 and TNF-α are reported to induce blood–brain barrier breakdown

(Blamire et al., 2000, Cardoso et al., 2010, Quagliarello et al., 1991), as well as to trigger the

release of toxic substances, such as .NO from the vascular endothelium (Bonmann et al.,

1997), allowing the entrance of leucocytes into the brain parenchyma, which will contribute to

neuronal injury.

In addition, several studies demonstrated the association between inflammation and

generation of ROS/RNS, leading to multiple organ dysfunction (Bian and Murad, 2001, Sener

et al., 2005). .NO is recognized as a mediator and regulator of inflammatory responses. It

was first reported that mouse macrophages produce nitrite and nitrate in response to

bacterial lipopolysaccharide (Stuehr and Marletta, 1985). However, although high levels of .NO produced in response to inflammatory stimuli can have deleterious effects, this molecule

is also important in cellular signalling, having an important role in the amelioration of the

pathogenesis of inflammation (Korhonen et al., 2005). Furthermore, .NO and induction of

Chapter I __________________________________________________________________________

24

NOS are involved in apoptosis induced by inflammatory mediators in neuronal cells (Hemmer

et al., 2001, Heneka et al., 1998, Thomas et al., 2008).

The mitogen-activated protein kinases (MAPKs) and the transcription factor nuclear

factor κB (NF- κB) are among the main effectors that participate in inflammatory signalling

pathways. MAPKs are divided into three major subfamilies, according to structural

differences between them: the p38 kinase, the c-Jun N-terminal kinases 1 and 2 (JNK1/2)

and the extracellular signal-regulated kinases 1 and 2 (ERK1/2), as reviewed by Roux and

Blenis (2004). In general, p38 and JNK1/2 are more responsive to environmental stress and

pro-inflammatory cytokines, being designated as stress-activated protein kinases (SAPKs),

while ERK1/2 are mostly activated in response to mitogens and growth factors (Kyriakis and

Avruch, 2001). NF- κB is an important transcription factor responsible for modulation of the

host immune and inflammatory response (O'Neill and Kaltschmidt, 1997). It will be given

preferential attention to JNK1/2, since their activation is discussed in the present thesis.

JNK1/2 become activated in response to toxic stimulus, such as ROS (Luo et al., 1998,

Marques et al., 2003) and pro-inflammatory cytokines TNF-α and IL-1, pointing these SAPKs

as strong effectors of neuronal apoptosis (Mielke and Herdegen, 2000, Tibbles and

Woodgett, 1999). The activation of JNK1/2 enzyme is related to toxicity in developing

neurons, since overexpression of activated c-Jun was shown to produce apoptosis and

suppression of this protein protected against neuronal death induced by deprivation of nerve

growth factor in sympathetic and hippocampal neurons (Estus et al., 1994, Ham et al., 1995,

Schlingensiepen et al., 1993). In addition, .NO-induced JNK phosphorylation is observed in

models of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s (Katsuki et al.,

2006, Marques et al., 2003).

3.3. Neuronal susceptibility to inflammation In spite of being essential to tissue survival, repair and recovery, extensive, prolonged or

unregulated inflammation is highly detrimental. During the last decades it has been observed

that not only cells of the immune system participate actively in the inflammation, but also

cells belonging to the CNS are a fundamental part of this process, especially glial cells, as

indicated in section 3.1. The neurons have a minor participation in inflammatory processes,

however they have the ability to express class I molecules, to produce several cytokines like

IFN-γ and also to induce apoptosis of T cells through the Fas receptor (FasR) interaction

(Chavarria and Alcocer-Varela, 2004).

TNF-α is the pro-inflammatory cytokine most characterized in neurologic diseases. This

cytokine directly affects every neural cell, by inducing the release of other cytokines in glial

cells (Allan and Rothwell, 2003), several chemokines (Croitoru-Lamoury et al., 2003) and


25

.NO (Madrigal et al., 2002). TNF-α has been demonstrated to induce neuronal apoptosis in

human brain cell cultures and animal models through TNFR1 signalling (Yang et al., 2002)

and further production of other inflammatory and/or neurotoxic molecules such as ONOO- by

induction of iNOS in glial cells (Combs et al., 2001). In addition, post-treatment with TNF-α

potentiates NMDA-mediated toxicity in organotypic hippocampal slice cultures (Wilde et al.,

2000). TNF-α produced by glial cells was also found to damage neural precursor cells and to

inhibit neurite elongation and branching during development and regeneration (Sheng et al.,

2005). Furthermore, it is reported that during aging, both TNF-α production and TNF-α-

induced apoptosis are increased (Gupta, 2002).

Other cytokine that is widely accepted for its role in neuronal injury is IL-1β (as referred

in section 3.2). In fact, increased expression of this cytokine in CNS is observed after a

variety of brain insults, and administration of exogenous IL-1β to animals undergoing

ischemic or excitotoxic challenges leads to a dramatic increase in the resulting cell death

(Allan and Rothwell, 2003). In addition, administration of the selective IL-1 receptor

antagonist (IL-1ra) clearly inhibits the extent of cell death induced by ischemic, traumatic or

excitotoxic injury in the mouse brain (Rothwell and Luheshi, 2000).

3.4. Death signalling pathways The principal mechanisms implicated in the death signalling cascades of the CNS cells

include the alteration of Ca2+ homeostasis, oxidative and nitrosative stress, the accumulation

of extracellular neurotransmitters, as glutamate and the activation of signalling cascades

(Neumar, 2000), as schematically represented in Figure I.10.

Chapter I __________________________________________________________________________

26

Figure I.10 - Major mechanisms of nerve cell death upon an insult. Overstimulation of N-methyl-D-aspartate receptors (NMDAR) by an accumulation of extracellular glutamate leads to an accumulation of intracellular Ca2+. Elevated intracellular Ca2+ triggers DNA fragmentation, reactive oxygen and nitrogen species (ROS/RNS) formation, calpein activation and reduction of mitochondrial membrane potential with cytochrome c (Cyt c) release. Released Cyt c, together with protease activating factor 1 (Apaf 1) and pro-caspase 9 constitute the apoptossome. This association results in activation of caspase-9 that will activate effector caspases, such as caspase-3. Engagement of the membrane receptors TNF-α receptor 1 (TNFR1) or Fas receptor (FasR), activates specific apoptotic effectors, such as caspase-8 or -10. Activated caspase-8 propagates the apoptotic signal by activating downstream caspases through proteolytic cleavage, as well as by triggering mitochondrial pathway through cleavage and activation of pro-apoptotic Bid into tBid, which in turn promotes mitochondria dysfunction with Cyt c release. Like Bid, other proteins of Bcl-2 family will modulate mitochondrial apoptotic pathways by enhancing (Bax) or preventing (Bcl-2) the formation of mitochondrial permeability transition pore and the release of Cyt c. Dysfunctional mitochondria is also a source of ROS/RNS. ROS/RNS may directly promote DNA oxidation.

When ATP depletion occurs, there is a neuronal depolarization, with substantial release

of glutamate at the synaptic cleft (Santos et al., 1996). ATP depletion also inhibits re-uptake

of glutamate by glial cells (Di Monte et al., 1999), leading to extracellular accumulation of

glutamate. Accumulation of glutamate in the synaptic cleft results in excitotoxic phenomenon

and neuronal death (Santos et al., 1996). Glutamate will bind to NMDA receptors on adjacent

neurons, leading to an increase in intracellular Ca2+ within them, which may result in

neurodegeneration (Mailly et al., 1999). This increase in Ca2+ concentrations can induce

ROS/RNS

Ca2+

Ca2+

DNA f ragmentation

Bid

Caspase-8/10

Bcl2tBid Bax

Cyt c

Pro-Caspase-9

Apaf 1 Cyt c

Caspase-3/7

Caspase-9

ROS/RNS

Caspase-12

Calpein activation

Glutamate

Cyt c


27

massive production of .NO, by activation of nNOS. Excessive elevation of intracellular Ca2+

also leads to the activation of hydrolytic enzymes and triggers mitochondrial permeability

transition and activation of many enzymes, including phospholipases and calpains. Calpain

activation is coupled to execution of caspase-independent apoptosis in cerebellar granule

neurons (Volbracht et al., 2005). Furthermore, cysteine proteases, including caspases are

also sensitive to the redox balance of the cell (Blomgren et al., 2007). In addition,

mitochondrial dysfunction induced by increased production of ROS/RNS is accompanied by

activation of caspase-3 and DNA fragmentation (Gilland et al., 1998, Puka-Sundvall et al.,

2000). This mitochondrial dysfunction may facilitate the release of proapoptotic factors from

the intermembrane space of the mitochondria to the cytosol, such as cytochrome c,

apoptosis-inducing factor 1 (Apaf 1), endonuclease G, SMAC/Diablo and HtrA2/Omi

(Ravagnan et al., 2002). Release of cytochrome c interacts with Apaf 1 and dATP/ATP to

form the apoptosome, leading to activation of pro-caspase-9 into the initiator caspase-9

(Acehan et al., 2002), which in turn cleaves and activates pro-caspase-3, the most abundant

effector caspase in the brain, and consequent activation of the apoptotic cell death

(Matapurkar and Lazebnik, 2006). The apoptotic pathway may also be triggered by the

engagement of the membrane receptors TNFR1 or FasR, often referred as “death

receptors”, since they are coupled to specific apoptotic effectors, such as caspase-8 or -10

(Walczak and Krammer, 2000). Activated caspase-8 propagates the apoptotic signal by

activating downstream caspases through proteolytic cleavage, as well as by triggering

mitochondrial pathway through cleavage and activation of pro-apoptotic Bid into tBid, which

in turn promotes mitochondria dysfunction and cytochrome c release (Adams, 2003).

The role of the inflammatory caspases (mainly caspase-1, also called IL-1 converting

enzyme) in apoptosis is not clear, however they may contribute to brain injury after ischemia

through their pro-inflammatory actions (Blomgren et al., 2007).

4. Bilirubin induced neurological damage and risk factors involved

4.1. Neonatal hyperbilirubinemia In fetal life, bilirubin production begins as early as 12 weeks’ gestation. In this period,

bilirubin clearance is made through its passage to maternal circulation. At birth, this placental

protection is suddenly lost and an accumulation of UCB takes place (Brito et al., 2006).

Neonatal hyperbilirubinemia occurs due to the three main reasons: (i) bilirubin

overproduction because in neonatal life there is a shortened red blood cell lifespan; (ii)

decreased bilirubin conjugation in the liver, due to immaturity of newborns’ hepatic

Chapter I __________________________________________________________________________

28

machinery; (iii) impaired bilirubin excretion because of the absence of bacterial flora (Porter

and Dennis, 2002).

Neonatal hyperbilirubinemia is a very common condition in the neonatal period, with total

serum bilirubin levels above to 5 mg/dl. This condition occurs in up to 60% of full term

newborns and 80% of preterms (Dennery et al., 2001). Commonly designated as neonatal

jaundice, this condition is characterized by accumulation of unconjugated bilirubin (UCB) in

the skin and mucous membranes, responsible for the yellow-orange coloration observed in

jaundiced babies (Stevenson et al., 2001).

Although most of newborn infants have mild to moderate elevated serum UCB levels

within the first days of life, a condition known as “physiologic jaundice”, higher levels of UCB,

known as “pathologic jaundice” cause nerve cell damage, a condition called UCB

encephalopathy, that may lead to adverse neurological outcomes (Hansen, 2002). In fact,

moderate degrees of hyperbilirubinemia may be a starting point to the appearance of long-

term neurodevelopment disabilities (Dalman and Cullberg, 1999, Soorani-Lunsing et al.,

2001). Neurologic dysfunctions reported to be related with elevated concentrations of UCB in

neonatal period include risk of otoxoxicity and hearing loss (de Vries et al., 1985), as well as

visual acuity or mild-to-moderate cerebral palsy (Sampath et al., 2005) in extremely low-birth-

weight infants. Changes in the auditory brainstem response were also found in rhesus

monkeys during the intravenous infusion of UCB (Ahlfors et al., 1986). In addition, high

levels of UCB can constitute the basis for chronic to permanent sequelae, or even death

(Ostrow et al., 2004, Shapiro, 2005). As reviewed by Hansen (2000), the term kernicterus

was first used by Schmorl in 1904 to describe the yellow staining of some brain regions,

notably basal ganglia and medulla oblongata, observed in postmortem analysis of brains of

term neonates. Statistically, around 70% of infants with kernicterus dye within seven days,

and the ~30% survivors commonly develop irreversible sequelae such as auditory

dysfunction, mental retardation and choreoathetoid cerebral palsy (Blanckaert and Fevery,

1990).

4.2. Prematurity as a risk factor of neonatal hyperbilirubinemia The risk of bilirubin-induced neurologic dysfunction is particularly enhanced in premature

newborns due to the higher rates of UCB production because of the shorter life span of their

red blood cells. This fact contributes to an increased UCB production, since this molecule

results from the degradation of heme proteins (Blanckaert and Fevery, 1990). In addition,

prematures present some metabolic deficiencies at the level of excretion pathways, that will

account for the decreased UCB clearance from the organism (Stevenson et al., 2001,


29

Watchko, 2006). Moreover, cerebral palsy was found in preterm infants with risk of

kernicterus, in spite of relatively low total serum bilirubin levels (Gkoltsiou et al., 2008).

Prematurity is frequently associated with hypoalbuminemia (Cartlidge and Rutter, 1986),

which will contribute to increased levels of free UCB, since bilirubin is released in circulation

reversibly bound to albumin, until reaching into the liver in order to be metabolized. If

concentrations of albumin are lower, a higher rate of free UCB easily crosses the blood brain

barrier, which is also immature and permeable and presents a reduced content in tight

junctions and pericytes, together with a more fragile brain vasculature in preterm newborns

(Ballabh et al., 2004). Furthermore, premature infants present a higher incidence of neonatal

pathophysiological processes such as hypoxia-ischemia insult and cerebral hemorrhage that

can contribute for their increased vulnerability to brain damage in comparison to the full term

infants (Volpe, 1997). Hypoxic-ischemic conditions may lead to development of acidosis

(O'Shea, 2002), contributing to an environment with lower pH, which favors cellular

deposition of UCB (Ostrow et al., 1994).

It should be noticed that in preterms is complicated to establish a threshold at which

UCB interferes with neurodevelopment outcome, since these infants are commonly clinically

ill, with other perinatal complications. Thus, UCB-induced neurotoxicity may be more

pronounced that in full term ones, even at relatively low levels of UCB (Oh et al., 2003). In

fact, low weight premature infants present decreased albumin concentration and a lower

affinity and/or capacity for UCB binding (Cashore, 1980, Kaplan and Hammerman, 2005).

4.3. Sepsis-associated neonatal hyperbilirubinemia Premature newborns are also more susceptible to some insults that are described as

risk factors for UCB-induced encephalopathy. In fact, prematurity is frequently associated

with sepsis, which is responsible for the alteration of blood brain barrier permeability through

the release of great amounts of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6

(Goldenberg and Andrews, 1996). In addition, a correlation between infection and the

increased risk of UCB-induced neurotoxicity is reported: (i) in an animal model of sepsis, it

was shown that serum concentration of both total and free bilirubin was increased, promoting

a net accumulation of UCB in the brain (Hansen, 1993); (ii) pro-inflammatory cytokines were

reported to increase blood-brain-barrier permeability, allowing UCB entrance in the brain

(Petty and Lo, 2002), and to exacerbate UCB-induced cytotoxicity in different cell lines, such

as in neuroblastoma, glioblastoma, umbilical vein endothelial, liver cell and mouse fibroblasts

(Ngai and Yeung, 1999), as well as in astrocytes (Fernandes et al., 2004).

The association between inflammation and generation of ROS/RNS described in section

3.2 should also be taken into account for the increased susceptibility in sepsis-associated

Chapter I __________________________________________________________________________

30

hyperbilirubinemia, since immature brain lacks antioxidant defences, such as catalase and

GPx, which may help to explain the differential susceptibility of the developing CNS to brain

injury (Chang et al., 2005). As discussed in section 3, although essential for survival in

response to tissue injury or infection, inflammatory response also causes neuronal damage,

through an increased production of pro-inflammatory cytokines, as well as .NO and low-

gestational-age newborns have a prominently increased risk of brain dysfunctions attributed

to cerebral-cortex damage, including excess of apoptosis and impairment of surviving

neurons (Leviton and Gressens, 2007). In addition, It has been suggested that infection

increases the risk for UCB encephalopathy (Dawodu et al., 1984) and presence of

inflammatory features, namely fever episodes and brain edema, were described during or

following moderate to severe hyperbilirubinemia (Kaplan and Hammerman, 2005). As

schematically represented in Figure I.11, inflammatory response of astrocytes and microglia

causes an increase of apoptotic neurons, which may produce neuropathological sequelae.

Interestingly, lipopolysaccharide exacerbates the release of TNF-α and IL-1β by cultured

astrocytes (Falcão et al., 2005). Because of the relevance for the present dissertation, this

issue will be further dissected into section 4.5.

.

Figure I.11 - Astrocytes and mostly microglia produce an inflammatory response in response to a toxic stimulus and may produce neuropathological sequelae.

astrocyte

Inflammatoryresponse

Apoptosis Degeneration

Developmental diseasemicroglia

Immatureneuron

Differentiatedneuron

Toxicstimulus


31

4.4. Differential neuronal vulnerability among brain regions Differential susceptibilities may be related with preferential deposition within brain

regions. In fact, UCB shows a specific deposition pattern when is able to cross the blood

brain barrier and enters into the brain. Kernicterus condition results from a specific pattern of

UCB deposition in the brain, mainly in basal ganglia, hippocampus lateral ventricular walls,

mid brain, pons, cerebellum and inferior cerebellar peduncles, and subthalamic nuclei,

together with brain edema (Ahdab-Barmada and Moossy, 1984, Perlman et al., 1997). The

tendency of bilirubin deposits in kernicterus localized in areas vulnerable to hypoxic ischemic

injury, such as the pyramidal cell layer of the hippocampus, raises the question of whether

hypoxic-ischemic injury is important to the development of the lesions of kernicterus

(Perlman et al., 1997). Purkinje cells in the cerebellum have also showed increased

susceptibility to bilirubin injury in Gunn rats, the well-established animal model for severe

hyperbilirubinemia (Conlee and Shapiro, 1997, Lin et al., 2005). Other studies have referred dissimilar vulnerability to toxic stimuli in different brain

regions show, such as ischemia and oxidative stress. Many factors have been proposed to

account for differential vulnerability of brain regions. This includes local differences in

synaptic input and in neurotransmitter released, differences in expression levels of specific

neurotransmitter receptors, differences in antioxidant defences, and differences in signaling

pathways (Xu et al., 2001). In vivo studies demonstrate that brief periods of global ischemia

cause selective neuronal loss, especially in the CA1 region of hippocampus (Papadopoulos

et al., 1997, Papadopoulos et al., 1998). Studies with rat primary cultures of neurons and

astrocytes isolated from cortex, striatum, or hippocampus revealed distinct profiles of

vulnerability when subjected to injury. While astrocytes from striatum showed increased

injury by oxygen and glucose deprivation, they were more resistant to H2O2 exposure or

glucose deprivation, since they presented higher levels of antioxidant defences, such as

increased glutathione levels and increased activities of GPx and SOD (Xu et al., 2001). In

addition, it was reported that antioxidant enzymes, such as xantine oxidase and catalase,

have maximum activity in cortex, followed by cerebellum and hippocampus in developing

mouse brain exposed to lead (Prasanthi et al., 2010) and glutathione peroxidase activity is

considered determinant in the recovery of the immature mouse brain subjected to traumatic

brain injury (Tsuru-Aoyagi et al., 2009).

In the neonatal brain, the basal ganglia are the most vulnerable region in term infants,

whereas in preterms the most susceptible one is the periventricular white matter region

(Barkovich et al., 1995). Neuronal damage after hypoxic-ischemic insult seems to affect

particularly hippocampal CA1 region, striatum and neocortical layers III, V, and VI in animal

models of both mature and immature brain (Guzzetta et al., 2000, Jiang et al., 2004). For

Chapter I __________________________________________________________________________

32

hypoxia-ischemia-induced regional vulnerability may account preferential distribution of

immature NMDA receptors, which corresponds to regions that preferentially express nNOS,

such as layers CA1 and CA3 of the hippocampus, pons and globus pallidus (Black et al.,

1995, Greenamyre et al., 1987, Mitani et al., 1998), regions described for preferential UCB

deposition (Ahdab-Barmada and Moossy, 1984, Hansen, 2000, Perlman et al., 1997). In

addition, regional differences in pro-oxidant and antioxidant defences (Candelario-Jalil et al.,

2001, Khan and Black, 2003), the Ca2+-induced mitochondrial permeability transition (Friberg

et al., 1999) and DNA damage (Cardozo-Pelaez et al., 2000) are observed between

hippocampus, cortex, and striatum. Among these possible mechanisms, it is suggested that

insufficient antioxidant defence during oxidative stress is a major contributor to regional

specificity in the immature brain after ischemia (Jiang et al., 2004).

4.5. Mechanisms underlying bilirubin-induced neurotoxicity Encephalopathy by UCB and kernicterus are the main complications of UCB-induced

neurotoxic effects on the newborn brain. However, accumulating evidence strongly suggests

that low concentrations of bilirubin have anti-oxidant properties (Stocker et al., 1987),

providing protection against injury resulting from oxidation (Doré et al., 2000, Doré et al.,

1999), as described in section 2.4.2. However, elevated levels of UCB in neonatal period

produce neurotoxic effects, as stated in section 4.1.

Several studies have been made in order to better understand the mechanisms

underlying UCB neurotoxicity. Experimentally, UCB participates in numerous toxic events

occurring in different study models. Pioneer studies of Ernster and Zetterstrom (1956)

showed that UCB inhibits respiration and uncouples oxidative phosphorylation in brain

homogenates or isolated mitochondria. The energy depletion was later on corroborated by

the reduced rates of glycolysis and decreased ATP levels observed in Gunn rats and in

newborn piglets (Hoffman et al., 1996, Park et al., 2001). However, discrepant findings were

reported in other studies that failed to document significant changes in brain glucose

metabolism or oxidative phosphorylation (Diamond and Schmid, 1967), whereas others

demonstrated that hyperbilirubinemia only disturbs brain energy metabolism in the presence

of additional factors that disrupt the blood brain barrier, such as hypoxia or hyperosmolarity

(Wennberg et al., 1991). In addition, UCB is reported to induce impairment in neurogenesis,

neuritogenesis and synaptogenesis of primary cultures from both cortical and hippocampal

neurons (Falcão et al., 2007b, Fernandes et al., 2009). Among the UCB-induced neurotoxic

effects are ionic imbalance (Brito et al., 2004), extracellular accumulation of glutamate and

release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 by both astrocytes (Falcão et

al., 2005, Falcão et al., 2006, Fernandes et al., 2004) and microglia (Gordo et al., 2006).


33

Inflammatory signalling pathways triggered by UCB includes the activation of MAPKs and

NF-κB (Fernandes et al., 2007a, Fernandes et al., 2006, Silva et al., 2010), as well as TNF-α

and IL-1β receptors signaling pathways (Fernandes et al., 2010).

Involvement of oxidative stress in the pathways of cellular demise by UCB was already

demonstrated in neocortical synaptosomes (Brito et al., 2004) and mature cultured neurons

(Brito et al., 2008a, Brito et al., 2008b). Particular attention has been given to the

involvement of .NO and induction of nNOS in UCB-induced neurotoxicity (Brito et al., 2010,

Mancuso et al., 2008), as well as in UCB-induced cytotoxicity in cultured oligodendrocytes,

pointed to be mediated by activation of iNOS and .NO production (Genc et al., 2003).

Interestingly, it has been recently reported that synaptic transmission failure observed in

auditory brainstem of Gunn rats occurs in neurons that are expressing high levels of nNOS,

whereas antagonism of this enzyme confers protection against hearing loss (Haustein et al.,

2010). These upstream events culminate in nerve cell death by both necrosis and apoptosis.

Indeed, neurons and astrocytes (Brites et al., 2009, Silva et al., 2002), oligodendrocytes

(Genc et al., 2003), endothelial cells (Akin et al., 2002) and microglia (Gordo et al., 2006,

Silva et al., 2010) show a concentration-dependent UCB-induced cell death by both

oncosis/necrosis and apoptotic processes. Other studies demonstrated that UCB interferes

with DNA and protein synthesis in Gunn rat model (Greenfield and Majumdar, 1974, Yamada

et al., 1977), as well as protein phosphorylation (Hansen et al., 1996). It is also established

that UCB directly interacts with mitochondria, influencing membrane lipid and protein

properties, redox status, and cytochrome c content (Rodrigues et al., 2002b, Rodrigues et

al., 2000). In addition, it was demonstrated that UCB induces apoptosis trough mitochondria-

caspase-3 pathway involving cytochrome c release, caspase-3 activation, and subsequent

poli (ADP-ribose) polymerase (PARP) cleavage in developing rat brain neurons (Rodrigues

et al., 2002a).

Comparison of nerve cell susceptibility to UCB showed that neurons and microglia are

more vulnerable to UCB-induced cell death and microglial cells present the most reactive

features, such as the highest levels of pro-inflammatory cytokines and glutamate release

(Brites et al., 2009). In addition, UCB-induced inflammatory response, extracellular

accumulation of glutamate and cell death were shown to be enhanced in both immature

neurons and astrocytes when compared to mature ones (Falcão et al., 2005, Falcão et al.,

2006), observations that provide a basis for the increased risk of hyperbilirubinemia in

premature neonates. Interestingly, the P-glycoprotein (Pgp) and the multidrug resistance

associated protein 1 (Mrp1), two ATP-dependent plasma membrane efflux pumps, were

pointed to be responsible for limiting UCB levels inside the nerve cell (Ostrow et al., 2004,

Watchko et al., 1998). In addition, these proteins are shown to increase along nerve cell

Chapter I __________________________________________________________________________

34

maturation (Falcão et al., 2007a, Tsai et al., 2002). Therefore, it seems likely that the limited

levels of Pgp and Mrp1 in the initial maturation cellular stages may have a role in the

increased vulnerability of immature nerve cells to UCB.

Finally, neonatal hyperbilirubinemia is considered a vulnerability factor for the

development of mental disorders (Dalman and Cullberg, 1999), such as schizophrenia

(Hayashida et al., 2009) and it is reported that structural abnormalities at cytoskeleton level

are produced by ROS generated by prolonged treatment with haloperidol, commonly used in

the treatment of schizophrenia (Benitez-King et al., 2010).

5. Promising molecules for modulation in hyperbilirubinemia Understanding the various molecular players involved in neurotoxicity induced by

hyperbilirubinemia or hyperbilirubinemia with associated inflammation will contribute to

identify adequate therapeutic targets. For the present thesis we focused on a better

understanding on the role of molecules involved in response to oxidative stress and whether

they represent important strategies to prevent neuronal injury in hyperbilirubinemia.

5.1. Glycoursodeoxycholic acid (GUDCA)

Ursodeoxycholic acid (UDCA), the 7β-hydroxy epimer of chenodeoxycholic acid, is an

endogenous bile acid that has been widely used for the treatment of hepatobiliary disorders

(Lazaridis et al., 2001) and is also considered an anti-apoptotic agent (Rodrigues and Steer,

2001). After oral administration, UDCA is conjugated with taurine and glycine in the liver,

originating tauroursodeoxycholic acid (TUDCA) and, mostly, glycoursodeoxycholic acid

(GUDCA), respectively (Lazaridis et al., 2001). Thus, GUDCA is the conjugate form of UDCA

with highest clinical relevance.

UDCA is able to suppress the production of pro-inflammatory cytokines by inactivation of

the NF-κB pathway in different cell types (Joo et al., 2004, Schoemaker et al., 2004, Shah et

al., 2006, Solá et al., 2003). In other studies UDCA or its conjugates were able to act as a

cytoprotective agent, by promoting the stabilization of the plasma and mitochondrial

membranes and preventing cellular apoptosis (Güldütuna et al., 1993, Solá et al., 2002).

Regarding neurotoxic effects of UCB, both UDCA and TUDCA are able to prevent form UCB-

induced neuronal apoptosis by inhibiting the Bax translocation to mitochondria, the

consequent mitochondrial depolarization, cytochrome c release, caspase-3 activation and

PARP cleavage (Rodrigues et al., 2000, Solá et al., 2002).

More recently, it was demonstrated that GUDCA prevents from cell death, as well as

from release of pro-inflammatory cytokines in astrocytes exposed to UCB. This

immunomodulatory effect is made at post-translational level, since it affects mainly the


35

activity of TNF-α- and IL-1β-converting enzymes (TACE and ICE, respectively), thus

preventing the maturation of this cytokines and their consequent release (Fernandes et al.,

2007b). Furthermore, GUDCA counteracts UCB-induced neuronal cell death and oxidative

stress, by inhibiting UCB-induced protein oxidation, lipid peroxidation and glutathione loss in

mature neurons (Brito et al., 2008a), as well as to induces a rapid and sustained decrease in

plasma UCB concentrations in Gunn rat model (Cuperus et al., 2009).

5.2. N-ω-nitro-L-arginine methyl ester hydrochloride (L-NAME)

L-NAME is an analog of L-arginine that inhibits NOS and subsequent .NO production in a

enantiomerically specific manner. L-NAME is structurally related to N-ω-monomethyl-L-

arginine (L-NMMA), which was previously used in studies of the cytotoxicity of activated

macrophages, before the discovery that .NO is involved in this process. L-NAME, unlike L-

NMMA, shows progressive and irreversible or only slowly reversible inhibition of brain NOS

following the initial binding as reviewed by Knowles and Moncada (1994). Analogues of

L-arginine are also muscarinic acetylcholine receptor antagonists (Buxton et al., 1993). As

mentioned in section 3.2, .NO and induction of NOS are involved in apoptotic pathways

induced by inflammatory mediators in neuronal cells. Therefore, .NO inhibitors represent

important strategies in the comprehension of cellular injury associated with inflammatory

processes. In fact, local elimination of nNOS in P7 rats resulted in a significant attenuation of

the damage after hypoxic-ischemic insult (Ferriero et al., 1995) and nNOS deficiency through

genetic targeting was also neuroprotective in neonatal mice (Ferriero et al., 1996). Other

experimental models of hypoxia-ischemia demonstrated that NOS inhibition reduced

apoptosis at the level of caspase-3 activation (Zhu et al., 2004) and conferred tissue

protection (Peeters-Scholte et al., 2002). The role of .NO in UCB-induced cytotoxicity was

demonstrated in primary cultures of oligodendrocytes (Genc et al., 2003) and, more recently,

in primary cultures of mature neurons concomitantly treated with UCB and L-NAME (Brito et

al., 2008a, Brito et al., 2010).

5.3. N-acetylcysteine (NAC)

As mentioned in section 1.1, NAC is a thiol compound that is converted to cysteine, an

important precursor of cellular glutathione (Dringen, 2000, Zachwieja et al., 2005).

Antioxidant effects of NAC embrace its action as a source of sulfydryl groups, promoting

glutathione biosynthesis and its supply for GPx. In addition, NAC reacts with ROS (Ocal et

al., 2004). There are several in vitro and in vivo studies supporting antioxidant effect of NAC.

Treatment with NAC was shown to confer neuroprotection in lead-induced lipid peroxidation

Chapter I __________________________________________________________________________

36

and inhibition of antioxidant enzyme activities in rats’ brain (Nehru and Kanwar, 2004). In

addition, supplementation of cultured hippocampal neurons subjected to hypoxia with NAC

resulted in a significant cytoprotection, decline in ROS generation, and higher antioxidant

levels similar to that of control cells (Jayalakshmi et al., 2005). NAC was also able to inhibit

DNA strand breaks induced by hypoxia. Moreover, NAC attenuated long-term depletion of

dopamine and lipid peroxidation in rat striatum subjected to hypothermia induced by

amphetamine (Wan et al., 2006).

Regarding NAC effects on hyperbilirubinemia models, treatment with NAC was able to

decrease lipid peroxidation in cerebral cortex, midbrain and cerebellum observed in

jaundiced rats (Karageorgos et al., 2006), as well as to protect against UCB-induced protein

oxidation, oxidative disruption and cell death in rat cultured mature cortical neurons (Brito et

al., 2008b).


37

6. Global aims of the thesis The main goal of the present work is to further dissect the cellular mechanisms of

neonatal neurotoxicity by hyperbilirubinemia. Oxidative stress, mitochondrial dysfunction and

consequent cell death will be the major features studied. Since prematurity and sepsis are

risk factors for hyperbilirubinemia, it will be used an experimental model that intends to mimic

conditions of a moderate to severe neonatal jaundice in prematures, alone or with

associatied inflammation. Regarding that bilirubin presents a specific deposition pattern in

the brain, regional susceptibility will also be evaluated in different areas. In addition, it will be

discussed the role of antioxidants or known modulators of oxidant species production in

prevention of neuronal injury in neonatal hyperbilirubinemia.

The major questions addressed in the present thesis are:

1. Does UCB interfere with mitochondrial function and energy metabolic pathways

through increased oxidative status in immature neurons? Can GUDCA counteract

such effects?

2. Does sepsis have an aggravating role in UCB-induced dysfunction in immature

neurons? Can .NO/NOS and JNK1/2 activation be considered signalling

determinants in this neuronal dysfunction?

3. Does UCB deposition specific pattern in the brain determine the differential

regional susceptibility to UCB-induced oxidative damage? Can DJ-1 protein

expression and glutathione content be considered potential modulators of this

neurotoxicity?

The studies developed to address these questions are described and discussed in the

three subsequent chapters.

In summary, they bring new insights into UCB effects on metabolic pathways, cellular

redox status and cell death in conditions mimicking a moderate to severe hyperbilirubinemia

in the early neonatal period. In addition, these results provide a basis for the commonly

indicated higher risk of UCB brain damage in a condition of inflammation. Furthermore, these

data provide specific features that may explain the differential susceptibility to UCB observed

in different brain areas. These advances may substantiate target-driven approaches to the

prevention and treatment of UCB-induced neurological damage, and provide fruitful

opportunities for future investigations.

Chapter I __________________________________________________________________________

38

7. References Cellular respiration (2007) In: http://fig.cox.miami.edu/Faculty/Dana/105F00_13.html, from

Department of Biology, University of Miami.

Acehan, D., Jiang, X., Morgan, D. G., Heuser, J. E., Wang, X. and Akey, C. W. (2002) Three-

dimensional structure of the apoptosome: implications for assembly, procaspase-9

binding, and activation. Mol Cell, 9, 423-432.

Adams, J. M. (2003) Ways of dying: multiple pathways to apoptosis. Genes Dev, 17, 2481-

2495.

Ahdab-Barmada, M. and Moossy, J. (1984) The neuropathology of kernicterus in the

premature neonate: diagnostic problems. J Neuropathol Exp Neurol, 43, 45-56.

Ahlfors, C. E., Bennett, S. H., Shoemaker, C. T., Ellis, W. G., Davis, S. L., Wennberg, R. P.

and Goetzman, B. W. (1986) Changes in the auditory brainstem response associated with

intravenous infusion of unconjugated bilirubin into infant rhesus monkeys. Pediatr Res, 20, 511-515.

Akin, E., Clower, B., Tibbs, R., Tang, J. and Zhang, J. (2002) Bilirubin produces apoptosis in

cultured bovine brain endothelial cells. Brain Res, 931, 168-175.

Allan, S. M. and Rothwell, N. J. (2003) Inflammation in central nervous system injury. Philos

Trans R Soc Lond B Biol Sci, 358, 1669-1677.

Allen, N. J. and Barres, B. A. (2005) Signaling between glia and neurons: focus on synaptic

plasticity. Curr Opin Neurobiol, 15, 542-548.

Allen, N. J. and Barres, B. A. (2009) Neuroscience: Glia - more than just brain glue. Nature,

457, 675-677.

Almeida, A., Almeida, J., Bolaños, J. P. and Moncada, S. (2001) Different responses of

astrocytes and neurons to nitric oxide: the role of glycolytically generated ATP in astrocyte

protection. Proc Natl Acad Sci U S A, 98, 15294-15299.

Almeida, A. and Bolaños, J. P. (2001) A transient inhibition of mitochondrial ATP synthesis

by nitric oxide synthase activation triggered apoptosis in primary cortical neurons. J

Neurochem, 77, 676-690.

Almeida, A., Delgado-Esteban, M., Bolanos, J. P. and Medina, J. M. (2002) Oxygen and

glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones

but not in astrocytes in primary culture. J Neurochem, 81, 207-217.

Almeida, A., Moncada, S. and Bolaños, J. P. (2004) Nitric oxide switches on glycolysis

through the AMP protein kinase and 6-phosphofructo-2-kinase pathway. Nat Cell Biol, 6, 45-51.

Anderson, C. M. and Swanson, R. A. (2000) Astrocyte glutamate transport: review of

properties, regulation, and physiological functions. Glia, 32, 1-14.


39

Angermüller, S., Islinger, M. and Völkl, A. (2009) Peroxisomes and reactive oxygen species,

a lasting challenge. Histochem Cell Biol, 131, 459-463.

Aruoma, O. I., Laughton, M. J. and Halliwell, B. (1989) Carnosine, homocarnosine and

anserine: could they act as antioxidants in vivo? Biochem J, 264, 863-869.

Ballabh, P., Braun, A. and Nedergaard, M. (2004) The blood-brain barrier: an overview:

structure, regulation, and clinical implications. Neurobiol Dis, 16, 1-13.

Barañano, D. E., Rao, M., Ferris, C. D. and Snyder, S. H. (2002) Biliverdin reductase: a

major physiologic cytoprotectant. Proc Natl Acad Sci U S A, 99, 16093-16098.

Barkovich, A. J., Westmark, K., Partridge, C., Sola, A. and Ferriero, D. M. (1995) Perinatal

asphyxia: MR findings in the first 10 days. AJNR Am J Neuroradiol, 16, 427-438.

Baumann, N. and Pham-Dinh, D. (2001) Biology of oligodendrocyte and myelin in the

mammalian central nervous system. Physiol Rev, 81, 871-927.

Beckman, K. B. and Ames, B. N. (1998) The free radical theory of aging matures. Physiol

Rev, 78, 547-581.

Bellinger, F. P., Madamba, S. and Siggins, G. R. (1993) Interleukin 1 beta inhibits synaptic

strength and long-term potentiation in the rat CA1 hippocampus. Brain Res, 628, 227-234.

Benitez-King, G., Ortiz-Lopez, L., Jimenez-Rubio, G. and Ramirez-Rodriguez, G. (2010)

Haloperidol causes cytoskeletal collapse in N1E-115 cells through tau

hyperphosphorylation induced by oxidative stress: Implications for neurodevelopment. Eur

J Pharmacol, 644, 24-31.

Bian, K. and Murad, F. (2001) Diversity of endotoxin-induced nitrotyrosine formation in

macrophage-endothelium-rich organs. Free Radic Biol Med, 31, 421-429.

Black, S. M., Bedolli, M. A., Martinez, S., Bristow, J. D., Ferriero, D. M. and Soifer, S. J.

(1995) Expression of neuronal nitric oxide synthase corresponds to regions of selective

vulnerability to hypoxia-ischaemia in the developing rat brain. Neurobiol Dis, 2, 145-155.

Blamire, A. M., Anthony, D. C., Rajagopalan, B., Sibson, N. R., Perry, V. H. and Styles, P.

(2000) Interleukin-1beta -induced changes in blood-brain barrier permeability, apparent

diffusion coefficient, and cerebral blood volume in the rat brain: a magnetic resonance

study. J Neurosci, 20, 8153-8159.

Blanckaert, N. and Fevery, J. (1990) Physiology and patophysiology of bilirubin metabolism.

In: Hepatology. A textbook of liver disease., pp. 254-303. WB Saunders, Philadelphia.

Block, M. L. and Hong, J. S. (2005) Microglia and inflammation-mediated neurodegeneration:

multiple triggers with a common mechanism. Prog Neurobiol, 76, 77-98.

Blomgren, K., Leist, M. and Groc, L. (2007) Pathological apoptosis in the developing brain.

Apoptosis, 12, 993-1010.

Chapter I __________________________________________________________________________

40

Bolaños, J. P., Almeida, A. and Moncada, S. (2010) Glycolysis: a bioenergetic or a survival

pathway? Trends Biochem Sci, 35, 145-149.

Bolaños, J. P., Peuchen, S., Heales, S. J., Land, J. M. and Clark, J. B. (1994) Nitric oxide-

mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes. J

Neurochem, 63, 910-916.

Bonmann, E., Suschek, C., Spranger, M. and Kolb-Bachofen, V. (1997) The dominant role of

exogenous or endogenous interleukin-1 beta on expression and activity of inducible nitric

oxide synthase in rat microvascular brain endothelial cells. Neurosci Lett, 230, 109-112.

Bouzier-Sore, A. K., Voisin, P., Canioni, P., Magistretti, P. J. and Pellerin, L. (2003) Lactate is

a preferential oxidative energy substrate over glucose for neurons in culture. J Cereb

Blood Flow Metab, 23, 1298-1306.

Brites, D., Fernandes, A., Falcão, A. S., Gordo, A. C., Silva, R. F. M. and Brito, M. A. (2009)

Biological risks for neurological abnormalities associated with hyperbilirubinemia. J

Perinatol, 29 Suppl 1, S8-13.

Brito, M. A., Brites, D. and Butterfield, D. A. (2004) A link between hyperbilirubinemia,

oxidative stress and injury to neocortical synaptosomes. Brain Res, 1026, 33-43.

Brito, M. A., Lima, S., Fernandes, A., Falcão, A. S., Silva, R. F. M., Butterfield, D. A. and

Brites, D. (2008a) Bilirubin injury to neurons: contribution of oxidative stress and rescue by

glycoursodeoxycholic acid. Neurotoxicology, 29, 259-269.

Brito, M. A., Rosa, A. I., Falcão, A. S., Fernandes, A., Silva, R. F. M., Butterfield, D. A. and

Brites, D. (2008b) Unconjugated bilirubin differentially affects the redox status of neuronal

and astroglial cells. Neurobiol Dis, 29, 30-40.

Brito, M. A., Rosa, A. I., Silva, R. F. M., Falcão, A. S., Fernandes, A. and Brites, D. (2007)

Oxidative stress and disruption of the nervous cell. In: Focus in Brain Research, pp. 1-33.

Nova Science Publishers, Inc., New York.

Brito, M. A., Silva, R. F. M. and Brites, D. (2006) Cell response to hyperbilirrubinemia: a

journey along key molecular events. In: New Trends in Brain Research, pp. 1-38. Nova

Science Publishers, Inc., New York.

Brito, M. A., Vaz, A. R., Silva, S. L., Falcão, A. S., Fernandes, A., Silva, R. F. M. and Brites,

D. (2010) N-methyl-D-aspartate receptor and neuronal nitric oxide synthase activation

mediate bilirubin-induced neurotoxicity. Mol Med, 16, 372-380.

Brown, G. C. and Cooper, C. E. (1994) Nanomolar concentrations of nitric oxide reversibly

inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS

Lett, 356, 295-298.

Burdo, J. R. and Connor, J. R. (2003) Brain iron uptake and homeostatic mechanisms: an

overview. Biometals, 16, 63-75.


41

Buxton, I. L., Cheek, D. J., Eckman, D., Westfall, D. P., Sanders, K. M. and Keef, K. D.

(1993) NG-nitro L-arginine methyl ester and other alkyl esters of arginine are muscarinic

receptor antagonists. Circ Res, 72, 387-395.

Calabrese, V., Lodi, R., Tonon, C. et al. (2005) Oxidative stress, mitochondrial dysfunction

and cellular stress response in Friedreich's ataxia. J Neurol Sci, 233, 145-162.

Candelario-Jalil, E., Mhadu, N. H., Al-Dalain, S. M., Martinez, G. and Leon, O. S. (2001)

Time course of oxidative damage in different brain regions following transient cerebral

ischemia in gerbils. Neurosci Res, 41, 233-241.

Cardoso, F. L., Brites, D. and Brito, M. A. (2010) Looking at the blood-brain barrier:

molecular anatomy and possible investigation approaches. Brain Res Rev, 64, 328-363.

Cardozo-Pelaez, F., Brooks, P. J., Stedeford, T., Song, S. and Sanchez-Ramos, J. (2000)

DNA damage, repair, and antioxidant systems in brain regions: a correlative study. Free

Radic Biol Med, 28, 779-785.

Cartlidge, P. H. and Rutter, N. (1986) Serum albumin concentrations and oedema in the

newborn. Arch Dis Child, 61, 657-660.

Cashore, W. J. (1980) Free bilirubin concentrations and bilirubin-binding affinity in term and

preterm infants. J Pediatr, 96, 521-527.

Chang, E. F., Claus, C. P., Vreman, H. J., Wong, R. J. and Noble-Haeusslein, L. J. (2005)

Heme regulation in traumatic brain injury: relevance to the adult and developing brain. J

Cereb Blood Flow Metab, 25, 1401-1417.

Chavarria, A. and Alcocer-Varela, J. (2004) Is damage in central nervous system due to

inflammation? Autoimmun Rev, 3, 251-260.

Chen, Y. and Swanson, R. A. (2003) Astrocytes and brain injury. J Cereb Blood Flow Metab,

23, 137-149.

Circu, M. L. and Aw, T. Y. (2010) Reactive oxygen species, cellular redox systems, and

apoptosis. Free Radic Biol Med, 48, 749-762.

Cleeter, M. W., Cooper, J. M., Darley-Usmar, V. M., Moncada, S. and Schapira, A. H. (1994)

Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial

respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett,

345, 50-54.

Clementi, E., Brown, G. C., Foxwell, N. and Moncada, S. (1999) On the mechanism by which

vascular endothelial cells regulate their oxygen consumption. Proc Natl Acad Sci U S A,

96, 1559-1562.

Combs, C. K., Karlo, J. C., Kao, S. C. and Landreth, G. E. (2001) beta-Amyloid stimulation of

microglia and monocytes results in TNFalpha-dependent expression of inducible nitric

oxide synthase and neuronal apoptosis. J Neurosci, 21, 1179-1188.

Chapter I __________________________________________________________________________

42

Conlee, J. W. and Shapiro, S. M. (1997) Development of cerebellar hypoplasia in jaundiced

Gunn rats: a quantitative light microscopic analysis. Acta Neuropathol, 93, 450-460.

Cotgreave, I. A. and Gerdes, R. G. (1998) Recent trends in glutathione biochemistry--

glutathione-protein interactions: a molecular link between oxidative stress and cell

proliferation? Biochem Biophys Res Commun, 242, 1-9.

Couraud, P. O. (1994) Interactions between lymphocytes, macrophages, and central nervous

system cells. J Leukoc Biol, 56, 407-415.

Croitoru-Lamoury, J., Guillemin, G. J., Boussin, F. D. et al. (2003) Expression of chemokines

and their receptors in human and simian astrocytes: evidence for a central role of TNF

alpha and IFN gamma in CXCR4 and CCR5 modulation. Glia, 41, 354-370.

Cuperus, F. J., Hafkamp, A. M., Havinga, R., Vitek, L., Zelenka, J., Tiribelli, C., Ostrow, J. D.

and Verkade, H. J. (2009) Effective treatment of unconjugated hyperbilirubinemia with oral

bile salts in Gunn rats. Gastroenterology, 136, 673-682 e671.

Daikhin, Y. and Yudkoff, M. (2000) Compartmentation of brain glutamate metabolism in

neurons and glia. J Nutr, 130, 1026S-1031S.

Dalman, C. and Cullberg, J. (1999) Neonatal hyperbilirubinaemia--a vulnerability factor for

mental disorder? Acta Psychiatr Scand, 100, 469-471.

Dawodu, A. H., Owa, J. A. and Familusi, J. B. (1984) A prospective study of the role of

bacterial infection and G6PD deficiency in severe neonatal jaundice in Nigeria. Trop

Geogr Med, 36, 127-132.

Dawson, V. L., Dawson, T. M., London, E. D., Bredt, D. S. and Snyder, S. H. (1991) Nitric

oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci U

S A, 88, 6368-6371.

de Arriba, S. G., Krugel, U., Regenthal, R. et al. (2006) Carbonyl stress and NMDA receptor

activation contribute to methylglyoxal neurotoxicity. Free Radic Biol Med, 40, 779-790.

De Marchis, S., Modena, C., Peretto, P., Migheli, A., Margolis, F. L. and Fasolo, A. (2000)

Carnosine-related dipeptides in neurons and glia. Biochemistry (Mosc), 65, 824-833.

de Vries, L. S., Lary, S. and Dubowitz, L. M. (1985) Relationship of serum bilirubin levels to

ototoxicity and deafness in high-risk low-birth-weight infants. Pediatrics, 76, 351-354.

Decker, E. A., Livisay, S. A. and Zhou, S. (2000) A re-evaluation of the antioxidant activity of

purified carnosine. Biochemistry (Mosc), 65, 766-770.

Delgado-Esteban, M., Almeida, A. and Bolaños, J. P. (2000) D-Glucose prevents glutathione

oxidation and mitochondrial damage after glutamate receptor stimulation in rat cortical

primary neurons. J Neurochem, 75, 1618-1624.

Dennery, P. A., Seidman, D. S. and Stevenson, D. K. (2001) Neonatal hyperbilirubinemia. N

Engl J Med, 344, 581-590.


43

Desagher, S., Glowinski, J. and Premont, J. (1996) Astrocytes protect neurons from

hydrogen peroxide toxicity. J Neurosci, 16, 2553-2562.

Di Monte, D. A., Tokar, I. and Langston, J. W. (1999) Impaired glutamate clearance as a

consequence of energy failure caused by MPP(+) in astrocytic cultures. Toxicol Appl

Pharmacol, 158, 296-302.

Diamond, I. and Schmid, R. (1967) Oxidative phosphorylation in experimental bilirubin

encephalopathy. Science, 155, 1288-1289.

Doré, S., Goto, S., Sampei, K. et al. (2000) Heme oxygenase-2 acts to prevent neuronal

death in brain cultures and following transient cerebral ischemia. Neuroscience, 99, 587-

592.

Doré, S., Takahashi, M., Ferris, C. D., Zakhary, R., Hester, L. D., Guastella, D. and Snyder,

S. H. (1999) Bilirubin, formed by activation of heme oxygenase-2, protects neurons

against oxidative stress injury. Proc Natl Acad Sci U S A, 96, 2445-2450.

Dringen, R. (2000) Metabolism and functions of glutathione in brain. Prog Neurobiol, 62, 649-

671.

Dringen, R., Gutterer, J. M. and Hirrlinger, J. (2000) Glutathione metabolism in brain

metabolic interaction between astrocytes and neurons in the defence against reactive

oxygen species. Eur J Biochem, 267, 4912-4916.

Dringen, R. and Hamprecht, B. (1996) Glutathione content as an indicator for the presence of

metabolic pathways of amino acids in astroglial cultures. J Neurochem, 67, 1375-1382.

Dringen, R., Pawlowski, P. G. and Hirrlinger, J. (2005) Peroxide detoxification by brain cells.

J Neurosci Res, 79, 157-165.

Du, Y. and Dreyfus, C. F. (2002) Oligodendrocytes as providers of growth factors. J Neurosci

Res, 68, 647-654.

Eggleston, L. V. and Krebs, H. A. (1974) Regulation of the pentose phosphate cycle.

Biochem J, 138, 425-435.

Elfering, S. L., Sarkela, T. M. and Giulivi, C. (2002) Biochemistry of mitochondrial nitric-oxide

synthase. J Biol Chem, 277, 38079-38086.

Ernster, L. and Zetterstrom, R. (1956) Bilirubin, an uncoupler of oxidative phosphorylation in

isolated mitochondria. Nature, 178, 1335-1337.

Estus, S., Zaks, W. J., Freeman, R. S., Gruda, M., Bravo, R. and Johnson, E. M., Jr. (1994)

Altered gene expression in neurons during programmed cell death: identification of c-jun

as necessary for neuronal apoptosis. J Cell Biol, 127, 1717-1727.

Falcão, A. S., Bellarosa, C., Fernandes, A., Brito, M. A., Silva, R. F. M., Tiribelli, C. and

Brites, D. (2007a) Role of multidrug resistance-associated protein 1 expression in the in

vitro susceptibility of rat nerve cell to unconjugated bilirubin. Neuroscience, 144, 878-888.

Chapter I __________________________________________________________________________

44

Falcão, A. S., Fernandes, A., Brito, M. A., Silva, R. F. M. and Brites, D. (2005) Bilirubin-

induced inflammatory response, glutamate release, and cell death in rat cortical

astrocytes are enhanced in younger cells. Neurobiol Dis, 20, 199-206.


induced immunostimulant effects and toxicity vary with neural cell type and maturation

state. Acta Neuropathol, 112, 95-105.

Falcão, A. S., Silva, R. F. M., Pancadas, S., Fernandes, A., Brito, M. A. and Brites, D.

(2007b) Apoptosis and impairment of neurite network by short exposure of immature rat

cortical neurons to unconjugated bilirubin increase with cell differentiation and are

additionally enhanced by an inflammatory stimulus. J Neurosci Res, 85, 1229-1239.

Fernandes, A., Barateiro, A., Falcão, A. S., Silva, S. L., Vaz, A. R., Brito, M. A., Silva, R. F.

M. and Brites, D. (2010) Astrocyte reactivity to unconjugated bilirubin requires TNF-α and

IL-1β receptor signalling pathways. Glia, in press.

Fernandes, A., Falcão, A. S., Abranches, E., Bekman, E., Henrique, D., Lanier, L. M. and

Brites, D. (2009) Bilirubin as a determinant for altered neurogenesis, neuritogenesis, and

synaptogenesis. Dev Neurobiol, 69, 568-582.

Fernandes, A., Falcão, A. S., Silva, R. F. M., Brito, M. A. and Brites, D. (2007a) MAPKs are

key players in mediating cytokine release and cell death induced by unconjugated bilirubin

in cultured rat cortical astrocytes. Eur J Neurosci, 25, 1058-1068.

Fernandes, A., Falcão, A. S., Silva, R. F. M., Gordo, A. C., Gama, M. J., Brito, M. A. and

Brites, D. (2006) Inflammatory signalling pathways involved in astroglial activation by

unconjugated bilirubin. J Neurochem, 96, 1667-1679.

Fernandes, A., Silva, R. F. M., Falcão, A. S., Brito, M. A. and Brites, D. (2004) Cytokine

production, glutamate release and cell death in rat cultured astrocytes treated with

unconjugated bilirubin and LPS. J Neuroimmunol, 153, 64-75.

Fernandes, A., Vaz, A. R., Falcão, A. S., Silva, R. F. M., Brito, M. A. and Brites, D. (2007b)

Glycoursodeoxycholic Acid and interleukin-10 modulate the reactivity of rat cortical

astrocytes to unconjugated bilirubin. J Neuropathol Exp Neurol, 66, 789-798.

Ferriero, D. M., Holtzman, D. M., Black, S. M. and Sheldon, R. A. (1996) Neonatal mice

lacking neuronal nitric oxide synthase are less vulnerable to hypoxic-ischemic injury.

Neurobiol Dis, 3, 64-71.

Ferriero, D. M., Sheldon, R. A., Black, S. M. and Chuai, J. (1995) Selective destruction of

nitric oxide synthase neurons with quisqualate reduces damage after hypoxia-ischemia in

the neonatal rat. Pediatr Res, 38, 912-918.

Finkel, T. (2000) Redox-dependent signal transduction. FEBS Lett, 476, 52-54.


45

Forsyth, R. J. (1996) Astrocytes and the delivery of glucose from plasma to neurons.

Neurochem Int, 28, 231-241.

Friberg, H., Connern, C., Halestrap, A. P. and Wieloch, T. (1999) Differences in the activation

of the mitochondrial permeability transition among brain regions in the rat correlate with

selective vulnerability. J Neurochem, 72, 2488-2497.

Gal, S., Zheng, H., Fridkin, M. and Youdim, M. B. (2005) Novel multifunctional

neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative

diseases. In vivo selective brain monoamine oxidase inhibition and prevention of MPTP-

induced striatal dopamine depletion. J Neurochem, 95, 79-88.

García-Nogales, P., Almeida, A. and Bolaños, J. P. (2003) Peroxynitrite protects neurons

against nitric oxide-mediated apoptosis. A key role for glucose-6-phosphate

dehydrogenase activity in neuroprotection. J Biol Chem, 278, 864-874.

García-Nogales, P., Almeida, A., Fernández, E., Medina, J. M. and Bolaños, J. P. (1999)

Induction of glucose-6-phosphate dehydrogenase by lipopolysaccharide contributes to

preventing nitric oxide-mediated glutathione depletion in cultured rat astrocytes. J

Neurochem, 72, 1750-1758.

Genc, S., Genc, K., Kumral, A., Baskin, H. and Ozkan, H. (2003) Bilirubin is cytotoxic to rat

oligodendrocytes in vitro. Brain Res, 985, 135-141.

Ghafourifar, P. and Cadenas, E. (2005) Mitochondrial nitric oxide synthase. Trends

Pharmacol Sci, 26, 190-195.

Ghafourifar, P. and Richter, C. (1997) Nitric oxide synthase activity in mitochondria. FEBS

Lett, 418, 291-296.

Ghibelli, L., Fanelli, C., Rotilio, G., Lafavia, E., Coppola, S., Colussi, C., Civitareale, P. and

Ciriolo, M. R. (1998) Rescue of cells from apoptosis by inhibition of active GSH extrusion.

FASEB J, 12, 479-486.

Gilland, E., Puka-Sundvall, M., Hillered, L. and Hagberg, H. (1998) Mitochondrial function

and energy metabolism after hypoxia-ischemia in the immature rat brain: involvement of

NMDA-receptors. J Cereb Blood Flow Metab, 18, 297-304.

Gkoltsiou, K., Tzoufi, M., Counsell, S., Rutherford, M. and Cowan, F. (2008) Serial brain MRI

and ultrasound findings: relation to gestational age, bilirubin level, neonatal neurologic

status and neurodevelopmental outcome in infants at risk of kernicterus. Early Hum Dev,

84, 829-838.

Goldenberg, R. L. and Andrews, W. W. (1996) Intrauterine infection and why preterm

prevention programs have failed. Am J Public Health, 86, 781-783.

Gonzalez-Zulueta, M., Ensz, L. M., Mukhina, G., Lebovitz, R. M., Zwacka, R. M., Engelhardt,

J. F., Oberley, L. W., Dawson, V. L. and Dawson, T. M. (1998) Manganese superoxide

Chapter I __________________________________________________________________________

46

dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity. J

Neurosci, 18, 2040-2055.

Gordo, A. C., Falcão, A. S., Fernandes, A., Brito, M. A., Silva, R. F. M. and Brites, D. (2006)

Unconjugated bilirubin activates and damages microglia. J Neurosci Res, 84, 194-201.

Görg, B., Qvartskhava, N., Voss, P., Grune, T., Häussinger, D. and Schliess, F. (2007)

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration. FEBS Lett,

581, 84-90.

Greenamyre, T., Penney, J. B., Young, A. B., Hudson, C., Silverstein, F. S. and Johnston, M.

V. (1987) Evidence for transient perinatal glutamatergic innervation of globus pallidus. J

Neurosci, 7, 1022-1030.

Greenfield, S. and Majumdar, A. P. (1974) Bilirubin encephalopathy: effect on protein

synthesis in the brain of the Gunn rat. J Neurol Sci, 22, 83-89.

Güldütuna, S., Zimmer, G., Imhof, M., Bhatti, S., You, T. and Leuschner, U. (1993) Molecular

aspects of membrane stabilization by ursodeoxycholate [see comment]. Gastroenterology,

104, 1736-1744.

Gupta, S. (2002) A decision between life and death during TNF-alpha-induced signaling. J

Clin Immunol, 22, 185-194.

Guzzetta, F., Deodato, F. and Rando, T. (2000) Brain ischemic lesions of the newborn.

Childs Nerv Syst, 16, 633-637.

Haberg, A., Qu, H., Haraldseth, O., Unsgard, G. and Sonnewald, U. (1998) In vivo injection

of [1-13C]glucose and [1,2-13C]acetate combined with ex vivo 13C nuclear magnetic

resonance spectroscopy: a novel approach to the study of middle cerebral artery

occlusion in the rat. J Cereb Blood Flow Metab, 18, 1223-1232.

Haden, D. W., Suliman, H. B., Carraway, M. S., Welty-Wolf, K. E., Ali, A. S., Shitara, H.,

Yonekawa, H. and Piantadosi, C. A. (2007) Mitochondrial biogenesis restores oxidative

metabolism during Staphylococcus aureus sepsis. Am J Respir Crit Care Med, 176, 768-

777.

Halliwell, B. (2006) Oxidative stress and neurodegeneration: where are we now? J

Neurochem, 97, 1634-1658.

Ham, J., Babij, C., Whitfield, J., Pfarr, C. M., Lallemand, D., Yaniv, M. and Rubin, L. L. (1995)

A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell

death. Neuron, 14, 927-939.

Hanisch, U. K. (2002) Microglia as a source and target of cytokines. Glia, 40, 140-155.

Hansen, T. W. (2000) Pioneers in the scientific study of neonatal jaundice and kernicterus.

Pediatrics, 106, E15.


47

Hansen, T. W., Mathiesen, S. B. and Walaas, S. I. (1996) Bilirubin has widespread inhibitory

effects on protein phosphorylation. Pediatr Res, 39, 1072-1077.

Hansen, T. W. R. (2002) Mechanisms of bilirubin toxicity: clinical implications. Clin Perinatol,

29, 765-778, viii.

Haskew-Layton, R. E., Payappilly, J. B., Smirnova, N. A. et al. (2010) Controlled enzymatic

production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an

Nrf2-independent pathway. Proc Natl Acad Sci U S A.

Haustein, M. D., Read, D. J., Steinert, J. R., Pilati, N., Dinsdale, D. and Forsythe, I. D. (2010)

Acute hyperbilirubinaemia induces presynaptic neurodegeneration at a central

glutamatergic synapse. J Physiol.

Hayashida, M., Miyaoka, T., Tsuchie, K. et al. (2009) Hyperbilirubinemia-related behavioral

and neuropathological changes in rats: a possible schizophrenia animal model. Prog

Neuropsychopharmacol Biol Psychiatry, 33, 581-588.

Hayton, S. M. and Muller, D. P. (2004) Vitamin E in neural and visual function. Ann N Y Acad

Sci, 1031, 263-270.

Hemmer, K., Fransen, L., Vanderstichele, H., Vanmechelen, E. and Heuschling, P. (2001) An

in vitro model for the study of microglia-induced neurodegeneration: involvement of nitric

oxide and tumor necrosis factor-alpha. Neurochem Int, 38, 557-565.

Heneka, M. T., Loschmann, P. A., Gleichmann, M., Weller, M., Schulz, J. B., Wullner, U. and

Klockgether, T. (1998) Induction of nitric oxide synthase and nitric oxide-mediated

apoptosis in neuronal PC12 cells after stimulation with tumor necrosis factor-

alpha/lipopolysaccharide. J Neurochem, 71, 88-94.

Herrero-Mendez, A., Almeida, A., Fernández, E., Maestre, C., Moncada, S. and Bolaños, J.

P. (2009) The bioenergetic and antioxidant status of neurons is controlled by continuous

degradation of a key glycolytic enzyme by APC/C-Cdh1. Nat Cell Biol, 11, 747-752.

Hoffman, D. J., Zanelli, S. A., Kubin, J., Mishra, O. P. and Delivoria-Papadopoulos, M. (1996)

The in vivo effect of bilirubin on the N-methyl-D-aspartate receptor/ion channel complex in

the brains of newborn piglets. Pediatr Res, 40, 804-808.

Itoh, Y., Esaki, T., Shimoji, K., Cook, M., Law, M. J., Kaufman, E. and Sokoloff, L. (2003)

Dichloroacetate effects on glucose and lactate oxidation by neurons and astroglia in vitro

and on glucose utilization by brain in vivo. Proc Natl Acad Sci U S A, 100, 4879-4884.

Jayalakshmi, K., Sairam, M., Singh, S. B., Sharma, S. K., Ilavazhagan, G. and Banerjee, P.

K. (2005) Neuroprotective effect of N-acetyl cysteine on hypoxia-induced oxidative stress

in primary hippocampal culture. Brain Res, 1046, 97-104.

Chapter I __________________________________________________________________________

48

Jiang, X., Mu, D., Manabat, C., Koshy, A. A., Christen, S., Tauber, M. G., Vexler, Z. S. and

Ferriero, D. M. (2004) Differential vulnerability of immature murine neurons to oxygen-

glucose deprivation. Exp Neurol, 190, 224-232.

Jolivet, R., Magistretti, P. J. and Weber, B. (2009) Deciphering neuron-glia

compartmentalization in cortical energy metabolism. Front Neuroenergetics, 1, 4.

Joo, S. S., Won, T. J. and Lee, D. I. (2004) Potential role of ursodeoxycholic acid in

suppression of nuclear factor kappa B in microglial cell line (BV-2). Arch Pharm Res, 27, 954-960.

Kajta, M., Trotter, A., Lason, W. and Beyer, C. (2006) Impact of 17beta-estradiol on cytokine-

mediated apoptotic effects in primary hippocampal and neocortical cell cultures. Brain

Res, 1116, 64-74.

Kanski, J., Behring, A., Pelling, J. and Schoneich, C. (2005) Proteomic identification of 3-

nitrotyrosine-containing rat cardiac proteins: effects of biological aging. Am J Physiol

Heart Circ Physiol, 288, H371-381.

Kaplan, M. and Hammerman, C. (2005) Understanding severe hyperbilirubinemia and

preventing kernicterus: adjuncts in the interpretation of neonatal serum bilirubin. Clin Chim

Acta, 356, 9-21.

Karageorgos, N., Patsoukis, N., Chroni, E., Konstantinou, D., Assimakopoulos, S. F. and

Georgiou, C. (2006) Effect of N-acetylcysteine, allopurinol and vitamin E on jaundice-

induced brain oxidative stress in rats. Brain Res, 1111, 203-212.

Kasischke, K. A., Vishwasrao, H. D., Fisher, P. J., Zipfel, W. R. and Webb, W. W. (2004)

Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis.

Science, 305, 99-103.

Katsuki, H., Okawara, M., Shibata, H., Kume, T. and Akaike, A. (2006) Nitric oxide-producing

microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain

slice culture. J Neurochem, 97, 1232-1242.

Khan, J. Y. and Black, S. M. (2003) Developmental changes in murine brain antioxidant

enzymes. Pediatr Res, 54, 77-82.

Kletzien, R. F., Harris, P. K. and Foellmi, L. A. (1994) Glucose-6-phosphate dehydrogenase:

a "housekeeping" enzyme subject to tissue-specific regulation by hormones, nutrients,

and oxidant stress. FASEB J, 8, 174-181.

Knowles, R. G. and Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem J, 298 ( Pt 2), 249-258.

Knowles, R. G., Palacios, M., Palmer, R. M. and Moncada, S. (1989) Formation of nitric

oxide from L-arginine in the central nervous system: a transduction mechanism for

stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci U S A, 86, 5159-5162.


49

Konsman, J. P., Drukarch, B. and Van Dam, A. M. (2007) (Peri)vascular production and

action of pro-inflammatory cytokines in brain pathology. Clin Sci (Lond), 112, 1-25.

Korhonen, R., Lahti, A., Kankaanranta, H. and Moilanen, E. (2005) Nitric oxide production

and signaling in inflammation. Curr Drug Targets Inflamm Allergy, 4, 471-479.

Kowaltowski, A. J. (2000) Alternative mitochondrial functions in cell physiopathology: beyond

ATP production. Braz J Med Biol Res, 33, 241-250.

Kyriakis, J. M. and Avruch, J. (2001) Mammalian mitogen-activated protein kinase signal

transduction pathways activated by stress and inflammation. Physiol Rev, 81, 807-869.

Laranjinha, J. and Ledo, A. (2007) Coordination of physiologic and toxic pathways in

hippocampus by nitric oxide and mitochondria. Front Biosci, 12, 1094-1106.

Lazaridis, K. N., Gores, G. J. and Lindor, K. D. (2001) Ursodeoxycholic acid 'mechanisms of

action and clinical use in hepatobiliary disorders'. J Hepatol, 35, 134-146.

Leviton, A. and Gressens, P. (2007) Neuronal damage accompanies perinatal white-matter

damage. Trends Neurosci, 30, 473-478.

Lin, S., Wei, X., Bales, K. R., Paul, A. B., Ma, Z., Yan, G., Paul, S. M. and Du, Y. (2005)

Minocycline blocks bilirubin neurotoxicity and prevents hyperbilirubinemia-induced

cerebellar hypoplasia in the Gunn rat. Eur J Neurosci, 22, 21-27.

Liu, J., Killilea, D. W. and Ames, B. N. (2002) Age-associated mitochondrial oxidative decay:

improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain

by feeding old rats acetyl-L- carnitine and/or R-alpha -lipoic acid. Proc Natl Acad Sci U S

A, 99, 1876-1881.

Lu, S. C. (2009) Regulation of glutathione synthesis. Mol Aspects Med, 30, 42-59.

Lucius, R. and Sievers, J. (1996) Postnatal retinal ganglion cells in vitro: protection against

reactive oxygen species (ROS)-induced axonal degeneration by cocultured astrocytes.

Brain Res, 743, 56-62.

Luo, Y., Umegaki, H., Wang, X., Abe, R. and Roth, G. S. (1998) Dopamine induces

apoptosis through an oxidation-involved SAPK/JNK activation pathway. J Biol Chem, 273, 3756-3764.

Madrigal, J. L., Hurtado, O., Moro, M. A., Lizasoain, I., Lorenzo, P., Castrillo, A., Bosca, L.

and Leza, J. C. (2002) The increase in TNF-alpha levels is implicated in NF-kappaB

activation and inducible nitric oxide synthase expression in brain cortex after

immobilization stress. Neuropsychopharmacology, 26, 155-163.

Magistretti, P. J. (2006) Neuron-glia metabolic coupling and plasticity. J Exp Biol, 209, 2304-

2311.

Chapter I __________________________________________________________________________

50

Mailly, F., Marin, P., Israel, M., Glowinski, J. and Premont, J. (1999) Increase in external

glutamate and NMDA receptor activation contribute to H2O2-induced neuronal apoptosis.

J Neurochem, 73, 1181-1188.

Makar, T. K., Nedergaard, M., Preuss, A., Gelbard, A. S., Perumal, A. S. and Cooper, A. J.

(1994) Vitamin E, ascorbate, glutathione, glutathione disulfide, and enzymes of

glutathione metabolism in cultures of chick astrocytes and neurons: evidence that

astrocytes play an important role in antioxidative processes in the brain. J Neurochem, 62, 45-53.

Mancuso, C., Capone, C., Ranieri, S. C., Fusco, S., Calabrese, V., Eboli, M. L., Preziosi, P.,

Galeotti, T. and Pani, G. (2008) Bilirubin as an endogenous modulator of neurotrophin

redox signaling. J Neurosci Res, 86, 2235-2249.

Mark, R. J., Lovell, M. A., Markesbery, W. R., Uchida, K. and Mattson, M. P. (1997) A role for

4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion

homeostasis and neuronal death induced by amyloid beta-peptide. J Neurochem, 68, 255-

264.

Marques, C. A., Keil, U., Bonert, A., Steiner, B., Haass, C., Muller, W. E. and Eckert, A.

(2003) Neurotoxic mechanisms caused by the Alzheimer's disease-linked Swedish

amyloid precursor protein mutation: oxidative stress, caspases, and the JNK pathway. J

Biol Chem, 278, 28294-28302.

Marx, C. E., Jarskog, L. F., Lauder, J. M., Lieberman, J. A. and Gilmore, J. H. (2001)

Cytokine effects on cortical neuron MAP-2 immunoreactivity: implications for

schizophrenia. Biol Psychiatry, 50, 743-749.

Matapurkar, A. and Lazebnik, Y. (2006) Requirement of cytochrome c for apoptosis in human

cells. Cell Death Differ, 13, 2062-2067.

Merrill, J. E., Murphy, S. P., Mitrovic, B., Mackenzie-Graham, A., Dopp, J. C., Ding, M.,

Griscavage, J., Ignarro, L. J. and Lowenstein, C. J. (1997) Inducible nitric oxide synthase

and nitric oxide production by oligodendrocytes. J Neurosci Res, 48, 372-384.

Mielke, K. and Herdegen, T. (2000) JNK and p38 stresskinases--degenerative effectors of

signal-transduction-cascades in the nervous system. Prog Neurobiol, 61, 45-60.

Mitani, A., Watanabe, M. and Kataoka, K. (1998) Functional change of NMDA receptors

related to enhancement of susceptibility to neurotoxicity in the developing pontine

nucleus. J Neurosci, 18, 7941-7952.

Mizui, T., Kinouchi, H. and Chan, P. H. (1992) Depletion of brain glutathione by buthionine

sulfoximine enhances cerebral ischemic injury in rats. Am J Physiol, 262, H313-317.

Moncada, S. and Bolaños, J. P. (2006) Nitric oxide, cell bioenergetics and

neurodegeneration. J Neurochem, 97, 1676-1689.


51

Navarro, A. and Boveris, A. (2007) The mitochondrial energy transduction system and the

aging process. Am J Physiol Cell Physiol, 292, C670-686.

Nehru, B. and Kanwar, S. S. (2004) N-acetylcysteine exposure on lead-induced lipid

peroxidative damage and oxidative defence system in brain regions of rats. Biol Trace

Elem Res, 101, 257-264.

Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry, 4th Ed. Worth

Publishers, New York.

Neumar, R. W. (2000) Molecular mechanisms of ischemic neuronal injury. Ann Emerg Med,

36, 483-506.

Ngai, K. C. and Yeung, C. Y. (1999) Additive effect of tumor necrosis factor-alpha and

endotoxin on bilirubin cytotoxicity. Pediatr Res, 45, 526-530.

Nicholls, D. G. and Ferguson, S. J. (2002) Bioenergetics, 3rd Ed. Academic Press, New York.

Nohl, H., Gille, L. and Staniek, K. (2005) Intracellular generation of reactive oxygen species

by mitochondria. Biochem Pharmacol, 69, 719-723.

O'Neill, L. A. and Kaltschmidt, C. (1997) NF-kappa B: a crucial transcription factor for glial

and neuronal cell function. Trends Neurosci, 20, 252-258.

O'Shea, T. M. (2002) Cerebral palsy in very preterm infants: new epidemiological insights.

Ment Retard Dev Disabil Res Rev, 8, 135-145.

Ocal, K., Avlan, D., Cinel, I., Unlu, A., Ozturk, C., Yaylak, F., Dirlik, M., Camdeviren, H. and

Aydin, S. (2004) The effect of N-acetylcysteine on oxidative stress in intestine and

bacterial translocation after thermal injury. Burns, 30, 778-784.

Oh, W., Tyson, J. E., Fanaroff, A. A. et al. (2003) Association between peak serum bilirubin

and neurodevelopmental outcomes in extremely low birth weight infants. Pediatrics, 112, 773-779.

Ostrow, J. D., Mukerjee, P. and Tiribelli, C. (1994) Structure and binding of unconjugated

bilirubin: relevance for physiological and pathophysiological function. J Lipid Res, 35, 1715-1737.

Ostrow, J. D., Pascolo, L., Brites, D. and Tiribelli, C. (2004) Molecular basis of bilirubin-

induced neurotoxicity. Trends Mol Med, 10, 65-70.

Packer, L. and Cadenas, E. (2007) Oxidants and antioxidants revisited. New concepts of

oxidative stress. Free Radic Res, 41, 951-952.

Panegyres, P. K. and Hughes, J. (1998) The neuroprotective effects of the recombinant

interleukin-1 receptor antagonist rhIL-1ra after excitotoxic stimulation with kainic acid and

its relationship to the amyloid precursor protein gene. J Neurol Sci, 154, 123-132.

Chapter I __________________________________________________________________________

52

Papadopoulos, M. C., Koumenis, I. L., Dugan, L. L. and Giffard, R. G. (1997) Vulnerability to

glucose deprivation injury correlates with glutathione levels in astrocytes. Brain Res, 748, 151-156.

Papadopoulos, M. C., Koumenis, I. L., Yuan, T. Y. and Giffard, R. G. (1998) Increasing

vulnerability of astrocytes to oxidative injury with age despite constant antioxidant

defences. Neuroscience, 82, 915-925.

Park, W. S., Chang, Y. S., Chung, S. H., Seo, D. W., Hong, S. H. and Lee, M. (2001) Effect

of hypothermia on bilirubin-induced alterations in brain cell membrane function and energy

metabolism in newborn piglets. Brain Res, 922, 276-281.

Peeters-Scholte, C., Koster, J., Veldhuis, W. et al. (2002) Neuroprotection by selective nitric

oxide synthase inhibition at 24 hours after perinatal hypoxia-ischemia. Stroke, 33, 2304-

2310.

Pellerin, L., Bouzier-Sore, A. K., Aubert, A., Serres, S., Merle, M., Costalat, R. and

Magistretti, P. J. (2007) Activity-dependent regulation of energy metabolism by astrocytes:

an update. Glia, 55, 1251-1262.

Pellerin, L. and Magistretti, P. J. (1994) Glutamate uptake into astrocytes stimulates aerobic

glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad

Sci U S A, 91, 10625-10629.

Perlman, J. M., Rogers, B. B. and Burns, D. (1997) Kernicteric findings at autopsy in two sick

near term infants. Pediatrics, 99, 612-615.

Petty, M. A. and Lo, E. H. (2002) Junctional complexes of the blood-brain barrier:

permeability changes in neuroinflammation. Prog Neurobiol, 68, 311-323.

Poli, G., Cadenas, E. and Packer, L. (2000) Free Radicals in Brain Pathophysiology. Marcel

Dekker, Inc., New York.

Porras, O. H., Loaiza, A. and Barros, L. F. (2004) Glutamate mediates acute glucose

transport inhibition in hippocampal neurons. J Neurosci, 24, 9669-9673.

Porter, M. L. and Dennis, B. L. (2002) Hyperbilirubinemia in the term newborn. Am Fam

Physician, 65, 599-606.

Prasanthi, R. P., Devi, C. B., Basha, D. C., Reddy, N. S. and Reddy, G. R. (2010) Calcium

and zinc supplementation protects lead (Pb)-induced perturbations in antioxidant enzymes

and lipid peroxidation in developing mouse brain. Int J Dev Neurosci, 28, 161-167.

Puka-Sundvall, M., Wallin, C., Gilland, E., Hallin, U., Wang, X., Sandberg, M., Karlsson, J.,

Blomgren, K. and Hagberg, H. (2000) Impairment of mitochondrial respiration after

cerebral hypoxia-ischemia in immature rats: relationship to activation of caspase-3 and

neuronal injury. Brain Res Dev Brain Res, 125, 43-50.


53

Purves, D., Augustine, G. J., Fitzpatrick, D., Hall, W. C., LaMantia, A., McNamara, J. O. and

Williams, S. M. (2004) Studying the Nervous Systems of Humans and Other Animals. In:

Neuroscience, 3rd Ed. Sinauer Associates Inc., Sunderland.

Pyne-Geithman, G. J., Morgan, C. J., Wagner, K., Dulaney, E. M., Carrozzella, J., Kanter, D.

S., Zuccarello, M. and Clark, J. F. (2005) Bilirubin production and oxidation in CSF of

patients with cerebral vasospasm after subarachnoid hemorrhage. J Cereb Blood Flow

Metab, 25, 1070-1077.

Quagliarello, V. J., Wispelwey, B., Long, W. J., Jr. and Scheld, W. M. (1991) Recombinant

human interleukin-1 induces meningitis and blood-brain barrier injury in the rat.

Characterization and comparison with tumor necrosis factor. J Clin Invest, 87, 1360-1366.

Raivich, G., Bohatschek, M., Kloss, C. U., Werner, A., Jones, L. L. and Kreutzberg, G. W.

(1999) Neuroglial activation repertoire in the injured brain: graded response, molecular

mechanisms and cues to physiological function. Brain Res Brain Res Rev, 30, 77-105.

Ravagnan, L., Roumier, T. and Kroemer, G. (2002) Mitochondria, the killer organelles and

their weapons. J Cell Physiol, 192, 131-137.

Rice, M. E. (2000) Ascorbate regulation and its neuroprotective role in the brain. Trends

Neurosci, 23, 209-216.

Rodrigues, C. M. and Steer, C. J. (2001) The therapeutic effects of ursodeoxycholic acid as

an anti-apoptotic agent. Expert Opin Investig Drugs, 10, 1243-1253.

Rodrigues, C. M. P., Solá, S. and Brites, D. (2002a) Bilirubin induces apoptosis via the

mitochondrial pathway in developing rat brain neurons. Hepatology, 35, 1186-1195.

Rodrigues, C. M. P., Solá, S., Brito, M. A., Brites, D. and Moura, J. J. (2002b) Bilirubin

directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat

mitochondria. J Hepatol, 36, 335-341.

Rodrigues, C. M. P., Solá, S., Silva, R. F. M. and Brites, D. (2000) Bilirubin and amyloid-β

peptide induce cytochrome c release through mitochondrial membrane permeabilization.

Mol Med, 6, 936-946.

Rothwell, N. J. and Luheshi, G. N. (2000) Interleukin 1 in the brain: biology, pathology and

therapeutic target. Trends Neurosci, 23, 618-625.

Roux, P. P. and Blenis, J. (2004) ERK and p38 MAPK-activated protein kinases: a family of

protein kinases with diverse biological functions. Microbiol Mol Biol Rev, 68, 320-344.

Sampath, V., Bowen, J. and Gibson, F. (2005) Risk factors for adverse neurodevelopment in

extremely low birth weight infants with normal neonatal cranial ultrasound. J Perinatol, 25, 210-215.

Chapter I __________________________________________________________________________

54

Sánchez-Carbente, M. R., Castro-Obregon, S., Covarrubias, L. and Narvaez, V. (2005)

Motoneuronal death during spinal cord development is mediated by oxidative stress. Cell

Death Differ, 12, 279-291.

Santos, M. S., Moreno, A. J. and Carvalho, A. P. (1996) Relationships between ATP

depletion, membrane potential, and the release of neurotransmitters in rat nerve

terminals. An in vitro study under conditions that mimic anoxia, hypoglycemia, and

ischemia. Stroke, 27, 941-950.

Schlingensiepen, K. H., Schlingensiepen, R., Kunst, M., Klinger, I., Gerdes, W., Seifert, W.

and Brysch, W. (1993) Opposite functions of jun-B and c-jun in growth regulation and

neuronal differentiation. Dev Genet, 14, 305-312.

Schoemaker, M. H., Conde de la Rosa, L., Buist-Homan, M., Vrenken, T. E., Havinga, R.,

Poelstra, K., Haisma, H. J., Jansen, P. L. and Moshage, H. (2004) Tauroursodeoxycholic

acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival

pathways. Hepatology, 39, 1563-1573.

Sener, G., Toklu, H., Kapucu, C., Ercan, F., Erkanli, G., Kacmaz, A., Tilki, M. and Yegen, B.

C. (2005) Melatonin protects against oxidative organ injury in a rat model of sepsis. Surg

Today, 35, 52-59.

Shah, S. A., Volkov, Y., Arfin, Q., Abdel-Latif, M. M. and Kelleher, D. (2006) Ursodeoxycholic

acid inhibits interleukin 1 beta [corrected] and deoxycholic acid-induced activation of NF-

kappaB and AP-1 in human colon cancer cells. Int J Cancer, 118, 532-539.

Shapiro, S. M. (2005) Definition of the clinical spectrum of kernicterus and bilirubin-induced

neurologic dysfunction (BIND). J Perinatol, 25, 54-59.

Sharpe, M. A. and Cooper, C. E. (1998) Reactions of nitric oxide with mitochondrial

cytochrome c: a novel mechanism for the formation of nitroxyl anion and peroxynitrite.

Biochem J, 332 ( Pt 1), 9-19.

Sheng, W. S., Hu, S., Ni, H. T., Rowen, T. N., Lokensgard, J. R. and Peterson, P. K. (2005)

TNF-alpha-induced chemokine production and apoptosis in human neural precursor cells.

J Leukoc Biol, 78, 1233-1241.

Sheu, K. F. and Blass, J. P. (1999) The alpha-ketoglutarate dehydrogenase complex. Ann N

Y Acad Sci, 893, 61-78.

Sies, H. (1997) Oxidative stress: oxidants and antioxidants. Exp Physiol, 82, 291-295.

Silva, R. F. M., Rodrigues, C. M. P. and Brites, D. (2002) Rat cultured neuronal and glial cells

respond differently to toxicity of unconjugated bilirubin. Pediatr Res, 51, 535-541.

Silva, S. L., Vaz, A. R., Barateiro, A., Falcão, A. S., Fernandes, A., Brito, M. A., Silva, R. F.

M. and Brites, D. (2010) Features of bilirubin-induced reactive microglia: From

phagocytosis to inflammation. Neurobiol Dis, 40, 663-675.


55

Solá, S., Brito, M. A., Brites, D., Moura, J. J. and Rodrigues, C. M. (2002) Membrane

structural changes support the involvement of mitochondria in the bile salt-induced

apoptosis of rat hepatocytes. Clin Sci (Lond), 103, 475-485.

Solá, S., Ma, X., Castro, R. E., Kren, B. T., Steer, C. J. and Rodrigues, C. M. P. (2003)

Ursodeoxycholic acid modulates E2F-1 and p53 expression through a caspase-

independent mechanism in transforming growth factor beta1-induced apoptosis of rat

hepatocytes. J Biol Chem, 278, 48831-48838.

Soorani-Lunsing, I., Woltil, H. A. and Hadders-Algra, M. (2001) Are moderate degrees of

hyperbilirubinemia in healthy term neonates really safe for the brain? Pediatr Res, 50, 701-705.

Stevenson, D. K., Dennery, P. A. and Hintz, S. R. (2001) Understanding newborn jaundice. J

Perinatol, 21 Suppl 1, S21-24; discussion S35-29.

Stocker, R., Yamamoto, Y., McDonagh, A. F., Glazer, A. N. and Ames, B. N. (1987) Bilirubin

is an antioxidant of possible physiological importance. Science, 235, 1043-1046.

Streit, W. J. (2002) Microglia as neuroprotective, immunocompetent cells of the CNS. Glia,

40, 133-139.

Stuehr, D. J. and Marletta, M. A. (1985) Mammalian nitrate biosynthesis: mouse

macrophages produce nitrite and nitrate in response to Escherichia coli

lipopolysaccharide. Proc Natl Acad Sci U S A, 82, 7738-7742.

Suárez, I., Bodega, G. and Fernández, B. (2002) Glutamine synthetase in brain: effect of

ammonia. Neurochem Int, 41, 123-142.

Suliman, H. B., Welty-Wolf, K. E., Carraway, M., Tatro, L. and Piantadosi, C. A. (2004)

Lipopolysaccharide induces oxidative cardiac mitochondrial damage and biogenesis.

Cardiovasc Res, 64, 279-288.

Szeto, H. H. (2006) Mitochondria-targeted peptide antioxidants: novel neuroprotective

agents. AAPS J, 8, E521-531.

Tammariello, S. P., Quinn, M. T. and Estus, S. (2000) NADPH oxidase contributes directly to

oxidative stress and apoptosis in nerve growth factor-deprived sympathetic neurons. J

Neurosci, 20, RC53.

Thomas, T., Timmer, M., Cesnulevicius, K., Hitti, E., Kotlyarov, A. and Gaestel, M. (2008)

MAPKAP kinase 2-deficiency prevents neurons from cell death by reducing

neuroinflammation--relevance in a mouse model of Parkinson's disease. J Neurochem,

105, 2039-2052.

Tibbles, L. A. and Woodgett, J. R. (1999) The stress-activated protein kinase pathways. Cell

Mol Life Sci, 55, 1230-1254.

Chapter I __________________________________________________________________________

56

Tsai, C. E., Daood, M. J., Lane, R. H., Hansen, T. W., Gruetzmacher, E. M. and Watchko, J.

F. (2002) P-glycoprotein expression in mouse brain increases with maturation. Biol

Neonate, 81, 58-64.

Tsuru-Aoyagi, K., Potts, M. B., Trivedi, A. et al. (2009) Glutathione peroxidase activity

modulates recovery in the injured immature brain. Ann Neurol, 65, 540-549.

Vannucci, S. J. and Hagberg, H. (2004) Hypoxia-ischemia in the immature brain. J Exp Biol,

207, 3149-3154.

Vaughn, A. E. and Deshmukh, M. (2008) Glucose metabolism inhibits apoptosis in neurons

and cancer cells by redox inactivation of cytochrome c. Nat Cell Biol, 10, 1477-1483.

Vezzani, A., Conti, M., De Luigi, A., Ravizza, T., Moneta, D., Marchesi, F. and De Simoni, M.

G. (1999) Interleukin-1beta immunoreactivity and microglia are enhanced in the rat

hippocampus by focal kainate application: functional evidence for enhancement of

electrographic seizures. J Neurosci, 19, 5054-5065.

Volbracht, C., Chua, B. T., Ng, C. P., Bahr, B. A., Hong, W. and Li, P. (2005) The critical role

of calpain versus caspase activation in excitotoxic injury induced by nitric oxide. J

Neurochem, 93, 1280-1292.

Volpe, J. J. (1997) Brain injury in the premature infant--from pathogenesis to prevention.

Brain Dev, 19, 519-534.

Walczak, H. and Krammer, P. H. (2000) The CD95 (APO-1/Fas) and the TRAIL (APO-2L)

apoptosis systems. Exp Cell Res, 256, 58-66.

Wan, F. J., Tung, C. S., Shiah, I. S. and Lin, H. C. (2006) Effects of alpha-phenyl-N-tert-butyl

nitrone and N-acetylcysteine on hydroxyl radical formation and dopamine depletion in the

rat striatum produced by d-amphetamine. Eur Neuropsychopharmacol, 16, 147-153.

Watchko, J. F. (2006) Hyperbilirubinemia and bilirubin toxicity in the late preterm infant. Clin

Perinatol, 33, 839-852; abstract ix.

Watchko, J. F., Daood, M. J. and Hansen, T. W. (1998) Brain bilirubin content is increased in

P-glycoprotein-deficient transgenic null mutant mice. Pediatr Res, 44, 763-766.

Wendel, M. and Heller, A. R. (2010) Mitochondrial function and dysfunction in sepsis. Wien

Med Wochenschr, 160, 118-123.

Wender, R., Brown, A. M., Fern, R., Swanson, R. A., Farrell, K. and Ransom, B. R. (2000)

Astrocytic glycogen influences axon function and survival during glucose deprivation in

central white matter. J Neurosci, 20, 6804-6810.

Wennberg, R. P., Johansson, B. B., Folbergrova, J. and Siesjo, B. K. (1991) Bilirubin-

induced changes in brain energy metabolism after osmotic opening of the blood-brain

barrier. Pediatr Res, 30, 473-478.


57

Wilde, G. J., Pringle, A. K., Sundstrom, L. E., Mann, D. A. and Iannotti, F. (2000) Attenuation

and augmentation of ischaemia-related neuronal death by tumour necrosis factor-alpha in

vitro. Eur J Neurosci, 12, 3863-3870.

Wrona, M. Z. and Dryhurst, G. (1998) Oxidation of serotonin by superoxide radical:

implications to neurodegenerative brain disorders. Chem Res Toxicol, 11, 639-650.

Xu, L., Sapolsky, R. M. and Giffard, R. G. (2001) Differential sensitivity of murine astrocytes

and neurons from different brain regions to injury. Exp Neurol, 169, 416-424.

Yamada, N., Sawasaki, Y. and Nakajima, H. (1977) Impairment of DNA synthesis in Gunn rat

cerebellum. Brain Res, 126, 295-307.

Yan, L., Ge, H., Li, H. et al. (2004) Gender-specific proteomic alterations in glycolytic and

mitochondrial pathways in aging monkey hearts. J Mol Cell Cardiol, 37, 921-929.

Yang, G. Y., Liu, X. H., Kadoya, C., Zhao, Y. J., Mao, Y., Davidson, B. L. and Betz, A. L.

(1998) Attenuation of ischemic inflammatory response in mouse brain using an adenoviral

vector to induce overexpression of interleukin-1 receptor antagonist. J Cereb Blood Flow

Metab, 18, 840-847.

Yang, L., Lindholm, K., Konishi, Y., Li, R. and Shen, Y. (2002) Target depletion of distinct

tumor necrosis factor receptor subtypes reveals hippocampal neuron death and survival

through different signal transduction pathways. J Neurosci, 22, 3025-3032.

Yarian, C. S., Toroser, D. and Sohal, R. S. (2006) Aconitase is the main functional target of

aging in the citric acid cycle of kidney mitochondria from mice. Mech Ageing Dev, 127, 79-

84.

Yi, J. H. and Hazell, A. S. (2006) Excitotoxic mechanisms and the role of astrocytic glutamate

transporters in traumatic brain injury. Neurochem Int, 48, 394-403.

Yoneyama, M., Kawada, K., Gotoh, Y., Shiba, T. and Ogita, K. (2010) Endogenous reactive

oxygen species are essential for proliferation of neural stem/progenitor cells. Neurochem

Int, 56, 740-746.

Zachwieja, J., Zaniew, M., Bobkowski, W., Stefaniak, E., Warzywoda, A., Ostalska-Nowicka,

D., Dobrowolska-Zachwieja, A., Lewandowska-Stachowiak, M. and Siwinska, A. (2005)

Beneficial in vitro effect of N-acetyl-cysteine on oxidative stress and apoptosis. Pediatr

Nephrol, 20, 725-731.

Zhu, C., Wang, X., Qiu, L., Peeters-Scholte, C., Hagberg, H. and Blomgren, K. (2004)

Nitrosylation precedes caspase-3 activation and translocation of apoptosis-inducing factor

in neonatal rat cerebral hypoxia-ischaemia. J Neurochem, 90, 462-471.

Chapter II

II. Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in immature cortical neurons. Assessment of

the protective effects of glycoursodeoxycholic acid

Ana Rita Vaz1, Maria Delgado-Esteban2, Maria Alexandra Brito1, Juan P. Bolaños3, Dora Brites1, Angeles Almeida2,3

1Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL),

Faculdade de Farmácia, University of Lisbon, Av. Professor Gama Pinto, Lisbon

1649-003, Portugal.

2Unidad de Investigación, Hospital Universitario de Salamanca, Instituto de Estudios

de Ciencias de la Salud de Castilla y León, Salamanca 37007, Spain.

3Departmento de Bioquímica y Biologia Molecular, University of Salamanca, Instituto

de Neurociencias de Castilla y León, Salamanca 37007, Spain.

Journal of Neurochemistry (2010) 112, 56–65.

Acknowledgements The skilful assistance of Mrs. Monica Resch is acknowledged. We are grateful to Dr.

Margarida Silva for her advice concerning ATP measurements. Supported by RENEVAS,

Fondo de Investigación Sanitaria (FIS06/0794) and Junta de Castilla y León (to A.A),

SAF2007-61492, CONSOLIDER RosasNet CSD2007-00020, SA066A07 and Red

Terapia Celular-ISCIII (to J.P.B.), grants POCI/SAU/MMO/55955/2004, PTDC/SAU-

NEU/64385/2006, POCI 2010 and FEDER (to D.B.) and BD/30292/2006 (to A.R.V.) from

Fundação para a Ciência e a Tecnologia, Lisbon, Portugal

UCB, mitochondrial dysfunction and GUDCA __________________________________________________________________________

61

Abstract

High levels of unconjugated bilirubin (UCB) may initiate encephalopathy in neonatal life,

mainly in premature infants. The molecular mechanisms of this bilirubin-induced neurologic

dysfunction (BIND) are not yet clarified and no neuroprotective strategy is currently

worldwide accepted. Here, we show that UCB, at conditions mimicking those of

hyperbilirubinemic newborns (50 µM UCB in the presence of 100 µM human serum albumin),

rapidly (within 1 h) inhibited cytochrome c oxidase activity and ascorbate-driven oxygen

consumption in 3 days in vitro rat cortical neurons. This was accompanied by a bioenergetic

and oxidative crisis, and apoptotic cell death, as judged by the collapse of the inner

mitochondrial membrane potential, increased glycolytic activity, superoxide anion radical

production and ATP release, as well as disruption of glutathione redox status. Furthermore,

the antioxidant compound glycoursodeoxycholic acid, fully abrogated UCB-induced

cytochrome c oxidase inhibition and significantly prevented oxidative stress, metabolic

alterations and cell demise. These results suggest that the neurotoxicity associated with

neonatal bilirubin-induced encephalopathy occur through a deregulation of energy

metabolism, and supports the notion that glycoursodeoxycholic acid may be useful in the

treatment of BIND.

Keywords: bilirubin neurotoxicity, glycolysis, glycoursodeoxycholic acid, mitochondrial

dysfunction, oxidative stress, respiratory chain.

Chapter II __________________________________________________________________________

62

1. Introduction As a consequence of the short half-life of fetal erythrocytes, and of the limited ability of

the neonate to conjugate and excrete bilirubin, newborn infants often show increased levels

of serum unconjugated bilirubin (UCB). This condition, known as the physiologic jaundice, is

usually resolved by the end of the first week of life without treatment (Ostrow et al., 2003,

Reiser, 2004). However, severe hyperbilirubinemia in neonates with prematurity and/or

systemic illnesses such as hemolytic disease, acidosis, and hypoxemia enhances their risk

for bilirubin-induced neurologic dysfunction (BIND) (McDonald et al., 1998, Ostrow et al.,

2004, Shapiro, 2005). In fact, in vitro experiments revealed that immature neurons have an

increased susceptibility to UCB (Falcão et al., 2006). Although the molecular mechanisms of

BIND remain to be fully clarified, it was shown to involve immunostimulation, accumulation of

extracellular glutamate, oxidative stress, apoptosis and loss of cell viability (Brites et al.,

2009, Brito et al., 2008b). This excitotoxic-like situation prompted us to hypothesize whether

UCB would exert the death of immature nerve cells through an impairment of energy

metabolism.

Many previous studies have examined the effects of UCB on cerebral energy status but

the results have been equivocal. In fact, initial reports of an inhibition of respiration and

uncoupling of oxidative phosphorylation, observed in brain homogenates or isolated

mitochondria, pointed to mitochondrial dysfunction as an important element of UCB toxicity

(Mustafa et al., 1969, Ernster and Zetterström, 1956, Day, 1954). The energy depletion was

later on corroborated by the reduced rates of glycolysis and decreased ATP levels observed

in Gunn rats and in newborn piglets (Katoh-Semba, 1976, McCandless and Abel, 1980,

Hoffman et al., 1996, Park et al., 2001). However, discrepant findings were reported in other

studies that failed to document significant changes in brain glucose metabolism or oxidative

phosphorylation (Diamond and Schmid, 1967, Brann et al., 1987), whereas others

demonstrated that hyperbilirubinemia only disturbs brain energy metabolism in the presence

of additional factors that disrupt the blood brain barrier, such as hypoxia or hyperosmolarity

(Ives et al., 1988, Ives et al., 1989, Wennberg et al., 1991). Thus, further studies are needed

to clarify the effects of UCB on brain energy and glucose metabolism in nerve cells,

particularly in poor differentiated neurons. This would provide a valuable contribute to the

current understanding of the neuropathological effects of UCB, as neuronal energy

metabolism is determinant for processes as neurotransmitter release, neurite outgrowth and

cell survival (Mattson and Liu, 2002), which are impaired by UCB. Moreover, energy

hypometabolism is one of the most consistent and earliest abnormalities seen in mild

cognitive impairment (Atamna and Frey, 2007), which is particularly relevant in premature

jaundiced infants.


63

Recent data indicate that ursodeoxycholic acid and its glycine-conjugated species are

able to prevent UCB-induced apoptosis and loss of cell viability, oxidative stress and

immunostimulation (Brito et al., 2008a, Fernandes et al., 2007) and induces a rapid and

sustained decrease in plasma UCB concentrations in Gunn rats, the well-established animal

model for severe hyperbilirubinemia (Cuperus et al., 2009). Thus, we decided to test the

neuroprotective effects of glycoursodeoxycholic acid (GUDCA) in our model of immature

neurons.

In this study we show that UCB at a UCB to HSA molar ratio that can be found in the

plasma of moderately jaundiced neonates (Brito et al., 2006), rapidly and selectively inhibits

the activity of cytochrome c oxidase, the terminal component of the mitochondrial respiratory

chain, in immature neurons; this led to an impairment in oxygen consumption, inner-

mitochondrial membrane potential (ΔѰm) collapse and apoptosis. These phenomena were

associated with an increase in cellular oxidized glutathione, production of superoxide anion

radical (O2.-) and a decrease in NADPH. Pretreatment of neurons with GUDCA prior to

exposure to UCB prevented inhibition of cytrochrome c oxidase activity together with

preservation of glutathione and NADPH status. These data indicate that cytochrome c

oxidase inhibition may be involved in the neurotoxicity associated with BIND and strongly

indicates the possible therapeutic potential of GUDCA in the treatment of this disorder.

2. Materials and Methods

2.1. Chemicals Neurobasal medium, B-27 supplement, Hanks’ balanced salt solution (HBSS-1), Hanks’

balanced salt solution without Ca2+ and Mg2+ (HBSS-2), gentamicin (50 mg/mL),

tetramethylrhodamine (TMRE), MitoSOX Red and trypsin (0.025%) were acquired from

Invitrogen (Carlsbad, CA). Human serum albumin (HSA) (fraction V, fatty acid free), carbonyl

cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), ubiquinone-5 (coenzyme Q1, CoQ1),

2,2,4-trimethyl-1,3-pentanediol (TMPD), sulfosalicyclic acid and 2-vinylpyridine were

purchased from Sigma Chemical Co (St Louis, MO). UCB was also from Sigma and purified

as previously described (McDonagh, 1979). Cytochrome c was obtained from Roche

Diagnostics (Heidelberg, Germany), and it was reduced with sodium ascorbate (Sigma) just

before use and passed through Sephadex G-25 M (PD-10 columns, Amersham Pharmacia

Biotech, Uppsala, Sweden) to remove excess ascorbate. GUDCA, as well as Caspases 3

and 9 substrates, Ac-DEVD-pNA and Ac-LEHD-pNA, respectively, were purchased from

Chapter II __________________________________________________________________________

64

Calbiochem (Darmstadt, Germany). Other substrates, inhibitors, enzymes, and coenzymes

were purchased from Sigma, Roche Diagnostics or Merck (Darmstadt, Germany).

2.2. Neurons in primary culture

Animal care followed the European Legislation on Protection of Animals Used for

Experimental and Scientific Purposes (EU directive L0065, 22/07/2003). Neurons were

isolated from fetuses of 16-17-day pregnant Wistar rats, as described previously (Silva et al.,

2002). The fetuses were collected in HBSS-1, the brain cortex was mechanically fragmented,

and the fragments transferred to a 0.025% (w/v) trypsin in HBSS-2 solution and incubated for

15 min at 37ºC. After trypsinization, cells were washed twice in HBSS-2 containing 10% (v/v)

FBS, and resuspended in Neurobasal medium supplemented with 0.5 mM L-glutamine, 25

μM L-glutamic acid, 2% B-27 supplement, and 0.12 mg/mL gentamicin. Finally, cells were

seeded on poly-D-lysine coated tissue culture plates at a density of 2.5 x 105 cells/cm2 and

maintained at 37ºC in a humidified atmosphere of 5% CO2. In this work we used neurons at 3

days in vitro (DIV).

2.3. Treatment of neurons

Neurons were incubated in Neurobasal medium without (control) or with 50 μM UCB

(from a 10 mM stock solution) in the presence of 100 μM HSA (UCB/HSA molar ratio of 0.5)

for 1 h at 37ºC. Stock UCB solutions were extemporarily prepared in 0.1 M NaOH under the

dark and the pH adjusted to 7.4 using 0.1 M HCl. When appropriate, neurons were pre-

incubated with GUDCA (50 µM) 1 h prior to UCB addition.

2.4. Determination of the mitochondrial respiratory chain complex activities and citrate

synthase Neurons plated on 60 cm2 Petri dishes were washed with ice-cold PBS and surviving

cells were collected by trypsinization, centrifuged and resuspended in 300 μL of 0.1 M

potassium phosphate buffer (pH 7.0). Cell suspensions (containing about 7-8 mg of

protein/mL) were frozen and thawed three times to ensure cell lysis. Enzyme activities were

determined in the cell lysates using an Uvikon XL spectrophotometer (Secomam, Domont,

France). NADH-CoQ1 reductase (complex I; EC 1.6.99.3) activity was measured as

described by Ragan et al. (1987). The activity of succinate-cytochrome c reductase (complex

II-III; EC 1.8.3.1) was determined following the method of King (1967). Cytochome c oxidase

(complex IV, EC 1.9.3.1) activity was assessed as described by Wharton and Tzagoloff

(1967). Citrate synthase (EC 4.1.3.7) activity was assessed as referred by Shepherd and

Garland (1969). Protein concentrations were determined by the method of Lowry et al.


65

(1951). All enzyme activities were expressed as nanomoles per minute per milligram of

protein, except for cytochrome c oxidase, which was expressed as the first-order rate

constant (k) per minute per milligram of protein (Almeida and Bolaños, 2001).

2.5. Detection of superoxide anion radical (O2.-)

After incubation in 9.6 cm2 wells, neurons were incubated in PBS containing MitoSox-

Red (2 μM) for 30 min, washed with PBS and fluorescence assessed by flow cytometry,

using MitoSox-Red method (Invitrogen), as previously described (Mukhopadhyay et al.,

2007). For the determination of mitochondrial superoxide by flow cytometry, the

measurements were carried out using FACScalibur flow cytometer (15 mW argon ion laser

tuned at 488 nm; CellQuest software, Becton Dickinson Biosciences) and the data collected

at 585/42 nm (FL2) and 670LP (FL3) channel. In this study, the data were presented in the

FL2 channel. Antimycin A at 20 µM was used for 15 minutes as a mitochondrial superoxide

generator.

2.6. Determination of oxygen consumption

Oxygen consumption was determined with a Clark-type electrode (Rank Brothers,

Cambridge, UK). Briefly, after the incubation period, neurons plated on 60 cm2 Petri dishes

were collected by trypsinization, centrifuged, rinsed once with buffered Hank’s solution and

resuspended in 500 μL of Hank’s solution (without glucose). Cell suspensions were kept on

ice until used for oxygen consumption (within 1 h). The rates of oxygen consumption were

calculated from the slopes (monitored for at least 15 min per trace), and expressed as

nanomoles of oxygen consumed per minute per 106 cells (Almeida et al., 1998). TMPD was

used together with ascorbate to assure the reduced form of cytochrome c.

2.7. ∆Ѱm measurements

For fluorescence measurements, neurons incubated in 9.6 cm2 wells were stained as

previously described (Almeida et al., 1999) with minor modifications. Briefly, neurons were

incubated in Hanks’ solution containing 1 μg/mL of tetramethylrhodamine (TMRE) for 30 min

at 37ºC. Excess dye was removed by washing cells twice with buffered Hanks’ solution and

covered with 1 mL of Hanks’solution. For each Petri dish, four fluorescence

microphotographs were taken with an inverted microscope with a fluorescein filter (excitation

filter 480-490 nm; emission filter 510-530 nm) and the intensity of fluorescence was

quantified using an image analyser system (NIH Image Program). The representative

selected area was always the same for all experimental conditions studied. The fluorescence

intensity corresponding to control cells was arbitrarily assigned a value of 100%

Chapter II __________________________________________________________________________

66

fluorescence. The 0% ΔѰm value was obtained when cells were loaded with TMRE in the

presence 10 μM of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) as

previously referred (Almeida and Bolaños, 2001). The monotonic decrease of fluorescence

with FCCP assures that we are measuring mitochondrial membrane potential and not the

plasma membrane potential where a different pattern of fluorescence would be observed

(Farkas et al., 1989).

2.8. Metabolite determinations

Neurons (about 3x107) cultured in 60 cm2 Petri dishes and treated as abovementioned

were rapidly washed with ice-cold PBS, scrapped off with 0.3 M HClO4 and neutralized with 2

M KHCO3 to pH 6.5. The perchlorate precipitate was removed by centrifugation, and

fructose-6-phosphate (F6P) and fructose-1,6-bisphosphate (F1,6P2) concentrations were

measured in the supernatants as previously described (Almeida et al. 2004); intracellular

lactate concentrations were determined in the same neutralized extracts as previously

mentioned (Gutmann and Wahlefeld, 1974).

For the assessment of fructose-2,6-bisphosphate (F2,6P2), cell extracts were lysed in

0.1 M sodium hydroxide and centrifuged (20,000g for 20 min) as previously described

(Almeida et al., 2004). Briefly, an aliquot of the homogenate was used for protein

determination and the remaining sample was heated at 80°C (5 min), centrifuged (20,000g

for 20 min) and the resulting supernatant used for the determination of F2,6P2 concentrations

(Kawaguchi et al., 2001, Van Schaftingen et al., 1982).

For intracellular ATP evaluation, neurons were cultured in 2 cm2 wells and treated as

above. At the end of the incubation period, cells were rapidly washed with ice-cold PBS,

scrapped off with 2 x 0.5 mL of 0.3 M HClO4, and neutralized with 0.5 mL of 2 M KHCO3 at

pH 6.5. The perchlorate precipitate was removed by centrifugation, and ATP was determined

in the supernatants by chemiluminescence using a commercially available kit following the

manufacturer’s instructions. The released ATP was considered to be that found in the culture

medium and the quantification was an adaptation of the method previously described by

Silva et al. (1997). Briefly, after incubation period, supernatants were collected, placed on ice

and exposed to 2 M HClO4. A solution of 2 M KHCO3 was used to restore pH at 6.5. The

perchlorate precipitate was removed by centrifugation, and ATP levels were measured

fluorimetrically in the protein-free supernatants.

For glutathione measurement, neurons incubated in 9.6 cm2 wells were washed with ice-

cold PBS and immediately collected by scrapping off with 0.5 mL of 1% (w/v) sulfosalicyclic

acid. Cell lysates were centrifuged at 13,000 g for 5 min at 4ºC, and the supernatants used

for glutathione determinations on the same day. Total glutathione content (GSx, i.e. the


67

amount of GSH plus two times the amount of GSSG) and oxidized glutathione (GSSG) were

measured and calculated as previously described (Dringen and Hamprecht, 1996, García-

Nogales et al., 1999). GSX and GSSG concentrations were expressed as nanomoles per

milligram of protein.

NADPH concentrations were measured accordingly with García-Nogales et al. (1999). In

brief, neurons incubated in 2 cm2 wells were washed with ice-cold PBS, and collected in 200

μL of 0.5 M KOH in 50% (v/v) ethanol. Cell lysates were neutralized (pH 7.8) with 200 μL of

0.5 M triethanolamine/0.5 M potassium phosphate and centrifuged at 13,000 g for 2 min at

4ºC. A 50-μL aliquot of the supernatant was immediately used for NADPH determination

(Wulff, 1985), with the exception that NADH was oxidized by incubation of the samples with

0.5 mU/μL lactate dehydrogenase and 1 mM pyruvate (Klingerberg, 1985).

2.9. Assessment of apoptotic cell death by flow citometry

Allophycocyanin (APC)-conjugated annexin-V and 7-amino-actinomycin D (7-AAD)

(Apoptosis Assay Kit; Becton Dickinson Biosciences, San Jose, CA, USA) were used to

quantitatively determine the percentage of apoptotic cells by flow cytometry. After incubation

in 2 cm2 wells, neurons were stained with annexin-V-APC and 7-AAD, following the

manufacturer’s instructions, and they were analysed on a FACScalibur flow cytometer (15

mW argon ion laser tuned at 488 nm; CellQuest software, Becton Dickinson Biosciences).

The annexin V-APC-stained cells that were 7-AAD negative were considered apoptotic

(Almeida et al., 2004).

2.10. Analysis of apoptotic cell death by 4'-6-diamidino-2-phenylindole (DAPI) nuclear

staining Neurons were fixed with 4% (v/v, in PBS) paraformaldehyde for 30 min at room

temperature, rinsed with PBS and incubated with DAPI (30 µM, Sigma). After 10 min, cells

were washed three times with PBS and their nuclei examined under a fluorescence

microscope by an author blinded to the test. A total of ~200 cells per condition in three

different cultures were quantified, and the results were expressed as the percentage of

condensed or fragmented nuclei.

2.11. Caspase-3 and -9 activity assays

Activities of caspase-3 and -9 were measured by a colorimetric method (Calbiochem,

Darmstadt, Germany). Cells were harvested, washed with ice-cold PBS and lysed for 30

minutes on ice in the lysis buffer [50 mM HEPES (pH 7.4); 100 mM NaCl; 0.1% (w/v)

CHAPS; 1 mM DTT; 0.1 mM EDTA]. The lysate was centrifuged at 10,000 g for 10 min at

Chapter II __________________________________________________________________________

68

4ºC and the supernatants were collected and stored at -80ºC. Protein concentrations were

determined as aforementioned. The activity of caspases 3 and 9 was determined in cell

lysates by enzymatic cleavage of chromophore p-nitroanilide (pNA) from the substrate Ac-

YVAD-pNA, according to manufacturer’s instructions. The proteolytic reaction was carried

out in protease assay buffer buffer [50 mM HEPES (pH 7.4); 100 mM NaCl; 0.1% (w/v)

CHAPS; 10 mM DTT; 0.1 mM EDTA; 10% (v/v) glicerol], containing 2 mM substrate Ac-

DEVD-pNA for caspase-3 and Ac-LEHD-pNA for caspase-9. Following incubation of the

reaction mixtures for 2 h at 37ºC, the formation of pNA was measured at λ= 405 nm with a

reference filter of 620 nm.

2.12. Statistical analysis

Measurements from individual cultures were performed in triplicate. The results are

expressed as the mean ± SEM values for the number of culture preparations indicated in the

legends. Statistical analysis of the results was performed by one-way analysis of variance

followed by the least significant difference multiple range test, and p<0.05 was accepted as

statistically significant in all cases.

3. Results

3.1. UCB selectively impairs cytochrome c oxidase activity in immature neurons, which is prevented by GUDCA

To investigate the possible role of UCB on mitochondrial function in immature nerve

cells, 3 DIV cortical neurons were incubated with UCB at conditions mimicking neonatal

jaundice (50 µM UCB + 100 µM HSA) that had previously shown to induce oxidative injury

and cell death in mature neurons (Brites et al., 2009, Brito et al., 2008b). Cells were collected

1 h after treatment for the analysis of the activities of the mitochondrial respiratory chain

complexes. Activity of NADH-CoQ1 (Fig. II.1A) was unaffected and that of succinate-

cytochrome c reductase (Fig. II.1B), although slightly reduced, did not significantly change

after neuronal exposure to UCB. In contrast, cytochrome c oxidase activity (Fig. II.1C) was

inhibited by UCB in approximately 50% (p<0.01). Finally, since the activity of citrate synthase

(Fig. II.1D) was also unchanged, this indicates that no differences in mitochondrial

enrichment in our study model account for the decreased complex activity. Since GUDCA

has shown to have neuroprotective effects through the prevention of mitochondrial swelling

we have tested the beneficial effects of this bile acid on the UCB-induced alterations on the


69

mitochondrial respiratory chain. GUDCA revealed to be able to completely reverse the

inhibition of the cytochrome c oxidase activity (Fig. II.1C).

Figure II.1 - Unconjugated bilirubin (UCB) selectively impairs cytochrome c oxidase activity in immature neurons and glycoursodeoxycholic acid (GUDCA) exerts a preventive effect. Neurons at 3 days in vitro were incubated for 1 h with UCB (50 µM) plus human serum albumin (100 µM). When indicated, neurons were pre-treated with GUDCA (50 µM) for 1 h. After incubation, neurons were used for enzyme activity determinations, as indicated in Methods. UCB did not alter NADH-CoQ1 reductase - Complex I (A) as well as succinate-cytochrome c reductase - Complex II-III (B), but inhibited cytochrome c oxidase activity that was prevented by GUDCA (C). Additionally, no changes were noticed in citrate synthase activity (D). **p<0.01 vs. control; ##p<0.01 vs. UCB.

3.2. UCB produces oxidative stress in immature neurons, which is prevented by GUDCA

Next we explored the oxidative status of immature neurons exposed to UCB. We

observed that UCB markedly induced the production of reactive oxygen species (ROS),

namely O2.- (Fig. II.2A) and oxidized glutathione (Fig. II.2B), as well as decreased NADPH

0

50

100

150

200C

itrat

e sy

ntha

se a

ctiv

ity(n

mol

/min

/mg

prot

ein)

0.0

0.2

0.4

0.6

0.8

Com

plex

IV a

ctiv

ity(k

/min

/mg

prot

ein)

0

2

4

6

8

Com

plex

II a

ctiv

ity(n

mo/

min

/mg

prot

ein)

0

2

4

6

8C

ompl

ex I

activ

ity(n

mo/

min

/mg

prot

ein)

A B

**

##

C D

Chapter II __________________________________________________________________________

70

concentrations (Fig. II.2C). Moreover, pre-incubation of neurons with GUDCA efficiently

prevented all these oxidative events caused by UCB.

Figure II.2 - Unconjugated bilirubin (UCB) produces oxidative stress in immature neurons and glycoursodeoxycholic acid (GUDCA) exerts a preventive effect. Neurons at 3 days in vitro were treated as in Figure II.1. After incubation, neurons were used for metabolite assessments as indicated in Methods. UCB-induced oxidative stress through the increase in superoxide anion radical production, as indicated by a higher MitoSox fluoresence intensity (A), oxidized glutathione (B), as revealed by the increase in GSSG/GSx ratio, and the decrease in NADPH concentrations (C) that were prevented by GUDCA. **p<0.01 and *p<0.05 vs. control; #p<0.05 vs. UCB.

3.3. UCB impairs cellular oxygen consumption and collapses ΔѰm in immature

neurons and GUDCA exerts a preventive effect In view that the mitochondrial respiratory chain thresholds for oxygen consumption

(Davey et al., 1998), it could be speculated that the level of cytochrome c oxidase inhibition

caused by UCB might not be enough to impair the mitochondrial function. To elucidate this,

we first determined the rate of oxygen consumption in the dissociated neurons previously

incubated with UCB. As shown in Figure II.3A, UCB significantly reduced the rates of oxygen

consumption using glucose, succinate or ascorbate as substrates, effects that were

significantly prevented by GUDCA. The effects on O2 consumption are related to

mitochondria, since both succinate and ascorbate reproduced the same results as with

glucose. In addition, in all cases antimycin or potassium cyanide abolished O2 consumption

driven by succinate or ascorbate, respectively (data not shown). The inhibition of oxygen

consumption from ascorbate indicates that UCB-inhibition of cytochrome c oxidase, affects

cell respiration. To further test this possibility, we assessed the ΔѰm as an index of the

mitochondrial inner membrane integrity; as depicted in Figure II.3B, UCB caused the

collapse of ΔѰm, and GUDCA showed ability to restore mitochondrial integrity.

0

1

2

3

4

5

GS

SG

/GS

x x

100

0

50

100

150

NA

DP

H(p

mo/

mg

prot

ein)

0

10

20

Mito

Sox

Red

fluo

resc

ence

(arb

itrar

y un

its)

*#

A

*

#

B

**#

C


71

Figure II.3 - Unconjugated bilirubin (UCB) impairs cellular oxygen consumption and collapses ΔѰm in immature neurons and glycoursodeoxycholic acid (GUDCA) exerts a preventive effect. Neurons at 3 days in vitro were treated as in Figure II.1. After incubation, neurons were either subjected to ΔѰm assessment in the plates or collected for oxygen consumption determinations as indicated in Methods. UCB inhibited the rate of glucose-, succinate- and ascorbate-driven oxygen consumption (A) that was prevented by GUDCA. UCB decreased ΔѰm (B), as assessed by TMRE staining (left panels) and fluorescence quantification (right panel), effects that were prevented by GUDCA **p<0.01 and *p<0.05; #p<0.05 and ##p<0.01 vs. UCB (scale bar 50 µm).

3.4. UCB increases extracellular ATP content, glycolysis and F2,6P2 levels in immature neurons, which are counteracted by GUDCA Curiously, although UCB-induced impairment of mitochondrial respiratory chain function

was not accompanied by a reduction of the intracellular ATP levels (data not shown), an

increase in the concentrations of extracellular ATP (Fig. II.4A) was obtained. In addition, it

was also observed an increase in the concentration of intracellular lactate (Fig. II.4B), as well

0

20

40

60

80

100

120

A

2.8

2.1

1.4

0.7

0

nmol

O2/m

in/1

06ne

uron

s

*##

Oxygen consumption f rom glucose

2.8

2.1

1.4

0.7

0

nmol

O2/m

in/1

06ne

uron

s

*## 2.8

2.1

1.4

0.7

0nmol

O2/m

in/1

06ne

uron

s

*

#

Oxygen consumption f rom succinate

Oxygen consumption f rom ascorbate

B

∆Ψ

m

(% o

f con

trol)

**

##

Chapter II __________________________________________________________________________

72

as in F1,6P2/F6P ratio (Fig. II.4C), suggesting an activation of glycolysis, which was further

supported by the concomitant elevated levels of F2,6P2 (Fig. II.4D). Noticeably, all these

effects were abolished by GUDCA.

Figure II.4 - Unconjugated bilirubin (UCB) increases extracellular ATP content, glycolysis and fructose-2,6-bisphosphate (F2,6P2) levels in immature neurons, and glycoursodeoxycholic acid (GUDCA) exerts a preventive effect. Neurons at 3 days in vitro were treated as in Figure II.1. After incubation, extracellular ATP was evaluated, and neurons were lysed for intracellular lactate, fructose-1,6-bisphosphate (F1,6P2), fructose-6-phosphate (F6P) and F2,6P2 measurements, as indicated in Methods. UCB triggered an increase in ATP release (A), intracellular lactate concentration (B), F1,6P2/F6P (C) and F2,6P2 concentration (D), which were prevented by GUDCA. **p<0.01 and *p<0.05 vs. control; ##p<0.01 and #p<0.05 vs. UCB.

3.5. UCB triggers apoptotic cell death in immature neurons, which is prevented by GUDCA Finally, we sought to investigate whether the mitochondrial impairment triggered by UCB

was associated with neurotoxicity. As shown in Figure II.5A, UCB enhanced the proportion of

annexin V+/7-AAD- neurons, as assessed by flow cytometry; it also triggered an increase in

the proportion of condensed or fragmented nuclei, as visualized with DAPI by fluorescence

0.00

0.05

0.10

0.15

0.0

0.5

1.0

1.5

2.0

F1,6

P2/

F6P

0

10

20

30

Ext

race

llula

r ATP

(nm

ol/m

g pr

otei

n)

0.0

1.0

2.0

3.0

4.0F2

,6P

2

(nm

ol/m

g pr

otei

n)

A B

**

##

C D

#

*

*

#

**

##

Intra

cellu

lar

Lact

ate

(µm

ol/m

g pr

otei

n)


73

microscopy (Fig. II.5B). Cell death by apoptosis was further corroborated by the increase in

the activation of caspase-3 (Fig. II.5C). Similar increase in the activation of caspase-9

indicates the involvement of mitochondria in this process. Such effects were again

completely counteracted by GUDCA. We can then speculate that the increased glycolytic

rate (Fig. II4) is a failed attempt to compensate the mitochondrial impairment. These results

support the notion that UCB causes nerve cell death by apoptosis, mainly in immature

neurons, and confirms that GUDCA efficiently protects cells against this type of neurotoxicity.

4. Discussion Here we show, for the first time, that brief exposure (1 h) of primary cortical immature

neurons to UCB in conditions that have relevance to the clinical manifestations of BIND (50

µM UCB + 100 µM HSA) inhibits mitochondrial respiratory chain, at the level of cytochrome c

oxidase complex. Mitochondrial dysfunction by UCB appears to involve .NO, accordingly with

previous studies (Brito et al., 2008a, Mancuso et al., 2008) reporting that neuronal oxidative

dysruption by UCB is abrogated by inhibition of neuronal NO synthase (nNOS). .NO is

capable of rapidly and reversibly inhibit the mitochondrial respiratory chain and may be

implicated in the cytotoxic effects in the CNS (Bolaños et al., 1994, Brown and Cooper, 1994,

Cleeter et al., 1994). Additionally, inhibition of the mitochondrial transport chain at the level of

complex IV can further produce O2.- from O2, a finding also observed in this study (Fig.

II.2A). Thus, inhibition of mitochondrial cytochrome c oxidase by .NO can lead to the

formation of both .NO and O2.- and thereby lead to the formation of ONOO- (Sharpe and

Cooper, 1998). Interestingly, we previously demonstrated that UCB induces protein oxidation

and lipid peroxidation, while diminishes the thiol antioxidant defences, events that were

correlated with the extent of cell death, and that GUDCA primarily acts as an antioxidant at

protecting neurons against UCB-induced oxidative stress (Brito et al., 2008a). The present

study extended our previous ones by showing that UCB decreases NADPH concentrations

(Fig. II.2C) in immature neurons, in adition to glutathione oxidation (Fig. II.2B) and O2.-

production, confirming oxidative stress by UCB.

Chapter II __________________________________________________________________________

74

Figure II.5 – Unconjugated bilirubin (UCB) triggers apoptotic death in immature neurons and glycoursodeoxycholic acid (GUDCA) exerts a preventive effect. Neurons at 3 days in vitro were treated as in Figure II.1. After incubation, neurons were subjected to assessment of apoptotic death by flow cytometry (annexin V+/7-AAD-), nuclear condensation or fragmentation in DAPI-stained cells, and caspase-3 and -9 activities, as indicated in Methods. UCB increased neuronal apoptosis as measured by the percentage of annexin V+/7-AAD- cells (A) (left panel shows a typical diagram; right panel represents the quantification) or fragmented or condensed nuclei (B) (left panel show a typical microphotograph of the DAPI-stained cells; right panel represents the quantification), which was prevented by GUDCA. Data was corroborated by the increase in the activation of caspase-3 and caspase-9 (C) pointing to the involvement of mitochondria. GUDCA was able to markedly prevent this effect. **p<0.01 and *p<0.05 vs. control; ##p<0.01 vs. UCB (scale bar 20 µm).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Cas

pase

9 a

ctiv

ity (f

old

vs.c

ontro

l)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Cas

pase

3 a

ctiv

ity (f

old

vs.c

ontro

l)

0

5

10

15

20

Con

dens

ed o

r fra

gmen

ted

nucl

ei(%

)

0

5

10

15

20

25

30

Apo

ptot

ic n

euro

ns(%

Ann

exin

+ /7A

AD

- )

A

B

*

##

C

**

##

##

**

##

**

Control

UCB


75

Moreover, pre-incubation of neurons with GUDCA efficiently prevented these oxidative

events, thus highlighting the antioxidant properties of the bile acid in this paradigm. In

addition, GUDCA abolished the inhibition of cytochrome c oxidase caused by UCB (Fig.

II.1C), indicating that the mitochondrial respiratory chain damage caused by UCB would be a

free radical-mediated process. To the best of our knowledge, this is the first evidence

reporting the ability of GUDCA to completely restore the activity of cytochrome c oxidase

when impaired.

Once a defective respiratory function also includes decreased oxygen consumption, we

explored the effects of UCB at such level, since contradictory results were previously found

by several authors using neither relevant physiological concentrations nor purified UCB

(Diamond and Schmid, 1967, Ernster and Zetterström, 1956, Mustafa et al., 1969). Since

glucose provides NAD-linked electrons through NADH-CoQ1 reductase, and succinate FAD-

linked ones through succinate dehydrogenase, the inhibition of cell respiration from these

substrates by UCB is compatible with, but does not demonstrate, inhibition at the terminal

complex, cytochrome c oxidase. However, the inhibition of oxygen consumption from

ascorbate, which directly supplies electrons to cytochrome c oxidase, confirms that the

inhibition at the level of this complex by UCB affects cell respiration. This was reinforced by

the collapse of ΔѰm produced by UCB indicating decreased mitochondrial membrane

potential and mitochondrial dysfunction, which was again prevented when neurons were pre-

treated with GUDCA before the exposure to UCB. Permeabilization of the mitochondrial

membrane by UCB and increased efflux of cytochrome c were previously observed in

isolated mitochondria from the brain and liver of adult male Wistar rats (Rodrigues et al.,

2002, Rodrigues et al., 2000). Such mitochondrial impairment by UCB was in the present

study accompanied by an increase in intracellular lactate concentrations and F1,6P2/F6P

ratio, suggesting an activation of glycolysis by stimulation of 6-phosphofructo-1-kinase

(Pfk1), i.e. a master regulator of this pathway (Uyeda, 1979); this notion was further

supported by the observed increased levels of F2,6P2, i.e. the positive effector of Pfk1 (Van

Schaftingen et al., 1982). Moreover, intracellular ATP content was unchanged, whereas

extracellular levels were increased. Altogether, these results suggest that UCB disrupts the

mitochondrial function in immature neurons leading to up-regulation of glycolysis, which

reflects the natural metabolic response of cells to cytochrome c oxidase deficiency (Almeida

et al., 2004, Bolaños et al., 1994) and may determine the unchanged intracellular ATP levels.

In fact, it is conceivable that the up-regulation of glycolysis in these still immature neuronal

cells may provide sufficient ATP as a self-protective attempt to support the bioenergetic

crisis, as previously demonstrated for astrocytes exposed to injurious stimuli (Bolaños et al.,

2004). In parallel, the release of ATP by neurons has been suggested to be determined by

Chapter II __________________________________________________________________________

76

the production of .NO and oxidative stress and to be associated with neuronal cell death by

apoptosis, in that ATP is critical to inducing several apoptotic events (Figueroa et al., 2006,

López et al., 2006). Noticeably, all these effects were prevented by GUDCA.

Another new finding on the mechanisms of UCB-induced oxidative stress is the

decrease of NADPH levels which may contribute to the altered glutathione redox status

(Dringen, 2000) observed in the presence of UCB. This result is consistent with the recently

reported notion that, in neurons, the activation of glycolysis leads to inhibition of the pentose-

phosphate pathway (PPP), causing glutathione oxidation (Herrero-Mendez et al., 2009).

Interestingly, through PPP, neurons maintain cytochrome c in a reduced status in order to

prevent its release and apoptotic death (Vaughn and Deshmukh, 2008). In good agreement

with this, GUDCA restored reduced glutathione levels in immature neurons, as it did with

differentiated cells (Brito et al., 2008a) and also re-established NADPH values reinforcing its

antioxidant capacity. An up-regulation of gamma-glutamyl cysteine synthetase, together with

the efficient scavenging of free radicals previously reported for the non conjugated form of

the bile acid (Lapenna et al., 2002, Rodriguez-Ortigosa et al., 2002, Serviddio et al., 2004)

shall contribute to the protective actions herewith found.

UCB induced mitochondrial dysfunction in 3 DIV neurons by selective inhibition of

cytochrome c oxidase activity, and decreased mitochondrial membrane potential, but

maintained intracellular ATP levels, ultimately leading to apoptotic cell death. The activation

of caspase-3, but mostly of caspase-9, points to the activation of the mitochondrial apoptotic

pathway in immature neurons after a short exposure to UCB in conditions mimicking an

acute neonatal jaundice. To the prevention of this neurotoxicity by GUDCA may account its

ability to re-establish the energy metabolism and redox status of the injured cell.

In conclusion, in this study we report evidence of UCB induced inhibition of cytochrome c

oxidase activity in immature rat neurons resulting in respiratory chain dysfunction, a

decrease in antioxidant defences and apoptosis, as schematically represented in Figure II.6.

The ability of GUDCA to ameliorate UCB induced mitochondrial respiratory chain dysfunction

and restore cellular antioxidant potential supports the efficacy of this compound as a

potential treatment for BIND.


77

Figure II.6 - Schematic representation of some important steps in neuronal injury by unconjugated bilirubin (UCB) and potential targets of glycoursodeoxycholic acid (GUDCA). UCB interaction with immature neurons causes the selective inhibition of complex IV (CIV) activity, impairs oxygen (O2) consumption, leads to a loss of mitochondrial membrane potential (ΔѰm) and superoxide anion radical production (O2

.-) production. UCB also induces the up-regulation of glycolysis and increased amounts of extracellular ATP, together with an inhibition of the pentose-phosphate pathway (PPP), as inferred by the decreased NADPH and increased oxidized glutathione (GSSG) levels. All these events shall contribute to the activation of caspases 9 and 3 and apoptotic cell death. GUDCA fully abrogated UCB-induced mitochondrial dysfunction, alterations in energy metabolism and disruption of the redox status, therefore counteraction immature nerve cell demise resulting from UCB exposure.

Chapter II __________________________________________________________________________

78

5. References

Almeida, A. and Bolaños, J. P. (2001) A transient inhibition of mitochondrial ATP synthesis

by nitric oxide synthase activation triggered apoptosis in primary cortical neurons. J

Neurochem, 77, 676-690.

Almeida, A., Bolaños, J. P. and Medina, J. M. (1999) Nitric oxide mediates glutamate-

induced mitochondrial depolarization in rat cortical neurons. Brain Res, 816, 580-586.

Almeida, A., Heales, S. J., Bolaños, J. P. and Medina, J. M. (1998) Glutamate neurotoxicity

is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione

depletion. Brain Res, 790, 209-216.

Almeida, A., Moncada, S. and Bolaños, J. P. (2004) Nitric oxide switches on glycolysis

through the AMP protein kinase and 6-phosphofructo-2-kinase pathway. Nat Cell Biol, 6,

45-51.

Atamna, H. and Frey, W. H., 2nd (2007) Mechanisms of mitochondrial dysfunction and

energy deficiency in Alzheimer's disease. Mitochondrion, 7, 297-310.

Bolaños, J. P., Cidad, P., García-Nogales, P., Delgado-Esteban, M., Fernandez, E. and

Almeida, A. (2004) Regulation of glucose metabolism by nitrosative stress in neural cells.

Mol Aspects Med, 25, 61-73.



Neurochem, 63, 910-916.

Brann, B. S. t., Stonestreet, B. S., Oh, W. and Cashore, W. J. (1987) The in vivo effect of

bilirubin and sulfisoxazole on cerebral oxygen, glucose, and lactate metabolism in

newborn piglets. Pediatr Res, 22, 135-140.

Brites, D., Fernandes, A., Falcão, A. S., Gordo, A. C., Silva, R. F. M. and Brito, M. A. (2009)

Biological risks for neurological abnormalities associated with hyperbilirubinemia. J

Perinatol, 29 Suppl 1, S8-13.

Brito, M. A., Lima, S., Fernandes, A., Falcao, A. S., Silva, R. F., Butterfield, D. A. and Brites,

D. (2008a) Bilirubin injury to neurons: contribution of oxidative stress and rescue by







Lett, 356, 295-298.


79




345, 50-54.

Cuperus, F. J., Hafkamp, A. M., Havinga, R., Vitek, L., Zelenka, J., Tiribelli, C., Ostrow, J. D.

and Verkade, H. J. (2009) Effective treatment of unconjugated hyperbilirubinemia with oral

bile salts in Gunn rats. Gastroenterology, 136, 673-682 e671.

Davey, G. P., Peuchen, S. and Clark, J. B. (1998) Energy thresholds in brain mitochondria.

Potential involvement in neurodegeneration. J Biol Chem, 273, 12753-12757.

Day, R. L. (1954) Inhibition of brain respiration in vitro by bilirubin: reversal if inhibition by

various means. Am J Dis Child, 88, 504-506.

Diamond, I. and Schmid, R. (1967) Oxidative phosphorylation in experimental bilirubin

encephalopathy. Science, 155, 1288-1289.


671.



Ernster, L. and Zetterström, R. (1956) Bilirubin, an uncoupler of oxidative phosphorylation in

isolated mitochondria. Nature, 178, 1335-1337.




Farkas, D. L., Wei, M. D., Febbroriello, P., Carson, J. H. and Loew, L. M. (1989)

Simultaneous imaging of cell and mitochondrial membrane potentials. Biophys J, 56,

1053-1069.

Fernandes, A., Vaz, A. R., Falcão, A. S., Silva, R. F. M., Brito, M. A. and Brites, D. (2007)



Figueroa, S., Oset-Gasque, M. J., Arce, C., Martinez-Honduvilla, C. J. and González, M. P.

(2006) Mitochondrial involvement in nitric oxide-induced cellular death in cortical neurons

in culture. J Neurosci Res, 83, 441-449.

García-Nogales, P., Almeida, A., Fernández, E., Medina, J. M. and Bolaños, J. P. (1999)

Induction of glucose-6-phosphate dehydrogenase by lipopolysaccharide contributes to

preventing nitric oxide-mediated glutathione depletion in cultured rat astrocytes. J

Neurochem, 72, 1750-1758.

Chapter II __________________________________________________________________________

80

Gutmann, I. and Wahlefeld, A. W. (1974) L-(+)-Lactate. Determination with Lactate

Dehydrogenase and NAD In: Methods of Enzymatic Analysis, Vol. 3, pp. 1464-1468.

Verlag Chemie GmbH, Weinheim.




Hoffman, D. J., Zanelli, S. A., Kubin, J., Mishra, O. P. and Delivoria-Papadopoulos, M. (1996)

The in vivo effect of bilirubin on the N-methyl-D-aspartate receptor/ion channel complex in

the brains of newborn piglets. Pediatr Res, 40, 804-808.

Ives, N. K., Bolas, N. M. and Gardiner, R. M. (1989) The effects of bilirubin on brain energy

metabolism during hyperosmolar opening of the blood-brain barrier: an in vivo study using

31P nuclear magnetic resonance spectroscopy. Pediatr Res, 26, 356-361.

Ives, N. K., Cox, D. W., Gardiner, R. M. and Bachelard, H. S. (1988) The effects of bilirubin

on brain energy metabolism during normoxia and hypoxia: an in vitro study using 31P

nuclear magnetic resonance spectroscopy. Pediatr Res, 23, 569-573.

Katoh-Semba, R. (1976) Studies on cellular toxicity of bilirubin: effect on brain glycolysis in

the young rat. Brain Res, 113, 339-348.

Kawaguchi, T., Veech, R. L. and Uyeda, K. (2001) Regulation of energy metabolism in

macrophages during hypoxia. Roles of fructose 2,6-bisphosphate and ribose 1,5-

bisphosphate. J Biol Chem, 276, 28554-28561.

King, T. E. (1967) Preparation of succinate cytochrome c reductase and the cytochrome b-c1

particle, and reconstitution of succinate cytochrome c reductase. Methods Enzymology,

10, 216-225.

Klingerberg, M. (1985) NADH/NADPH. UV-methods. In: Methods of Enzymatic Analysis, Vol.

7, pp. 251-271. Verlag Chemie GmbH, Weinheim.

Lapenna, D., Ciofani, G., Festi, D., Neri, M., Pierdomenico, S. D., Giamberardino, M. A. and

Cuccurullo, F. (2002) Antioxidant properties of ursodeoxycholic acid. Biochem Pharmacol,

64, 1661-1667.

López, E., Arce, C., Oset-Gasque, M. J., Canadas, S. and González, M. P. (2006) Cadmium

induces reactive oxygen species generation and lipid peroxidation in cortical neurons in

culture. Free Radic Biol Med, 40, 940-951.

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement

with the Folin phenol reagent. J Biol Chem, 193, 265-275.





81

Mattson, M. P. and Liu, D. (2002) Energetics and oxidative stress in synaptic plasticity and

neurodegenerative disorders. Neuromolecular Med, 2, 215-231.

McCandless, D. W. and Abel, M. S. (1980) The effect of unconjugated bilirubin on regional

cerebellar energy metabolism. Neurobehav Toxicol, 2, 81-84.

McDonagh, A. F. (1979) Bile pigments: Bilatrienes and 5,15-biladienes. In: The Porfirins, pp.

293-491. Academic Press, San Diego.

McDonald, J. W., Shapiro, S. M., Silverstein, F. S. and Johnston, M. V. (1998) Role of

glutamate receptor-mediated excitotoxicity in bilirubin-induced brain injury in the Gunn rat

model. Exp Neurol, 150, 21-29.

Mukhopadhyay, P., Rajesh, M., Hasko, G., Hawkins, B. J., Madesh, M. and Pacher, P.

(2007) Simultaneous detection of apoptosis and mitochondrial superoxide production in

live cells by flow cytometry and confocal microscopy. Nat Protoc, 2, 2295-2301.

Mustafa, M. G., Cowger, M. L. and King, T. E. (1969) Effects of bilirubin on mitochondrial

reactions. J Biol Chem, 244, 6403-6414.



Ostrow, J. D., Pascolo, L., Shapiro, S. M. and Tiribelli, C. (2003) New concepts in bilirubin

encephalopathy. Eur J Clin Invest, 33, 988-997.

Park, W. S., Chang, Y. S., Chung, S. H., Seo, D. W., Hong, S. H. and Lee, M. (2001) Effect

of hypothermia on bilirubin-induced alterations in brain cell membrane function and energy

metabolism in newborn piglets. Brain Res, 922, 276-281.

Ragan, C. I., Wilson, M. T., Darley-Usmar, V. M. and Lowe, P. N. (1987) Subfractionation of

mitochondria and isolation of the proteins of oxidative phosphorylation. In: Mitochondria: A

Practical Approach, pp. 79-112. IRL Press, London.

Reiser, D. J. (2004) Neonatal jaundice: physiologic variation or pathologic process. Crit Care

Nurs Clin North Am, 16, 257-269.

Rodrigues, C. M. P., Solá, S., Brito, M. A., Brites, D. and Moura, J. J. (2002) Bilirubin directly

disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat


Rodrigues, C. M. P., Stieers, C. L., Keene, C. D., Ma, X., Kren, B. T., Low, W. C. and Steer,

C. J. (2000) Tauroursodeoxycholic acid partially prevents apoptosis induced by 3-

nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability

transition. J Neurochem, 75, 2368-2379.

Rodriguez-Ortigosa, C. M., Cincu, R. N., Sanz, S., Ruiz, F., Quiroga, J. and Prieto, J. (2002)

Effect of ursodeoxycholic acid on methionine adenosyltransferase activity and hepatic

glutathione metabolism in rats. Gut, 50, 701-706.

Chapter II __________________________________________________________________________

82

Serviddio, G., Pereda, J., Pallardo, F. V. et al. (2004) Ursodeoxycholic acid protects against

secondary biliary cirrhosis in rats by preventing mitochondrial oxidative stress.

Hepatology, 39, 711-720.



Sharpe, M. A. and Cooper, C. E. (1998) Interaction of peroxynitrite with mitochondrial

cytochrome oxidase. Catalytic production of nitric oxide and irreversible inhibition of

enzyme activity. J Biol Chem, 273, 30961-30972.

Shepherd, D. and Garland, P. B. (1969) The kinetic properties of citrate synthase from rat

liver mitochondria. Biochem J, 114, 597-610.

Silva, M. F., Ruiter, J. P., Illst, L., Jakobs, C., Duran, M., de Almeida, I. T. and Wanders, R. J.

(1997) Valproate inhibits the mitochondrial pyruvate-driven oxidative phosphorylation in

vitro. J Inherit Metab Dis, 20, 397-400.



Uyeda, K. (1979) Phosphofructokinase. Adv Enzymol Relat Areas Mol Biol, 48, 193-244.

Van Schaftingen, E., Lederer, B., Bartrons, R. and Hers, H. G. (1982) A kinetic study of

pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application

to a microassay of fructose 2,6-bisphosphate. Eur J Biochem, 129, 191-195.

Vaughn, A. E. and Deshmukh, M. (2008) Glucose metabolism inhibits apoptosis in neurons

and cancer cells by redox inactivation of cytochrome c. Nat Cell Biol, 10, 1477-1483.

Wennberg, R. P., Johansson, B. B., Folbergrova, J. and Siesjo, B. K. (1991) Bilirubin-

induced changes in brain energy metabolism after osmotic opening of the blood-brain

barrier. Pediatr Res, 30, 473-478.

Wharton, D. C. and Tzagoloff, A. (1967) Cytochrome oxidase from beef heart mitochondria.

Methods Enzymology, 10, 245-250.

Wulff, K. (1985) NADH/NADPH. Luminometric method. In: Methods of Enzymatic Analysis,

Vol. 7, pp. 280-284. Verlag Chemie GmbH, Weinheim.

Chapter III

III. Pro-inflammatory cytokines intensify the activation of .NO/NOS,

JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin

Ana Rita Vaz, Sandra Leitão Silva, Andreia Barateiro, Adelaide Fernandes, Ana Sofia Falcão, Maria A Brito, Dora Brites

Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade

de Farmácia, University of Lisbon, Av. Professor Gama Pinto, Lisbon 1649-003,

Portugal.

Experimental Neurology (submitted).

Acknowledgements This work was supported by grants PPCDT/SAU/MMO/55955/2004 and PTDC/SAU-

NEU/64385/2006 (to D.B.) and BD/30292/2006 (to A.R.V.) from Fundação para a Ciência

e a Tecnologia, Lisbon, Portugal.

BIND is increased by inflammation __________________________________________________________________________

85

Abstract

Hyperbilirubinemia may lead to encephalopathy in neonatal life, particularly in premature

infants. Although the mechanisms were never established, clinicians commonly consider

sepsis as a risk factor for bilirubin-induced neurological dysfunction (BIND). Our previous

studies showed that elevated levels of unconjugated bilirubin (UCB) have immunostimulant

effects, which are potentiated by LPS, and that immature neural cells are more vulnerable to

UCB. The present study was undertaken to explore the role of nitric oxide (.NO)/NO synthase

(NOS), c-Jun N-terminal kinase (JNK) 1/2 and caspase activation in BIND, as well as the

additional effects of inflammation, in immature neurons incubated from 1 h to 24 h, at 37ºC.

UCB, at conditions mimicking those of jaundiced newborns (UCB/serum albumin = 0.5),

induced .NO production, nNOS expression and JNK1/2 activation in 3 days in vitro neuron

cultures. As a consequence of these events, mitochondrial and extrinsic pathways of

apoptosis were initiated, ultimately leading to neuronal dysfunction. Co-incubation with TNF-

α+IL-1β intensified the activation of .NO/NOS, JNK1/2, caspase-8, caspase-9 and caspase-3

by UCB. Cleavage of Bid and truncated Bid (tBid) up-regulation, as well as increased

cytotoxic potential, was also observed. Interestingly, both L-NAME (NOS inhibitor) and

SP600125 (JNK1/2 inhibitor) reversed the effects produced by UCB either alone, or in

association to pro-inflammatory cytokines. Taken together, our data reveal not only that

activation of .NO/NOS, JNK1/2 and caspase cascades are important determinants of BIND

but also that the association of TNF-α+IL-1β have cumulative effects. These events provide a

reason for the risk of sepsis in BIND and point to potential targets for therapeutic

intervention.

Keywords: neuronal .NO/NOS system; JNK1/2 signalling pathway; Caspase activation; TNF-

α+IL-1β; Bilirubin-induced neurological dysfunction (BIND)

.

Chapter III __________________________________________________________________________

86

1. Introduction Elevated levels of unconjugated bilirubin (UCB) are responsible for the clinical

manifestation of jaundice, a common condition in the neonatal period. Although normal (or

slightly increased) levels of UCB provide protection against injury resulting from oxidation

(Doré et al., 2000), elevated UCB concentrations cause nerve cell damage, leading to

adverse neurological outcomes (Hansen, 2002), ranging from minimal damage to chronic

and permanent sequelae, or even death (Ostrow et al., 2004, Shapiro, 2005). The risk of

bilirubin-induced neurologic dysfunction (BIND) is particularly enhanced in premature

newborns due to the higher rates of UCB production and the immaturity of the excretion

pathways (Stevenson et al., 2001, Watchko, 2006).

Although the determinants of vulnerability to BIND are only partially understood, it is well

known that UCB triggers the accumulation of extracellular glutamate (Falcão et al., 2006),

oxidative stress (Brito et al., 2008a, Brito et al., 2010, Brito et al., 2008b), as well as the

release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β

(IL-1β) and IL-6 by both astrocytes (Fernandes et al., 2004) and microglia (Fernandes et al.,

2006). Inflammatory signalling pathway involves the activation of mitogen-activated protein

kinases (MAPKs) and nuclear factor (NF)-kB (Fernandes et al., 2006, Silva et al., 2010).

These upstream events culminate in nerve cell death by both necrosis and apoptosis (Silva

et al., 2002). UCB directly interacts with mitochondria influencing membrane lipid and protein

properties, redox status, and cytochrome c content (Rodrigues et al., 2002b). In addition, we

also demonstrated that UCB induces apoptosis trough mitochondria-caspase-3 pathway

involving cytochrome c release, caspase-3 activation, and subsequent PARP cleavage in

developing rat brain neurons (Rodrigues et al., 2002a). Recent findings evidenced that UCB

inhibits cytochrome c oxidase activity, and induces both ATP release and disruption of

glutathione redox status in immature neurons (Vaz et al., 2010).

Interestingly, immature nerve cells are more susceptible than differentiated ones to

UCB-induced toxicity and release higher levels of glutamate and TNF-α providing a basis for

the increased susceptibility of premature newborns to UCB deleterious effects (Falcão et al.,

2006). In addition, LPS showed to exacerbate the release of TNF-α and IL-1β by immature

astrocytes (Falcão et al., 2005).

It has been suggested that infection increases the risk for UCB encephalopathy

(Dawodu et al., 1984) and presence of inflammatory features, namely fever episodes and

brain edema, were described during or following moderate to severe hyperbilirubinemia

(Kaplan and Hammerman, 2005). The inflammatory reaction is essential for survival in

response to tissue injury or infection, but it can also cause neuronal damage, since cytokines

are not only involved in neuroprotection but also in neurodegeneration processes (Konsman


87

et al., 2007). Pro-inflammatory cytokines, such as TNF-α and IL-1β, besides having

pleiotropic effects in the central nervous system, including their emerging role in

neurodevelopment (Marx et al., 2001) have also been described as mediators of neuronal

apoptosis (Kajta et al., 2006, Takahashi et al., 2008). Several studies have shown that

inflammation is associated with the enhanced generation of reactive oxygen species (ROS),

and/or reactive nitrogen species (Bian and Murad, 2001, Sener et al., 2005, Brito et al.,

2007). One of the oxidant species, nitric oxide (.NO), although important in cellular signalling,

has an important role in the pathogenesis of inflammation (Korhonen et al., 2005). In fact, .NO and induction of nitric oxide synthase (NOS) are involved in apoptosis induced by

inflammatory mediators in neuronal cells (Hemmer et al., 2001, Heneka et al., 1998, Thomas

et al., 2008). To add that exposure to UCB leads to increased expression of neuronal NOS

(nNOS) and production of .NO, cyclic guanosine 3',5'-monophosphate (cGMP) and ROS,

along with protein oxidation and depletion of glutathione (Brito et al., 2008b, Fernandes et al.,

2010). Therefore, .NO inhibitors represent important strategies to prevent cellular injury

associated with jaundice and inflammatory processes. Among the upstream signals leading

to neuronal degeneration, one may include the stress-activated protein kinases (SAPKs). In

fact, c-Jun N-terminal kinases 1/2 (JNK1/2) become activated in response to toxic stimulus,

such as the reactive nitrogen species (Luo et al., 1998, Marques et al., 2003) and the pro-

inflammatory cytokines TNF-α and IL-1, pointing these SAPKs as strong effectors of

neuronal apoptosis (Tibbles and Woodgett, 1999, Mielke and Herdegen, 2000). JNK 1/2

directly mediates the UCB-stimulation of TNF-α by astrocytes as we showed by the use of

SP600125, a JNK1/2 inhibitor (Fernandes et al., 2007). This feature may be relevant if we

consider that astrocytic activation has also been reported in several neurodegenerative

disorders and that this transition may be accompanied by dysfunction of astrocytes leading to

incorrect glia-to-neuron cross-talk (Rossi and Volterra, 2009).

In this study, we investigated if .NO/NOS and JNK 1/2 activation were signalling

determinants for caspase-8 activation and mitochondrial death pathway in UCB-induced

dysfunction of immature neurons. Most of all, we explored if the combination of the pro-

inflammatory cytokines TNF-α and IL-1β aggravate the functional de-regulation of immature

neurons produced by UCB and whether use the same cascade of mediators.

Chapter III __________________________________________________________________________

88


2.1. Chemicals Neurobasal medium, B-27 supplement (50X), Hanks’ balanced salt solution (HBSS-1),

Hanks’ balanced salt solution without Ca2+ and Mg2+ (HBSS-2), gentamicin (50 mg/mL), and

trypsin (0.025%) were acquired from Invitrogen (Carlsbad, CA). Recombinant rat IL-1β and

TNF-α were form R&D Systems Inc. (Minneapolis, MN, USA). Human serum albumin (HSA)

(fraction V, fatty acid free), N-ω-nitro-L-arginine methyl ester hydrochloride (L-NAME),

primary monoclonal antibody mouse anti-β-actin, N-1-naphthylethylenediamine,

sulfanilamide, sodium nitrite and MTT [3-(4,5-dimethylthiazol, 2-yl)-2,5-diphenyltetrazolium

bromide] were purchased from Sigma Chemical Co (St Louis, MO). UCB was also from

Sigma and purified as previously described (McDonagh, 1979). Nitrocellulose membrane,

Hyperfilm ECL and horseradish peroxidase-labelled sheep anti-mouse IgG were from

Amersham Biosciences (Piscataway, NJ, USA). Cell lysis buffer and LumiGLO® were

acquired from Cell Signalling (Beverly, MA, USA). Antibodies directed to phosphorylated

JNK1/2 (P-JNK1/2) and Bid were from Santa Cruz Biotechnology (Santa Cruz, CA, USA)

while antibody against neuronal NOS (nNOS) was from Becton Dickinson Biosciences (San

José, CA, USA). Caspases 3, 8 and 9 substrates, Ac-DEVD-pNA, Ac-IETD-pNA and Ac-

LEHD-pNA, respectively, were purchased from Calbiochem (Darmstadt, Germany). A

concentrated solution (10 mM) of the JNK1/2 inhibitor SP600125 (Calbiochem) was prepared

in dimethylsulfoxide.


Animal care followed the recommendations of European Convention for the Protection of

Vertebrate Animals Used for Experimental and other Scientific Purposes (Council Directive

86/609/EEC) and National Law 1005/92 (rules for protection of experimental animals).

Neurons were isolated from fetuses of 16-17-day pregnant Wistar rats, as previously

described (Silva et al., 2002). The fetuses were collected in Hanks’ balanced salt solution

(HBSS), the brain cortices were mechanically fragmented, and the fragments transferred to a

0.5 g/L trypsin in Ca2+ and Mg2+ free HBSS medium and incubated for 15 min at 37ºC. After

trypsinization, cells were washed twice in Ca2+ and Mg2+ free HBSS medium containing 10%

fetal bovine serum, and resuspended in Neurobasal medium supplemented with 0.5 mM L-

glutamine, 25 μM L-glutamic acid, 2% B-27 supplement, and 0.12 mg/mL gentamicin. Finally,

cells were seeded on poly-D-lysine coated tissue culture plates at a density of 2 x 105


89

cells/cm2 and maintained at 37ºC in a humidified atmosphere of 5% CO2. In this work we

used neurons at 3 days in vitro (DIV).

2.3. Treatment of neurons Immature neurons were incubated in Neurobasal medium without (control) or with 50 μM

UCB (Sigma Chemical Co, St Louis, MO, USA) in the presence of 100 μM HSA from 1 h to

24 h, at 37ºC. For co-incubation studies, neurons were co-incubated with recombinant 50

ng/mL TNF-α plus 50 ng/mL IL-1β (R&D Systems Inc., Minneapolis, MN, USA). Stock UCB

solutions were extemporarily prepared in 0.1 M NaOH under the dark and the pH adjusted to

7.4 using 0.1 M HCl. In parallel studies, cells were co-incubated with 100 µM N-ω-nitro-L-

arginine methyl ester hydrochloride (L-NAME) (Sigma Chemcial Co), a non-selective NOS

inhibitor or treated with 0.2 µM SP600125 (Calbiochem, Darmstadt, Germany), an inhibitor of

JNK1/2, 1 h prior to UCB addition, alone or in combination with TNF-α+IL-1β.

2.4. Quantification of nitrite levels Nitric oxide levels were estimated by measuring the concentrations of nitrites (NO2

-),

which are the resulting .NO metabolites. Briefly, supernatants free from cellular debris were

mixed with Griess reagent [1 part 1% (w/v) sulfanilamide in 5% H3PO4, 1 part 0.1% (w/v) N-1-

naphthylethylenediamine (v/v)] in 96-well tissue culture plates for 10 min at room

temperature in the dark. The absorbance at 540 nm was determined using a microplate

reader (Bio-Rad Laboratories, Hercules, CA, USA).

2.5. Western blot assay The intracellular forms of nNOS, Bid and phosphorylated P-JNK1/2 were determined by

Western blot analysis as usual in our laboratory (Fernandes et al., 2006). Briefly, cells were

washed in ice-cold PBS and lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM

NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 2.5 mM sodium pyrophosphate,

1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptine, 1 mM PMSF. The lysate was

sonicated for 20 s, centrifuged at 14.000 g for 10 min at 4ºC and the supernatants were

collected and stored at -80ºC. Protein concentrations were determined according to the

Bradford method (Bradford, 1976) using Bio-Rad’s Protein Assay reagent (Bio-Rad, CA,

USA). Equal amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide

gel electrophoresis and transferred to a nitrocellulose membrane. After blocking with 5% milk

solution, membranes were incubated with the primary antibody overnight at 4ºC [mouse anti-

nNOS (BD Biosciences, San José, CA, USA) diluted at 1:2.500, rabbit anti-Bid (Santa Cruz

Biotechnology, Santa Cruz, CA, USA) diluted at 1:500, mouse anti-P-JNK1/2 (Santa Cruz

Biotechnology) diluted at 1:200 or anti-β-actin (Sigma Chemical Co) diluted at 1:10.000], and

Chapter III __________________________________________________________________________

90

finally with horseradish peroxidase-labelled secondary antibody. Protein bands were

detected by LumiGLO® and visualized by autoradiography with Hyperfilm ECL.

2.6. Caspase activity determination Activities of caspase-3, -8 and -9 were measured by a colorimetric method (Calbiochem,

Darmstadt, Germany) as usual in our laboratory (Vaz et al., 2010). Cells were harvested,

washed with ice-cold PBS and lysed for 30 minutes on ice in the lysis buffer [50 mM HEPES

(pH 7.4), 100 mM NaCl, 0.1% (w/v) cholamidopropyldimethylammonio-1-propanesulfonate

(CHAPS), 1 mM DTT, 0.1 mM EDTA]. The lysate was centrifuged at 10,000 g for 10 min at

4ºC and the supernatants were collected and stored at -80ºC. Protein concentrations were

determined as aforementioned. The activity of caspases 3, 8 and 9 was determined in cell

lysates by enzymatic cleavage of chromophore p-nitroaniline (pNA) from the substrate Ac-

YVAD-pNA, according to manufacturer’s instructions. The proteolytic reaction was carried

out in protease assay buffer [50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% (w/v) CHAPS, 10

mM DTT, 0.1 mM EDTA, 10% (v/v) glicerol], containing 2 mM substrate Ac-DEVD-pNA for

caspase-3, Ac-IETD-pNA for caspase-8 and Ac-LEHD-pNA for caspase-9. Following

incubation of the reaction mixtures for 2 h at 37ºC, the formation of pNA was measured at

405 nm with a reference filter of 620 nm.

2.7. MTT reduction Cellular reduction of [3-(4,5-dimethylthiazol, 2-yl)-2,5-diphenyltetrazolium bromide]

(MTT) was measured in nerve cells as previously described by us (Silva et al., 2002). Briefly,

a stock solution of MTT at 5 mg/mL was freshly prepared and after the incubation periods,

supernatants were removed and cells were incubated for 1 h, at 37°C, with 0.5 mL of MTT at

0.5 mg/mL. After incubation, medium was discarded and MTT formazan crystals were

dissolved by addition of 1 mL isopropanol/HCl 0.04 M and gentle shaking for 15 min, at room

temperature. After centrifugation, absorbance values at 570 nm were determined in a

Unicam UV2 spectrophotometer (Unicam Limited, UV2, Cambridge, UK). Results were

expressed as percentage of control, which was considered as 100%.

2.8. Densitometry and statistical analysis The relative intensities of protein and nucleic acid bands were analysed using the

Quantity One (version 4.6) program (Bio-Rad, CA, USA). Results of, at least, three different

experiments were expressed as mean ± S.E.M. Significant differences between two groups

were determined by the two-tailed t-test performed on the basis of equal and unequal

variance as appropriate. Comparison of more than two groups was done by ANOVA using

Instat 3.05 (GraphPad Software, San Diego, CA, USA) followed by multiple comparisons


91

Bonferroni post-hoc correction. Mean values were considered statistically significant when P

values were lower than 0.05.

3. Results 3.1. UCB, alone or in combination with TNF-α+IL-1β, induces nNOS expression and

.NO production in immature neurons, which are counteracted by L-NAME To investigate the possible role of UCB -induced neuronal damage in immature nerve

cells, 3 DIV cortical neurons were incubated with UCB at conditions mimicking moderate to

severe neonatal jaundice (UCB to HSA molar ratio of 0.5) that had previously shown to

promote oxidative injury and cell death in 8 DIVs neurons (Brito et al., 2008a). Cells were

collected after treatment during 1 h to 12 h for the analysis of nNOS expression and

production of nitrites. We also investigated the aggravating effects of pro-inflammatory

cytokines TNF-α and IL-1β on UCB-induced oxidative stress. In order to evaluate the

importance of .NO signalling in oxidative damage resulting from UCB or UCB+TNF-α+IL-1β,

we used L-NAME to inhibit nNOS activity. We observed that UCB induced nNOS expression,

together with a raise in nitrite production in immature neurons. Co-incubation with

TNF-α+IL-1β, in concentrations that have been described to induce neuronal loss (Patel and

Brewer, 2008, Zhang et al., 2008), aggravated UCB-induced nNOS expression and nitrite

production in these cells (Fig. III.1A and III.1C). In parallel studies, co-incubation with 100 µM

L-NAME led to at least ~70% and ~60% inhibition in nNOS expression and nitrite production

(p<0.05), respectively (Fig. III.1B and III.1D).

3.2. Inhibition of nNOS by L-NAME prevents the cascade of apoptosis induced by UCB or UCB+TNF-α+ IL-1β in immature neurons To deepen characterize the mechanisms of neuronal apoptosis upon exposure of

immature neurons to UCB and pro-inflammatory cytokines, we evaluated the activity of

initiator caspases from mitochondrial (intrinsic) and death receptor (extrinsic) pathways,

caspases-9 and -8, respectively, as well as of the effector caspase-3. As shown in Figures

III.2A and III.2C UCB led to the activation of caspases-3 and -9, indicating the stimulation of

the intrinsic pathway of apoptosis. Interestingly, co-incubation with TNF-α+IL-1β significantly

enhanced this activation.

Chapter III __________________________________________________________________________

92

Figure III.1 - Unconjugated bilirubin (UCB) induces neuronal nitric oxide synthase (nNOS) expression and nitrite production in immature neurons, which are intensified by co-treatment with TNF-α+IL-1β and prevented by nNOS inhibition (L-NAME). Rat neurons cultured for 3 days in vitro were incubated with UCB (50 µM), alone or plus TNF-α+IL-1β (50 ng/mL each), in the presence of human serum albumin (100 μM), from 1 to 12 h at 37ºC. In sister experiments, cells were treated with L-NAME (100 μM). Expression levels of nNOS (A,B) were assessed by Western blot analysis and nitrite levels (C,D) were measured by the Griess reagent, as indicated in Methods. Results are mean (± SEM) from at least three independent experiments performed in duplicate. *p<0.05, **p<0.01 vs. control; #p<0.05, ##p<0.01 vs. UCB alone; §p<0.05, §§p<0.01 vs. respective condition without L-NAME.

To investigate whether .NO plays a role in neurotoxicity induced by UCB or UCB+TNF-

α+IL-1β, we evaluated caspase activation in cells co-incubated with L-NAME. Our results

demonstrated that inhibition of nNOS prevents activation of the effector caspase (Fig. III.2B),

as well as of the caspase from the intrinsic pathway (Fig. III.2D). In addition, UCB induced

the extrinsic pathway of apoptosis, as indicated by the activation of caspase-8, which was

further increased when cells were concomitantly exposed to TNF-α+IL-1β (Fig. III.3A). Once

more, capase-8 activity was significantly reduced in cells co-incubated with L-NAME (Fig.

III.3B). We also analyzed the cleavage of Bid, a Bcl-2 family member that is a specific

substrate of caspase-8 and plays a role in caspase 8-mediated mitochondrial damage and

0.0

0.5

1.0

1.5

2.0

nNO

S(f

old

chan

ge)

0.0

0.5

1.0

1.5

2.0nN

OS

(fol

d ch

ange

)

0.0

1.0

2.0

3.0

Nitr

ites

(μM

)

0.0

1.0

2.0

3.0

Nitr

ites

(μM

)

* **

**##**

##

**

##

**

§§

§§§

§§ §

§§

A B

****

*##**

##

**

#

**

§§§§

§

§§

§

§§

UCB (50 μM)TNF-α (50 ng/ml)+IL-1β (50 ng/ml)

1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +

C D


93

cell death (Li et al., 1998). As shown in Figure III.3C, the cleavage profile of Bid (22-kDa) into

truncated Bid (tBid, a 15-kDa fragment) occurred after the activation of caspase-8, as we

observed the activation of caspase-8 after 1 h treatment with UCB or UCB+TNF-α+IL-1β and

tBid significantly increased only after 4 h treatment (p<0.01 vs. 1 and 12 h of treatment). In

addition, co-incubation with L-NAME prevented tBid generation in cells treated with UCB or

UCB+TNF-α+IL-1β UCB (Fig. III.3D, p<0.05).

Figure III.2 - Unconjugated bilirubin (UCB) triggers the activation of caspases-3 and -9 in immature neurons, which is intensified by co-treatment with TNF-α+IL-1β and prevented by nNOS inhibition (L-NAME). Rat neurons cultured for 3 days in vitro were treated as in Figure III.1. Activation of caspases-3 (A,B) and -9 (C,D) was determined by using colorimetric substrate cleavage assays, as indicated in Methods. Results are mean (± SEM) from at least three independent experiments performed in duplicate. **p<0.01 vs. control; #p<0.05, ##p<0.01 vs. UCB alone; §p<0.05, §§p<0.01 vs. respective condition without L-NAME.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Cas

pase

-9 a

ctiv

ity(f

old

chan

ge)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Cas

pase

-9 a

ctiv

ity(f

old

chan

ge)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Cas

pase

-3 a

ctiv

ity(f

old

chan

ge)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Cas

pase

-3 a

ctiv

ity(f

old

chan

ge) **

** **

##**

##

**

##**

§§

§§ §§

§

§§

** ****

**

#

** #

**

§§

§§

§

§§

§§§§


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +

A B

C D

Chapter III __________________________________________________________________________

94

Figure III.3 - Unconjugated bilirubin (UCB) triggers the activation of caspase-8 and Bid in immature neurons, which is intensified by co-treatment with TNF-α+IL-1β and prevented by nNOS inhibition (L-NAME). Rat neurons cultured for 3 days in vitro were treated as in Figure III.1. Activation of caspase-8 (A,B) was determined by using colorimetric substrate cleavage assay and expression levels of truncated form of Bid, tBid (C,D), was assessed by Western blot analysis, as indicated in Methods. Results are mean (± SEM) from at least three independent experiments performed in duplicate. **p<0.01 vs. control; #p<0.05, ##p<0.01 vs. UCB alone; §p<0.01 vs. respective condition without L-NAME.

3.3. Inhibition of nNOS by L-NAME decreases P-JNK1/2 in immature neurons treated with UCB or UCB+TNF-α+IL-1β

In order to verify if oxidative stress occurs in parallel with JNK1/2 activation in our model, we

determined P-JNK1/2 expression, in the absence or presence of L-NAME. Our results

demonstrated that neuronal exposure to UCB increases P-JNK1/2 levels only after 4 h

treatment (p<0.01 and p<0.05 vs. 1 and 12 h of treatment, respectively), effect that was

increased by co-incubation with TNF-α+IL-1β (Fig. III.4A). Furthermore, inhibition of nNOS by

L-NAME decreased P-JNK1/2 expression (Fig. III.4B), indicating that JNK1/2 activation is

0.0

0.5

1.0

1.5tB

id(f

old

chan

ge)

0.0

0.5

1.0

1.5

tBid

(fol

d ch

ange

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Cas

pase

-8 a

ctiv

ity(f

old

chan

ge)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Cas

pase

-8 a

ctiv

ity(f

old

chan

ge)

****

**

#** #

** ##

**

§§§§

§§§§§§

§§

**

#

**

§§§§


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +

- 22 KDa

- 15 KDa

Bid -

tBid -

βact - - 42 KDa

Bid -

tBid -

βact -

- 22 KDa

- 15 KDa

- 42 KDa

A B

C D


95

mediated by production of .NO. To better understand whether JNK1/2 activation takes part in

neuronal dysfunction, we tested the effects of SP600125, a specific inhibitor of JNK1/2, on

cells treated with UCB or UCB+TNF-α+IL-1β. Immature neurons were pre-treated with 0.2

µM of the inhibitor for 1 h, followed by 4 h stimulation with UCB alone or in combination with

TNF-α+IL-1β. As shown in Figures III.4C and III.4D, SP600125 efficiently prevented the

appearance of P-JNK1/2 induced by UCB or UCB+TNF-α+IL-1β (~90%, p<0.05).

Figure III.4 - Unconjugated bilirubin (UCB) leads to activation of c-Jun N-terminal kinases 1/2 (JNK1/2) in immature neurons, which is intensified by co-treatment with TNF-α+IL-1β and prevented by inhibition of nNOS (L-NAME) or JNK1/2 (SP600125). Rat neurons cultured for 3 days in vitro were treated as in Figure 1 (A,B). In another set of experiments, cells were treated for 1 h with 0.2 μM SP600125 (C,D), prior to UCB (50 µM) exposure, alone or in combination with TNF-α+IL-1β (50 ng/mL each), in the presence of human serum albumin (100 μM), for 4 h at 37ºC. Activation of JNK1/2 was determined by measuring the expression levels of phosphorylated forms of this enzyme (P-JNK1/2) by Western blot analysis, as indicated in Methods. Results are mean (± SEM) from at least three independent experiments performed in duplicate. **p<0.01 vs. control; ##p<0.01 vs. UCB alone; §§p<0.01, §p<0.05 vs. respective condition without L-NAME; &&p<0.01 vs. respective condition without SP600125.

0.0

0.4

0.8

1.2

1.6

2.0

P-J

NK

1/2

(fol

d ch

ange

)

0.0

0.4

0.8

1.2

1.6

2.0

P-J

NK

1/2

(fol

d ch

ange

)

0.0

0.4

0.8

1.2

1.6

2.0

P-J

NK

1/2

(fol

d ch

ange

) **

##

**

§§

§

&&

- 55 KDa- 46 KDa

P-JNK1/2 -

βact - - 42 KDa

P-JNK1/2 -

βact -

- 55 KDa- 46 KDa

- 42 KDa


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


1 h

- + +- - +

4 h

- + +- - +

12 h

- + +- - +


- + +- - +

- + +- - +SP600125 (0.2 µM)


- + +

- - +

- + +

- - +

SP600125 (0.2 µM)

**

##

**

&&

- 55 KDa- 46 KDa

P-JNK1/2 -

βact - - 42 KDa

C D

A B

Chapter III __________________________________________________________________________

96

3.4. Inhibition of P-JNK1/2 by SP600125 prevents the cascade of apoptosis induced by UCB or UCB+TNF-α+IL-1β in immature neurons Having established that the appearance of P-JNK1/2 induced by UCB or UCB+TNF-

α+IL-1β is mediated, at least in part, by .NO production, we attempted to examine whether

JNK1/2 activation is also linked to the apoptotic events described previously. As shown in

Figure III.5, pre-treatment with SP600125 significantly reduced the activation of caspases-3,

-8 and -9, as well as activation of Bid into tBid in cells treated with UCB or UCB+TNF-α+IL-1β

(p<0.05), suggesting that, in our model, both intrinsic and extrinsic apoptotic pathways are

being initiated during JNK1/2 activation by bilirubin and cytokines.

3.5. Loss of neuronal functionality in immature cells exposed to UCB is increased by UCB+TNF-α+IL-1β and prevented by inhibition of nNOS and JNK1/2 activation To investigate whether induction of nNOS and JNK1/2 is implicated in neuronal

dysfunction, we assessed the effects of UCB, alone or in combination with TNF-α+IL-1β, on

the immature cell function in neurons treated with or without L-NAME or SP600125. Our

results demonstrated that the loss of functionality in immature neurons exposed to UCB for

24 h is increased by the pro-inflammatory cytokines TNF-α+IL-1β (Fig. III.6). In addition, this

neuronal dysfunction was prevented by co-incubation with L-NAME, as well as with

pretreatment with SP600125, suggesting both .NO and JNK1/2 as key elements in neuronal

dysfunction during hyperbilirubinemia with associated inflammation.


97

Figure III.5 - Inhibition of c-Jun N-terminal kinases 1/2 (JNK1/2) with SP600125 prevents the cascade of apoptosis induced either by unconjugated bilirubin (UCB) alone or by UCB co-treatment with TNF-α+IL-1β. Rat neurons cultured for 3 days in vitro were treated as in Figure III.4. Activation of caspases-3, -9 and -8 (A-C) was determined by using colorimetric substrate cleavage assays and expression levels of truncated form of Bid, tBid (D,E), was assessed by Western blot analysis, as indicated in Methods. Results are mean (± SEM) from at least three independent experiments performed in duplicate. **p<0.01 vs. control; ##p<0.01, #p<0.05 vs. UCB alone; 0.05, &&p<0.01 vs. respective condition without SP600125.

0.0

0.5

1.0

1.5

tBid

(fol

d ch

ange

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Cas

pse-

8 ac

tivity

(fol

d ch

ange

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Cas

pse-

9 ac

tivity

(fol

d ch

ange

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Cas

pse-

3 ac

tivity

(fol

d ch

ange

)


- + +- - +

- + +- - +SP600125 (0.2 µM)

**

##

**


- + +- - +

- + +- - +SP600125 (0.2 µM)


- + +- - +

- + +- - +SP600125 (0.2 µM)


- + +- - +

- + +- - +SP600125 (0.2 µM)

**

#

**

**

#

** **

#

**

&&&

&&&&

&&

&&&&&

A B

C D

- 22 KDa

- 15 KDa

Bid -

tBid -

βact - - 42 KDa


- + +

- - +

- + +

- - +

SP600125 (0.2 µM)

E

Chapter III __________________________________________________________________________

98

Figure III.6 - Loss of cell functionality in immature neurons exposed to unconjugated bilirubin (UCB) is intensified by co-treatment with TNF-α+IL-1β and prevented by inhibition of nNOS (L-NAME) or JNK1/2 (SP600125). Rat neurons cultured for 3 days in vitro were incubated with UCB (50 µM), alone or in combination with TNF-α+IL-1β (50 ng/mL each), in the presence of human serum albumin (100 μM), for 24 h at 37ºC. In one set of experiments, cells were treated with L-NAME (100 μM); in the other set, cells were pre-treated for 1 h with 0.2 μM SP600125. Cell functionality was evaluated by measuring 3-(4,5-dimethylthiazol, 2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, as indicated in Methods. Results are mean (± SEM) from at least three independent experiments performed in duplicate. **p<0.01 vs. control; ##p<0.01 vs. UCB alone; §p<0.05, §§p<0.01 vs. respective condition without L-NAME; &&p<0.01, &p<0.05 vs. respective condition without SP600125.

4. Discussion Previously, we demonstrated that UCB induces neurotoxicity, which differ from neural

cell type and maturation state, being neurons and immature cells the most susceptible ones

(Falcão et al., 2006, Brito et al., 2008b). Moreover, co-incubation with LPS showed to

enhance the demise of astrocytes (Fernandes et al., 2004), namely in immature cells (Falcão

et al., 2005). Based on these data, we investigated whether pro-inflammatory cytokines are

able to strengthen the oxidative stress exerted by UCB in immature neurons, and if the effect

is reproduced on the extrinsic and intrinsic signalling apoptotic pathways, as well as at the

level of neuronal dysfunction.

In this paper, we demonstrate that UCB induces oxidative damage in immature rat

neurons, as indicated by the cell function impairment and apoptosis associated with .NO

production. Interestingly, we previously reported an inhibition of mitochondrial cytochrome c

0

25

50

75

100

MTT

(% o

f Con

trol)


- + +- - +

SP600125 (0.2 µM)

L- NAME(100 µM)

- + +- - +

- + +- - +

**§

§§

**##

&&&


99

oxidase by UCB in the same in vitro model (Vaz et al., 2010), which can explain the

formation of .NO. In addition, we also observed that UCB decreases NADPH concentration

and increases glutathione oxidation and superoxide anion radical production, thus confirming

neuronal oxidative stress by UCB. Furthermore, UCB was shown to induce protein oxidation

and lipid peroxidation, and to diminish the antioxidant defences in mature neurons (8 DIV),

events that occur in parallel with necrotic cell death and could be due to low glutathione

stores (Brito et al., 2008a).

Here, we present evidence that mitochondrial-dependent and -independent apoptotic

pathways are induced in immature rat cultured neurons after UCB treatment. To the best of

our knowledge, this is the first evidence reporting UCB to induce neuronal extrinsic pathway

of apoptosis, in addition to that of mitochondria-mediated apoptosis observed in differentiated

neurons for UCB/HSA molar ratio of 3 (Rodrigues et al., 2002a). However, this molar ratio is

supposed to never occur in jaundiced babies, even during the worst pathological situations

and, therefore, we have repeated the experiments for the most suitable molar ratio of 0.5.

Interestingly, the same effect was significantly produced in immature neurons, after a short

exposure (1 h incubation) to UCB (Vaz et al., 2010). It is widely accepted that activated

caspase-8 propagates the apoptotic signal by activating downstream caspases through

proteolytic cleavage, as well as by triggering mitochondrial pathway through cleavage and

activation of pro-apoptotic Bid into tBid (Adams, 2003). In fact, in our model, activation of

caspase-8 begins early in time (at 1 h incubation), followed by tBid generation (at 4 h

incubation). This raises interesting possibilities as both caspase-8 and -9-initiated apoptotic

pathways converge independently to activate the execution phase of caspase-3 (Hengartner,

2000). In addition, we are tempted to propose that caspase-8/tBid-related route may play an

important role in UCB-induced neuronal cell death. The results herein obtained in immature

neurons are in line with our own previous observations showing that UCB induces intrinsic

signalling pathways by requiring Bax translocation to the mitochondria, mitochondrial

depolarization, release of cytochrome c and caspase-3 activation in isolated mitochondria

from the brain and liver of adult male Wistar rats (Rodrigues et al., 2002a, Rodrigues et al.,

2000). They also agree with other findings indicating activation of TNFR1 upon exposure of

astrocytes, to UCB reflecting the activation of the death receptor pathway (Fernandes et al.,

2006), and therefore the extrinsic cascade.

The present study also shows for the first time that pro-inflammatory cytokines TNF-α

and IL-1β intensify UCB-induced oxidative stress and apoptotic cell death by both

mitochondrial-dependent and –independent apoptotic pathways in immature neurons. These

observations are particularly important, since low-gestational-age newborns have a

prominently increased risk of brain dysfunction attributed to cerebral-cortex damage,

Chapter III __________________________________________________________________________

100

including excess of apoptosis and impairment of surviving neurons (Leviton and Gressens,

2007). With our data, we associate inflammation with the increased risk of UCB-induced

neurotoxicity. In fact, there are some reports in agreement with our finding: (i) in an animal

model of sepsis, it was shown that serum concentration of total and free bilirubin was

increased, promoting a net accumulation of UCB in the brain (Hansen et al., 1993); (ii) pro-

inflammatory cytokines were reported to increase blood-brain-barrier permeability (Petty and

Lo, 2002), allowing UCB entrance into the brain, and to exacerbate UCB-induced cytotoxicity

in different cell lines, such as in neuroblastoma (ATCC, HTB-10, SK-N-MC), glioblastoma

(ATCC, CRL 1690, T98G), umbilical vein endothelial (ATCC, CRL 1730, HUV-EC), liver cell

(ATCC, CCL 13) and mouse fibroblasts (L-929) (Ngai and Yeung, 1999, Yeung and Ngai,

2001). Thus, with our model, that mimics pro-inflammatory cytokine release from glial cells,

as we have previously reported for astrocytes and microglia exposed to UCB (Fernandes et

al., 2004, Fernandes et al., 2006), we can establish a relation between UCB-induced toxicity

and associated inflammation in immature neurons.

In this study we demonstrate that .NO production mediates, at least in part, neurotoxicity

in immature neurons exposed to UCB, alone or in combination with TNF-α+IL-1β. This

corroborates our recent observation in that oxidative stress and necrotic cell death induced

by UCB in mature neurons decreases by concomitant treatment with the NOS inhibitor, l-

NAME (Fernandes et al., 2010). Additionally, in P7 rats, local elimination of nNOS resulted in

a significant attenuation of the damage after hypoxic-ischemic insult (Ferriero et al., 1995)

and nNOS deficiency through genetic targeting was also neuroprotective in neonatal mice

(Ferriero et al., 1996). In other studies with hypoxia-ischemia models, NOS inhibition reduced

caspase-3 activation (Zhu et al., 2004) and conferred tissue protection (Peeters-Scholte et

al., 2002), further indicating that .NO production exerts cytotoxicity in the developing brain.

A very remarkable finding of our study is the JNK1/2 MAPK activation observed when

immature neurons are exposed to UCB, alone or in combination with TNF-α+IL-1β. The

activation of JNK1/2 is related to toxicity in developing neurons, since overexpression of

activated JNK1/2 was shown to produce apoptosis, as suppression of this protein protected

against neuronal death induced by deprivation of nerve growth factor in sympathetic and

hippocampal neurons (Estus et al., 1994, Ham et al., 1995, Schlingensiepen et al., 1993).

Interestingly, in our study, inhibition of .NO production led to a significant reduction in JKN1/2

activation. This observation is in good agreement with some reports on models of

neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, where inhibition of .NO-

induced JNK 1/2 phosphorylation conferred protection against neuronal cell death (Katsuki et

al., 2006, Marques et al., 2003).


101

Here, we observed a reduction of either the intrinsic and extrinsic caspase cascades by

using the selective JNK1/2 inhibitor, SP600125. Due to the fact that members of the

antiapoptotic Bcl-2 family proteins are inactivated through JNK1/2 phosphorylation (Inoshita

et al., 2002, Maundrell et al., 1997), we may conclude that in our model JNK1/2 activation is

altering mitochondrial function. In agreement with our thoughts, there are several reports on

dopaminergic neuron models, where a correlation between SAPKs activation and

neurotoxicity was established: (i) phosphorylation of p38 by oxidative stress was linked to

activation of both caspases-8- and -9 (Choi et al., 2004); and (ii) repression of JNK1/2

activation by transfection of a dominant negative mutant SEK1(Lys 3 Arg) blocked dopamine-

induced apoptosis (Luo et al., 1998). In this last study, antioxidants, such as N-acetylcysteine

(NAC) and catalase, blocked dopamine-induced JNK1/2 activation and subsequent

apoptosis, confirming that dopamine-induced oxidative stress is involved in JNK1/2 pathway.

More recently, apoptotic cascade mediated by caspase-3 activation has been demonstrated

to be JNK1/2 dependent in different neuronal cell models (Cerezo-Guisado et al., 2007,

Sahara et al., 2008). Therefore, inhibition of SAPKs signalling might represent a therapeutic

target in acute brain insults, such as neonatal hyperbilirubinemia and associated

inflammation.

In summary, the data obtained in the present study contributes to a better understanding

of the mechanisms underlying neurotoxicity in conditions mimicking a moderate to severe

hyperbilirubinemia in the early neonatal period. Actually, and as schematically represented in

Figure III.7, they demonstrate that UCB-induced neuronal dysfunction in 3 DIV neurons

results from activation of both the intrinsic and extrinsic pathways of apoptosis, that injury is

linked to oxidative stress, and that .NO signalling and JNK1/2 activation are key players. The

association of pro-inflammatory cytokines, TNF-α+IL-1β, to the condition of

hyperbilirubinemia significantly increased the cytotoxic potential of UCB through the same

cascade of mediators. Most important, the results provide supportive evidence for the

commonly indicated higher risk of UCB brain damage in a condition of infection, thus

justifying that treatment should be carried out in these conditions at lower levels of UCB than

those for well-appearing jaundiced neonates. These advances may substantiate target-

driven approaches to the prevention and treatment of UCB-induced neurological damage,

and provide fruitful opportunities for future investigations.

Chapter III __________________________________________________________________________

102

Figure III.7 - Schematic representation of the cellular targets involved in unconjugated bilirubin (UCB) injury to immature cortical neurons, all of them further stimulated by the combination of UCB with pro-inflammatory cytokines (TNF-α and IL-1β), and modulating effects by specific inhibitors. Lighter grey and small arrows indicate the effects of UCB, while darkness grey and large arrows point to the combined effects of UCB plus TNF-α+IL-1β; lines with blocked ends indicate steps that are being modulated. UCB interaction with neurons leads to neuronal nitric oxide synthase (nNOS) increased expression, nitric oxide (.NO) production and activation of c-Jun N-terminal kinase (JNK1/2). As a consequence of these events, mitochondrial and extrinsic pathways of apoptosis are initiated, with activation of caspases-9 and -8, respectively. Activation of caspase-9 further activates the effector capase-3, leading to neuronal apoptosis. Activation of caspase-8 triggers the cleavage of the pro-apoptotic Bid into the truncated form (tBid) that is translocated to mitochondria, thus propagating the apoptotic signal by activation of caspase-3. Ultimately, these events lead to neuronal dysfunction, as assessed by

3-(4,5-dimethylthiazol, 2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Co-incubation of UCB with TNF-α+IL-1β intensifies all the events activated by UCB alone. Inhibition of JNK1/2 activation by SP600125 conferred neuroprotection to immature neurons exposed either to UCB alone or UCB plus TNF-α+IL-1β, corroborating the involvement of this signaling pathway in the neuronal demise observed. Upstream inhibition of nNOS by N-ω-nitro-L-arginine methyl ester (L-NAME) prevents downstream events from occurring, thus pointing to .NO as a key mediator in UCB-induced neuronal dysfunction of immature neurons.

nNOS activation

JNK1/2 activation

NO production

Caspase-9Caspase-3

L- NAME

SP600125

Caspase-8

Bid

UCBTNF-α

IL-1β

UCB

tBid


103

5. References Adams, J. M. (2003) Ways of dying: multiple pathways to apoptosis. Genes Dev, 17, 2481-

2495.



Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-

254.













Cerezo-Guisado, M. I., Alvarez-Barrientos, A., Argent, R., Garcia-Marin, L. J., Bragado, M. J.

and Lorenzo, M. J. (2007) c-Jun N-terminal protein kinase signalling pathway mediates

lovastatin-induced rat brain neuroblast apoptosis. Biochim Biophys Acta, 1771, 164-176.

Choi, W. S., Eom, D. S., Han, B. S. et al. (2004) Phosphorylation of p38 MAPK induced by

oxidative stress is linked to activation of both caspase-8- and -9-mediated apoptotic

pathways in dopaminergic neurons. J Biol Chem, 279, 20451-20460.



Geogr Med, 36, 127-132.



592.

Estus, S., Zaks, W. J., Freeman, R. S., Gruda, M., Bravo, R. and Johnson, E. M., Jr. (1994)

Altered gene expression in neurons during programmed cell death: identification of c-jun

as necessary for neuronal apoptosis. J Cell Biol, 127, 1717-1727.

Chapter III __________________________________________________________________________

104







Fernandes, A., Barateiro, A., Falcão, A. S., Silva, S. L., Vaz, A. R., Brito, M. A., Silva, R. F.

M. and Brites, D. (2010) Astrocyte reactivity to unconjugated bilirubin requires TNF-a and

IL-1b receptor signalling pathways. Glia, in press.

Fernandes, A., Falcão, A. S., Silva, R. F. M., Brito, M. A. and Brites, D. (2007) MAPKs are















Ham, J., Babij, C., Whitfield, J., Pfarr, C. M., Lallemand, D., Yaniv, M. and Rubin, L. L. (1995)

A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell

death. Neuron, 14, 927-939.


29, 765-778, viii.

Hansen, T. W. R., Maynard, E. C., Cashore, W. J. and Oh, W. (1993) Endotoxemia and brain

bilirubin in the rat. Biol Neonate, 63, 171-176.







105



Hengartner, M. O. (2000) The biochemistry of apoptosis. Nature, 407, 770-776.

Inoshita, S., Takeda, K., Hatai, T., Terada, Y., Sano, M., Hata, J., Umezawa, A. and Ichijo, H.

(2002) Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to

oxidative stress. J Biol Chem, 277, 43730-43734.

Kajta, M., Trotter, A., Lason, W. and Beyer, C. (2006) Impact of 17beta-estradiol on cytokine-

mediated apoptotic effects in primary hippocampal and neocortical cell cultures. Brain

Res, 1116, 64-74.



Acta, 356, 9-21.

Katsuki, H., Okawara, M., Shibata, H., Kume, T. and Akaike, A. (2006) Nitric oxide-producing

microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain

slice culture. J Neurochem, 97, 1232-1242.

Konsman, J. P., Drukarch, B. and Van Dam, A. M. (2007) (Peri)vascular production and

action of pro-inflammatory cytokines in brain pathology. Clin Sci (Lond), 112, 1-25.



Leviton, A. and Gressens, P. (2007) Neuronal damage accompanies perinatal white-matter

damage. Trends Neurosci, 30, 473-478.

Li, H., Zhu, H., Xu, C. J. and Yuan, J. (1998) Cleavage of BID by caspase 8 mediates the

mitochondrial damage in the Fas pathway of apoptosis. Cell, 94, 491-501.


apoptosis through an oxidation-involved SAPK/JNK activation pathway. J Biol Chem, 273,

3756-3764.




Biol Chem, 278, 28294-28302.

Marx, C. E., Jarskog, L. F., Lauder, J. M., Lieberman, J. A. and Gilmore, J. H. (2001)

Cytokine effects on cortical neuron MAP-2 immunoreactivity: implications for

schizophrenia. Biol Psychiatry, 50, 743-749.

Maundrell, K., Antonsson, B., Magnenat, E. et al. (1997) Bcl-2 undergoes phosphorylation by

c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the

constitutively active GTP-binding protein Rac1. J Biol Chem, 272, 25238-25242.

Chapter III __________________________________________________________________________

106

McDonagh, A. F. (1979) Bile pigments: Bilatrienes and 5,15-biladienes. In: The Porfirins, pp.

293-491. Academic Press, San Diego.



Ngai, K. C. and Yeung, C. Y. (1999) Additive effect of tumor necrosis factor-alpha and

endotoxin on bilirubin cytotoxicity. Pediatr Res, 45, 526-530.



Patel, J. R. and Brewer, G. J. (2008) Age-related changes to tumor necrosis factor receptors

affect neuron survival in the presence of beta-amyloid. J Neurosci Res, 86, 2303-2313.



2310.

Petty, M. A. and Lo, E. H. (2002) Junctional complexes of the blood-brain barrier:

permeability changes in neuroinflammation. Prog Neurobiol, 68, 311-323.

Rodrigues, C. M. P., Solá, S. and Brites, D. (2002a) Bilirubin induces apoptosis via the


Rodrigues, C. M. P., Solá, S., Brito, M. A., Brites, D. and Moura, J. J. (2002b) Bilirubin

directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat




Mol Med, 6, 936-946.

Rossi, D. and Volterra, A. (2009) Astrocytic dysfunction: insights on the role in

neurodegeneration. Brain Res Bull, 80, 224-232.

Sahara, N., Murayama, M., Lee, B., Park, J. M., Lagalwar, S., Binder, L. I. and Takashima, A.

(2008) Active c-jun N-terminal kinase induces caspase cleavage of tau and additional

phosphorylation by GSK-3beta is required for tau aggregation. Eur J Neurosci, 27, 2897-

2906.

Schlingensiepen, K. H., Schlingensiepen, R., Kunst, M., Klinger, I., Gerdes, W., Seifert, W.

and Brysch, W. (1993) Opposite functions of jun-B and c-jun in growth regulation and

neuronal differentiation. Dev Genet, 14, 305-312.



Today, 35, 52-59.


107







phagocytosis to inflammation. Neurobiol Dis.



Takahashi, K., Funata, N., Ikuta, F. and Sato, S. (2008) Neuronal apoptosis and

inflammatory responses in the central nervous system of a rabbit treated with Shiga toxin-

2. J Neuroinflammation, 5, 11.




105, 2039-2052.


Mol Life Sci, 55, 1230-1254.

Vaz, A. R., Delgado-Esteban, M., Brito, M. A., Bolaños, J. P., Brites, D. and Almeida, A.

(2010) Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in

immature cortical neurons: assessment of the protective effects of glycoursodeoxycholic

acid. J Neurochem, 112, 56-65.



Yeung, C. Y. and Ngai, K. C. (2001) Cytokine- and endotoxin-enhanced bilirubin cytotoxicity.

J Perinatol, 21 Suppl 1, S56-58; discussion S59-62.

Zhang, R., Yamada, J., Hayashi, Y., Wu, Z., Koyama, S. and Nakanishi, H. (2008) Inhibition

of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons.

Biochem Biophys Res Commun, 372, 816-820.




Chapter IV

IV. Selective vulnerability of rat brain regions to unconjugated bilirubin

Ana Rita Vaz, Sandra Leitão Silva, Andreia Barateiro, Ana Sofia Falcão, Adelaide

Fernandes, Maria A Brito, Dora Brites

Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade

de Farmácia, University of Lisbon, Av. Professor Gama Pinto, Lisbon 1649-003,

Portugal.

Molecular and Cellular Neuroscience (submitted).

Acknowledgements This work was supported by grants PTDC/SAU-NEU/64385/2006 (to D.B.) and

BD/30292/2006 (to A.R.V.) from Fundação para a Ciência e a Tecnologia, Lisbon,

Portugal.

Hallmarks in hippocampal susceptibility to UCB __________________________________________________________________________

111

Abstract

Hippocampus is one of the brain regions most vulnerable to unconjugated bilirubin

(UCB) encephalopathy, although cerebellum also shows selective yellow staining in

kernicterus. We demonstrated that UCB induces oxidative stress in cortical neurons,

disruption of neuronal network dynamics, either in developing cortical or hippocampal

neurons, and that immature cortical neurons are more prone to UCB-induced injury.

Here, we studied features of oxidative stress and cell dysfunction induced by UCB in

immature rat neurons isolated from cortex, cerebellum and hippocampus. We also

explored whether oxidative damage and its regulation contribute to neuronal dysfunction

induced by hyperbilirubinemia, in terms of neurite extension and ramification, and cell

death. Our results show that UCB induces neuronal nitric oxide synthase expression, as

well as production of nitrites and cyclic guanosine monophosphate in immature neurons,

mainly in those from hippocampus. After exposure to UCB, hippocampal neurons

presented the highest content of reactive oxygen species, disruption of glutathione redox

status and cell death, when compared to those from cortex or cerebellum. In particular,

the results indicated that cells exposed to UCB undertake an adaptive response that

involves DJ-1, a multifunctional neuroprotective protein involved in cellular oxidation

status maintenance. However, longer neuronal exposure to UCB down-regulated DJ-1

expression, especially in hippocampal neurons. In addition, UCB induced impairment in

neurite outgrowth and branching, mainly in immature neurons from hippocampus.

Interestingly, pre-incubation with N-acetylcysteine, a precursor of glutathione synthesis,

conferred neuroprotection to UCB-induced oxidative stress and necrotic cell death, as

well as prevented DJ-1 down-regulation and neuritic impairment. Taken together, these

data point oxidative injury and disruption of neuritic network as hallmarks in hippocampal

susceptibility to UCB. Furthermore, it is suggested that local differences in glutathione

content may account to the different susceptibility found between brain regions exposed

to UCB.

Keywords: Cortex; cerebellum; DJ-1; hippocampus; immature neurons; oxidative and

nitrosative stress; neurite outgrowth and branching; unconjugated bilirubin.

Chapter IV __________________________________________________________________________

112

1. Introduction Hyperbilirubinemia, a very common condition in the neonatal period, characterized by

increased serum levels of unconjugated bilirubin (UCB) (Stevenson et al., 2001, Dennery et

al., 2001), is responsible for the clinical manifestation of jaundice. Although normal (or

slightly increased) levels of UCB provides protection against injury resulting from oxidation

(Doré et al., 2000), elevated UCB concentrations cause nerve cell damage, leading to

adverse neurological outcomes (Hansen, 2002), ranging from minor neurologic dysfunction

(Soorani-Lunsing et al., 2001) to chronic and permanent sequelae, or even death (Ostrow et

al., 2004, Shapiro, 2005). In addition, high levels of UCB are related with an augmented risk

for the appearance of long-term neurodevelopment disabilities (Dalman and Cullberg, 1999).

The risk of bilirubin-induced neurologic dysfunction is particularly enhanced in premature

newborns due to the higher rates of UCB production and the immaturity of the excretion

pathways (Stevenson et al., 2001, Watchko, 2006). Moreover, cerebral palsy is a common

condition in preterm infants at risk of kernicterus, in spite of the relatively low total serum

bilirubin levels (Gkoltsiou et al., 2008). However, little is known about mechanisms underlying

the increased vulnerability of selected regional neuronal populations in UCB-induced

neuronal damage. Hippocampus is one of the brain regions with preferential UCB

accumulation in severely jaundiced neonates who died with kernicterus, although cerebellum

and corpus striatum also showed selective yellow deposits (Ahdab-Barmada and Moossy,

1984, Hansen, 2000). This preferential deposition seems to be related with increased

vulnerability to hypoxic-ischemic injury (Perlman et al., 1997), as well as with the enhanced

vascularization and up-regulation of vascular endothelial growth factor (VEGF) that we have

recently observed in the hippocampus, cerebellum and striatum of a preterm infant with

sepsis who died with the diagnosis of kernicterus (personal communication Alexandra Brito,

2010).

In the last years, we demonstrated the involvement of oxidative stress in the

mechanisms underlying neuronal cell demise by clinically relevant concentrations of UCB

(Brito et al., 2004, Brito et al., 2008a, Brito et al., 2010). The UCB-induced dysfunction may

culminate in neuronal cell death (Silva et al., 2002, Falcão et al., 2006), which appears to be

mediated, at least in part, by perturbation of mitochondria (Rodrigues et al., 2002, Rodrigues

et al., 2000, Vaz et al., 2010). In addition, immature nerve cells were shown to be more

susceptible than more differentiated ones to UCB-induced toxicity (Falcão et al., 2006).

These findings provided a valuable contribute to the current understanding of the

neuropathological effects of UCB, as dysfunction and degeneration of neurons in several

neurological disorders are usually associated with reactive oxygen species (ROS) and/or

reactive nitrogen species (RNS) production (Mattson and Liu, 2002, Brito et al., 2007).


113

Recently, one of these oxidant species, nitric oxide (.NO), although important in cellular

signalling, showed to have a key role in the mechanisms of neurotoxicity by UCB, both in

mature (Brito et al., 2008a, Brito et al., 2010) and in immature neurons (personal

communication Ana Rita Vaz, 2010). RNS are also implicated in synapse injury (Sunico et

al., 2010) and early exposure to UCB provoked deleterious effects in neurogenesis,

neuritogenesis and synaptogenesis (Falcão et al., 2007, Fernandes et al., 2009), possibly

contributing to the development of mental illness in later life. Therefore, a better

understanding of the role of antioxidants and molecules involved in response to oxidative

stress represent important strategies to prevent neuronal injury by hyperbilirubinemia.

N-acetylcysteine (NAC) is a thiol compound that is converted to cysteine, an important

precursor of cellular glutathione (Zachwieja et al., 2005, Dringen, 2000). NAC is described by

its antioxidant effects in two ways: firstly, as a source of the cysteine aminoacid, it promotes

the biosynthesis of the tripeptide γ-l-glutamyl-l-cysteinylglycine, known as glutathione (GSH),

thus increasing GSH supply for glutathione peroxidise; secondly, as a source of sulfydryl

groups it promotes the reduction of ROS (Ocal et al., 2004). Several in vitro and in vivo

studies support the antioxidant effect of NAC. Treatment with NAC conferred neuroprotection

in lead-induced lipid peroxidation and in antioxidant enzyme activities deficiencies of rats’

brain (Nehru and Kanwar, 2004), as well as in hypoxia-induced oxidative stress in rat

cultured hippocampal neurons (Jayalakshmi et al., 2005). In addition, treatment with NAC

decreased lipid peroxidation in cerebral cortex, midbrain and cerebellum observed in

jaundiced rats (Karageorgos et al., 2006), and results from our group demonstrated that NAC

protects against UCB-induced protein oxidation in rat cultured cortical neurons (Brito et al.,

2008b).

DJ-1, also known as PARK7 [Parkinson disease (autosomal recessive, early onset) 7] is

a member of the peptidase C56 family of proteins, but is not known to exhibit proteolytic

activity. It functions as a redox-sensitive chaperone, like a sensor for oxidative stress,

protecting neurons against oxidative stress and cell death (Jin et al., 2005, Bonifati et al.,

2003). Recently, it was proposed that UCB-treated cells may undertake an adaptative

response that involves DJ-1 (Deganuto et al., 2010).

In this study, we investigated whether there is a dissimilar brain regional susceptibility to

UCB-induced oxidative damage and disruption of neurite outgrowth and branching in

immature neurons able to determine the selective pattern of UCB deposition and brain

damage in specific brain areas characteristic of kernicterus, such as cerebellum and

hippocampus. We also looked for potential mechanisms involved in UCB-induced

neurotoxicity modulation, namely DJ-1 protein expression and glutathione content.

Chapter IV __________________________________________________________________________

114


2.1. Chemicals Neurobasal medium, B-27 supplement (50X), Hanks’ balanced salt solution (HBSS),

Hanks’ balanced salt solution without Ca2+ and Mg2+ (Ca2+ and Mg2+ free HBSS), gentamicin

(50 mg/mL), and trypsin (2.5 g/L) were acquired from Invitrogen (Carlsbad, CA). Human

serum albumin (HSA) (fraction V, fatty acid free), NAC, dihydrorhodamine 123 (DHR 123),

3,8-diamino-5-(3-(diethyl-methylamino)propyl)-6-phenyl phenanthridinium diiodide,

sulfosalicyclic acid, and 2-vinylpyridine, Hoechst 33258 dye, 1-isobutyl-3-methylxanthine

(IBMX), primary monoclonal antibody mouse anti-β-actin, N-1-naphthylethylenediamine,

sulfanilamide and sodium nitrite were purchased from Sigma Chemical Co (St Louis, MO).

UCB was also from Sigma and purified as previously described (McDonagh 1979).

Nitrocellulose membrane, Hyperfilm ECL and horseradish peroxidase-labelled sheep anti-

mouse IgG were from Amersham Biosciences (Piscataway, NJ, USA). Cell lysis buffer,

LumiGLO® and antibody directed to caspase-3 were acquired from Cell Signalling (Beverly,

MA, USA).Cyclic guanosine monophosphate (cGMP) determination kit was from Enzo Life

Sciences (Plymouth Meeting, PA, USA). Antibodies directed to DJ-1 and microtubule

associated protein (MAP)-2 were from Chemicon (Temecula, CA, USA) while antibody

against neuronal nitric oxide synthase (nNOS) was from Becton Dickinson Biosciences (San

José, CA, USA).


Animal care followed the recommendations of European Convention for the Protection of

Vertebrate Animals Used for Experimental and other Scientific Purposes (Council Directive

86/609/EEC) and National Law 1005/92 (rules for protection of experimental animals).

Cortical, hippocampal and cerebellar neurons were isolated from fetuses of 16-17-day

pregnant Wistar rats, as previously described (Silva et al., 2002). Fetuses were collected in

HBSS medium, brain cortices, hippocampi and cerebella were mechanically fragmented, and

the fragments transferred to a 0.5 g/L trypsin in Ca2+ and Mg2+ free HBSS medium and

incubated for 15 min at 37ºC. After trypsinization, cells were washed twice in Ca2+ and Mg2+

free HBSS medium containing 10% fetal bovine serum, and resuspended in Neurobasal

medium supplemented with 0.5 mM L-glutamine, 25 μM L-glutamic acid, 2% B-27

supplement, and 0.12 mg/mL gentamicin. Finally, cells were seeded on poly-D-lysine coated

tissue culture plates at a density of 2 x 105 cells/cm2 and maintained at 37ºC in a humidified

atmosphere of 5% CO2. For hippocampal neurons, cells were first seeded in plating medium

(MEM with Earle’s salts supplemented with 10 mM HEPES, 10 mM sodium pyruvate, 1 mM


115

glutamine, 12.5 μM glutamate, 10% FBS and 0.6% glucose) and after 2 h, the media was

replaced with neuronal growth medium as abovementioned. In this work we used neurons at

3 days in vitro (DIV).

2.3. Treatment of neurons

Neurons from cortex, hippocampus and cerebellum were incubated in Neurobasal

medium without (control) or with 50 μM UCB in the presence of 100 μM HSA from 1 to 24 h,

at 37ºC. Stock UCB solutions were extemporarily prepared in 0.1 M NaOH under the dark

and the pH adjusted to 7.4 using 0.1 M HCl. In parallel studies, cells were incubated with

100 µM NAC, a precursor of glutathione synthesis, for 1 h prior to UCB addition.

2.4. Quantification of nitrite levels Nitric oxide levels were estimated by measuring the concentrations of nitrites (NO2

-),

which are the resulting .NO metabolites. Briefly, supernatants free from cellular debris were

mixed with Griess reagent [1 part 1% (w/v) sulfanilamide in 5% H3PO4, 1 part 0.1% (w/v) N-1-

naphthylethylenediamine (v/v)] in 96-well tissue culture plates for 10 min at room

temperature in the dark. The absorbance at 540 nm was determined using a microplate

reader (Bio-Rad Laboratories, Hercules, CA, USA).

2.5. Western blot assay The intracellular forms of nNOS, caspase-3 and DJ-1 were determined by Western blot

analysis as usual in our laboratory (Fernandes et al., 2006). Briefly, cells were washed in ice-

cold PBS and lysed in a buffer containing 20 mM Tris-HCl (pH 7.5); 150 mM NaCl; 1 mM

Na2EDTA; 1 mM EGTA; 1% (v/v) Triton X-100; 2.5 mM sodium pyrophosphate; 1 mM β-

glycerophosphate; 1 mM Na3VO4; 1 μg/mL leupeptine and 1 mM PMSF. The lysate was

sonicated for 20 s, centrifuged at 14000 g for 10 min at 4ºC and the supernatants were

collected and stored at -80ºC. Protein concentrations were determined according to the

Bradford method (Bradford, 1976) using Bio-Rad’s Protein Assay reagent (Bio-Rad, CA,

USA). Equal amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide

gel electrophoresis and transferred to a nitrocellulose membrane. After blocking with 5% milk

solution, membranes were incubated with the primary antibody overnight at 4ºC [mouse anti-

nNOS (1:2500), rabbit anti-caspase-3 (1: 1000), rabbit anti-DJ1 (1:200) or anti-β-actin

(1:5000)], and finally with horseradish peroxidase-labelled secondary antibody. Protein

bands were detected by LumiGLO® and visualized by autoradiography with Hyperfilm ECL.

Chapter IV __________________________________________________________________________

116

2.6. Determination of cGMP concentration:

For quantification of cGMP content, the phosphodiesterase inhibitor IBMX was included

in the incubation medium. Cell extracts collected from 9.6 cm2 wells were used for cGMP

determination using a commercially available kit from Enzo Life Sciences and measurements

were performed according to manufacturer’s instructions.

2.7. Glutathione measurement After incubation period, neurons were washed with ice-cold PBS and immediately

collected by scrapping off with 0.5 mL of 1% (w/v) sulfosalicyclic acid. Cell lysates were

centrifuged at 13,000 g for 5 min at 4ºC, and the supernatants used for glutathione

determinations. Total glutathione content (GSt, i.e. the amount of GSH plus two times the

amount of GSSG) and oxidized glutathione (GSSG) were measured and calculated as

previously described (Dringen and Hamprecht, 1996) and GSt and GSSG concentrations

were expressed as nanomoles per milligram of protein.

2.8. Assessment of ROS formation The nonfluorescent DHR 123 easily crosses cell membranes due to its lipophilicity and is

converted by ROS into rhodamine 123, a fluorescent compound that accumulates in

mitochondria and is considered as a sensitive indicator of ROS production in cell systems

(Gomes et al., 2005). To evaluate the production of ROS in neuronal cultures, cells were

seeded on glass coverslips placed in the 12-well culture plates. Cells were loaded, under

light protection, with 6 μM DHR 123 for 30 min at 37 °C, prior to cellular treatment. At the end

of the incubation period, cells were fixed with freshly prepared 4% paraformaldehyde in PBS,

and the nuclei immunostained with Hoechst 33258 dye. Cellular fluorescence was observed

using a fluorescence microscope (Axioskope®, Zeiss, Germany) and the intensity of the

fluorescence emission was quantified in at least six microscopic fields (×400) per sample

with an image analyzer software (ImageJ 1.29×, National Institutes of Health, USA) and

expressed as a percentage per total number of cells. Since UCB was referred as an

autofluorescent molecule (Özkan et al., 1995), a set of experiments was performed in

parallel, with no addition of DHR 123. The fact that no variations in the fluorescence intensity

were noticed in these control experiments guarantees that the rise in the fluorescence

intensity observed in the UCB-treated samples was due to ROS formation and not to UCB

interference.


117

2.9. Evaluation of cell death Necrotic-like cell death was assessed by monitoring the cellular uptake of the fluorescent

dye propidium iodide (PI). PI readily enters and stains non-viable cells, but cannot cross the

membrane of viable cells. This dye binds to double-stranded DNA and emits red

fluorescence (630 nm; absorbance 493 nm). Unpermeabilized adherent cells cultured on

coverslips were incubated with a 75 μM PI solution for 15 min in the absence of light.

Subsequently, cells were fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS and

the nuclei immunostained with Hoechst 33258 dye. Red-fluorescence and U.V. images of six

random microscopic fields (original magnification: 400×) were acquired per sample by using

a fluorescence microscope (Axioskope®, Zeiss, Germany) and the percentage of PI positive

cells was counted and expressed as a percentage per total number of cells.

2.10. Neurite Extension and Ramification For immunofluorescence detection of the cytoskeletal protein MAP-2, known to be

located mainly in dendrites and widely used as a neuritic marker (Hammond, 2001), cells

were fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS and a standard indirect

immunocytochemical technique was carried out using a mouse anti-MAP-2 antibody (1:100)

as the primary antibody and a horse FITC-labeled anti-mouse antibody (1:227) as the

secondary antibody. Fluorescence was visualized by using a fluorescence microscope

(Axioskope®, Zeiss, Germany). Green-fluorescence images of ten random microscopic fields

were acquired per sample. Evaluation of neurite extension and number of nodes from

individual neurons were determined using HCA-Vision Neurite Analysis software (Australia).

2.11. Densitometry and statistical analysis The relative intensities of protein and nucleic acid bands were analysed using the

Quantity One (version 4.6) program (Bio-Rad, CA, USA). Results of, at least, three different

experiments were expressed as mean ± SEM. Significant differences between two groups

were determined by the two-tailed t-test performed on the basis of equal and unequal

variance as appropriate. Comparison of more than two groups was done by ANOVA using

Instat 3.05 (GraphPad Software, San Diego, CA, USA), followed by multiple comparisons

Bonferroni post-hoc correction. Mean values were considered statistically significant when P

values were lower than 0.05.

Chapter IV __________________________________________________________________________

118

3. Results

3.1. UCB-induced nNOS expression and production of nitrites and cGMP is enhanced in immature hippocampal neurons as compared to cerebellar or cortical neurons To investigate whether different brain regions present particular susceptibilities to UCB-

induced neuronal damage, we used 3 DIVs neurons isolated from cortex, hippocampus and

cerebellum. Neuronal cells were incubated with 50 μM UCB plus 100 μM HSA, to produce a

free UCB concentration of ~100 nM (Ostrow et al., 2003, Weisiger et al., 2001), that mimics

moderate to severe neonatal jaundice, a condition already shown to induce oxidative stress

and cell death in immature cortical neurons (Vaz et al., 2010). We started by investigating

whether UCB up-regulates nNOS expression, as well as production of nitrites and cGMP, the

resulting product of soluble guanylate cyclase stimulation by .NO (Knowles et al., 1989), in

neurons exposed to UCB for 1 to 24 h. As shown in Figure IV.1A, UCB rapidly (at 1 h)

induced nNOS expression in cortical and hippocampal neurons, but not in cerebellar

neurons. The effect was already pronounced after 4 h of treatment (p<0.01 vs. respective

controls) and decreased at 24 h. Furthermore, we observed a raise in the production of

nitrites (Fig. IV.1B), as well as cGMP (Fig. IV.1C), after 1 h of treatment with UCB, in neurons

from all brain regions (p<0.01 for nitrites and p<0.05 for cGMP vs. respective controls). To

note that hippocampal neurons, namely at 4 h of incubation with UCB, showed to be the

most sensitive regarding nNOS expression (1.4- vs. 1.2- and 1.0- fold in cortical and

cerebellar neurons p<0.01), nitrite production (3.8- vs. 2.2-fold in cortical and cerebellum

neurons, p<0.01) and cGMP content (1.7- vs. 1.3-fold, in cortical and cerebellar neurons,

p<0.01 and p<0.05, respectively).

3.2. UCB-induced oxidative stress is highest in immature hippocampal neurons, probably as a result of the lowest levels of total glutathione Having observed that RNS are produced by immature neurons exposed to UCB, we

considered relevant to look for alterations in the oxidative status of neuronal cells from the

three mentioned brain regions. Firstly, we determined whether UCB induced mitochondrial

ROS production by evaluation of fluorescence intensity of rhodamine 123.

As shown in Figure IV.2A-B, UCB induced oxidative stress more markedly after 4 h

treatment in immature hippocampal and cerebellar neurons (p<0.01 vs. respective controls)

by cortical cells (p<0.05 vs. respective control).


119

Figure IV.1 - Unconjugated bilirubin (UCB)-induced neuronal nitric oxide synthase (nNOS) expression and production of nitrites and cyclic GMP (cGMP) is enhanced in immature hippocampal neurons as compared to cerebellar or cortical neurons. Primary neuron cultures from rat cortex, hippocampus and cerebellum, at 3 days in vitro, were incubated with either no addition (control) or with UCB (50 µM), in the presence of human serum albumin (100 μM), from 1 to 24 h at 37ºC. It was determined nNOS expression by western blot analysis (A), nitrites by Griess reagent (B) and cyclic GMP by a colorimetric kit (C). Results are mean (± SEM) from at least three independent experiments performed in duplicate. *p<0.05, **p<0.01 vs. respective control; $p<0.05, $$p<0.01 vs. cortical neurons; #p<0.05, ##p<0.01 vs. cerebellar neurons.

C

1h 4h 24hCortical neurons

1h 4h 24hCerebellarneurons

Hippocampalneurons

1h 4h 24h

Control

UCB 50 μM

0

1

2

3

4

Nitr

ites

(μM

)

0.0

0.5

1.0

1.5

nNO

S (f

old

vs.c

ontro

l)


Control

UCB 50 μM


Hippocampalneurons

1h 4h 24h

A



Hippocampalneurons

1h 4h 24h

Control

UCB 50 μM

B

***

** ****

$##

$$##

##

**

** **

**

**

$$##

$$##

##** **

* **

**

***$$#

*

*

0

5

10

15

20

cGM

P (p

mol

/mg

prot

ein)

Chapter IV __________________________________________________________________________

120

Figure IV.2 - Unconjugated bilirubin (UCB)-induced oxidative stress is highest in immature hippocampal neurons, as compared to the other brain regions. Primary neuron cultures from rat cortex, hippocampus and cerebellum, at 3 days in vitro, were treated as in Figure IV.1. Reactive oxygen species (ROS) content was evaluated by measuring fluorescence intensity of the cells (A) and expressed in arbitrary units (A.U.), resulting from fluorescence intensity per total number of cells (B). Scale bar represents 40 μm. Results are mean (± SEM) from at least three independent experiments performed in duplicate. *p<0.05, **p<0.01 vs. respective control; $$p<0.01 vs. cortical neurons; ##p<0.01 vs. cerebellar neurons.

Secondly, we measured glutathione content of the UCB-treated neurons. We observed

that UCB markedly disrupted glutathione homeostasis, as indicated in Fig. IV.3A by an

increase in GSSG/GSt ratio, mostly in hippocampal neurons after 4 h of treatment (p<0.01)

but also markedly either in cortical or cerebellar neurons (p<0.05). Therefore, we may

assume that hippocampal neurons were again the most reactive to UCB in terms of ROS

ACortical neurons Cerebellar neurons

Con

trol

UC

B 50

μM

Hippocampal neurons

0

20

40

60

RO

S p

rodu

ctio

n (A

.U.)

BControl

UCB 50 μM



Hippocampalneurons

1h 4h 24h

*

**$$##

*


121

production (2- vs. 1.4- and 1.7- fold in cortical and cerebellar neurons, respectively, p<0.01),

and disruption of glutathione homeostasis (5.3- vs. 2.3- and 2.1-fold in cortical and cerebellar

neurons, respectively, p<0.01), probably because they show, as indicated in Figure IV.3B,

the lowest levels of total glutathione in the absence of UCB (p<0.01 vs. cortical neurons and

p<0.05 vs. cerebellar neurons), whereas no significant changes were produced by UCB (data

not shown).

Figure IV.3 - Unconjugated bilirubin (UCB)-induced disruption of glutathione metabolism is particularly evident in immature hippocampal neurons, whose vulnerability appears to be determined by the lowest total glutathione levels relatively to the other brain regions. Primary neuron cultures from rat cortex, hippocampus and cerebellum, at 3 days in vitro, were treated as in Figure IV.1. Total (GSt) and oxidized (GSSG) glutathione were determined by an enzymatic assay and expressed as GSSG/GSx ratio (A) and as nmol/mg protein in the case of GSt (B). Results are mean (± SEM) from at least three independent experiments performed in duplicate. *p<0.05, **p<0.01 vs. respective control; $$p<0.01 vs. cortical neurons; #p<0.05, ##p<0.01 vs. cerebellar neurons.

3.3. UCB-induced neuronal death is higher in immature cells from hippocampus than in those from cortex or cerebellum We kept on determining whether UCB-induced oxidative stress courses in parallel with

cell death, by evaluating the necrotic-like cell death using PI uptake as an indicator of

membrane integrity and cell damage. We have also determined the possible involvement of

the apoptotic pathways by evaluating the relative levels of active form of caspase-3, an

effector caspase of apoptotic cascade (Fink and Cookson, 2005). As demonstrated in Figure

IV.4 A-B, UCB increased cellular uptake of PI in immature neurons, mainly after 4 h of

exposure (p<0.05 vs. respective control in cortical and cerebellar neurons and p<0.01 in

hippocampal ones). In Figure IV.4C, we verified an increase in the active form of caspase-3,

0

10

20

30

40

GS

t (nm

ol/m

g pr

otei

n)

Control



Hippocampalneurons

1h 4h 24h

$$

#

$$

#

BA

0

10

20

30

40

GS

SG

/GS

t x

100

Control

UCB 50 μM



Hippocampalneurons

1h 4h 24h

*

**$$

##

**

**

**

*

Chapter IV __________________________________________________________________________

122

also with peak levels at 4 h of treatment (p<0.01 vs. respective controls). Once more,

hippocampal neurons showed to be most sensitive to UCB, revealing higher necrosis (2.4-

vs. 1.5- and 1.7-fold in cortical and cerebellar neurons, p<0.05) and apoptosis (1.3- vs. 1.2-

fold in cortical and cerebellar neurons).

Figure IV.4 - Unconjugated bilirubin (UCB)-induced cell induced neuronal death is higher in cells from hippocampus than those from cortex or cerebellum. Primary neuron cultures from rat cortex, hippocampus and cerebellum, at 3 days in vitro, were treated as in Figure IV.1. Necrotic-like cell death was assessed by monitoring the cellular uptake of the fluorescent red dye propidium iodide (PI). PI+ cells are shown in pink, resulting from co-localization with nuclear staining with Hoechst 33258 dye, in blue (A) and expressed as a percentage per total number of cells (B). Scale bar represents 40 μm. Caspase-3 expression was determined by western blot analysis (C). Results are mean (± SEM) from at least three independent experiments performed in duplicate. *p<0.05, **p<0.01 vs. respective control; $p<0.05, $$p<0.01 vs. cortical neurons; #p<0.05, ##p<0.01 vs. cerebellar neurons.

0.0

0.5

1.0

1.5

Cas

pase

-3 (f

old

vs.c

ontro

l)

ACortical neurons Cerebellar neurons

Con

trol

UC

B 50

μM

Hippocampalneurons

Control

UCB 50 μM



Hippocampalneurons

1h 4h 24h

BControl

UCB 50 μM



Hippocampalneurons

1h 4h 24h

C

0

5

10

15

20

PI+

cells

(%)

**

****

*

$$##

**

#$**

**


123

3.4. UCB-induced neuronal oxidative stress and cell death in immature neurons is prevented by NAC To determine whether oxidative stress is related to UCB-induced neuronal death, we

evaluated ROS production, glutathione homeostasis and necrotic-like cell death in cells

treated with 100 µM NAC for 1 h, followed by 4 h incubation with UCB or not. NAC is a

cysteine donor, thus it promotes the synthesis of glutathione (Dringen, 2000, Zachwieja et

al., 2005). The concentration of this molecule was chosen based on our previous data

demonstrating that NAC prevents UCB-induced protein oxidation in 8 DIVs neurons (Brito et

al., 2008b). As stated in Table IV.1, pre-incubation with NAC significantly decreased ROS

production (p<0.05 vs. UCB-treated cortical neurons and p<0.01 vs. UCB-treated cerebellar

and hippocampal neurons), GSSG/GSt ratio (p<0.05 vs. UCB-treated cortical and cerebellar

neurons and p<0.01 vs. UCB-treated hippocampal neurons) and PI+ cells (p<0.05 vs. UCB-

treated cortical and hippocampal neurons and p<0.01 vs. UCB-treated cerebellar neurons).

Together, these results indicate that oxidative stress is involved in neuronal death by UCB.

3.5. UCB regulates DJ-1 protein expression in immature neurons, mainly in those from hippocampus, which is reverted by NAC There are several proteins that are differently expressed in response to toxic stimulus.

DJ-1, a protein involved in Parkinson ’s disease pathogenesis (Bonifati et al., 2003), is

particularly important as a redox-sensitive chaperone (Shendelman et al., 2004), protecting

neurons from oxidative stress and cell death (Lev et al., 2008, Lev et al., 2009), thus,

representing a putative protein for an adaptative response against UCB-induced oxidative

stress and neurotoxicity. Therefore, we evaluated DJ-1 protein expression in immature

neurons from the three regions, following incubation with UCB, in the absence or presence of

NAC. As observed in Figure IV.5A,C, 4 h treatment with UCB led to an increased expression

of DJ-1 in cortical and hippocampal neurons (respectively, p<0.05 and p<0.01 vs. respective

control), suggesting that DJ-1 protein expression is up-regulated, as an attempt to diminish

UCB-induced oxidative stress. However, after 24 h incubation, there is a significant decrease

of DJ1 expression in neurons from the three regions (p<0.05 vs. respective control for

cortical and cerebellar neurons and p<0.01 vs. respective control from hippocampal

neurons), suggesting that this effort to prevent oxidative stress is transient as it fails after

longer periods of neuronal exposure to UCB. Accordingly, with the abovementioned data,

hippocampal neurons are the most affected ones (36% reduction vs. 15% and 9% in cortical

and cerebellar neurons, respectively, p<0.01). In addition, this up or down-regulation of DJ-1

Chapter IV __________________________________________________________________________

124

is suppressed by NAC (Fig. IV.5B, D). These data support the notion that UCB-induced

oxidative stress mediates, at least in part, DJ-1 up-regulation.

Table IV.1. Oxidative stress and cell death by UCB is higher in immature hippocampal neurons than in cells from cortex and cerebellum, and is partially prevented by NAC.

Primary neuron cultures from rat cortex, hippocampus and cerebellum at, 3 days in vitro, were incubated with 50 µM unconjugated bilirubin (UCB), in the presence of 100 μM human serum albumin, for 24 h at 37ºC. In parallel experiments, cells were incubated with 100 mM N-acetyl cysteine (NAC), a precursor of glutathione synthesis for 1 h, prior to UCB addition. Reactive oxygen species (ROS) content was evaluated by measuring fluorescence intensity of the cells, total (GSt) and oxidized (GSSG) glutathione were determined by an enzymatic assay and necrotic-like cell death was assessed by monitoring the cellular uptake of the fluorescent red dye propidium iodide (PI). Results are expressed as fold change over respective control and are mean (± SEM) from at least three independent experiments performed in duplicate. *p<0.05, **p<0.01 vs. respective control; $p<0.05, $$p<0.01 vs. cortical neurons; ##p<0.01 vs. cerebellar neurons; &p<0.05, &&p<0.01 vs. respective condition without NAC.

Control UCB 50 μM NAC 100 μMUCB 50 μM

+ NAC 100 μM

Cortical neurons 1.00 ± 0.07 1.45 ± 0.10 0.99 ± 0.17 0.95 ± 0.14

ROS production Cerebellar

neurons 1.00 ± 0.14 1.68 ± 0.18 0.58 ± 0.08 0.75 ± 0.02

Hippocampal neurons 1.00 ± 0.24 2.01 ± 0.27 0.58 ± 0.03 0.96 ± 0.03


GSSG/GSt Cerebellar neurons 1.00 ± 0.10 2.13 ± 0.14 1.00 ± 0.04 0.82 ± 0.11



PI+ cells Cerebellar neurons 1.00 ± 0.15 1.66 ± 0.26 0.56 ± 0.10 0.62 ± 0.12


&

&&

&&

&

&

&&

&

&

&&

*

*

**$$##

**$$##

*

*

**

##$

*

*


125

Figure IV.5 - Early up-regulation of DJ-1 protein expression by unconjugated bilirubin (UCB) is reverted for longer periods of incubation, and suppressed in the presence of N-acetyl cysteine (NAC), namely in immature neurons from hippocampus. Primary neuron cultures from rat cortex, hippocampus and cerebellum at, 3 days in vitro, were incubated with UCB (50 µM), in the presence of human serum albumin (100 μM), from 1 to 24 h at 37ºC (A). In parallel experiments, cells were incubated with 100 μM NAC, a precursor of glutathione synthesis for 1 h, prior to UCB addition (B). DJ-1 expression was determined by western blot analysis. Representative results for 24 h incubation with UCB are indicated in the absence (C) or in the presence (D) of NAC. Results are mean (± SEM) from at least three independent experiments performed in duplicate. *p<0.05, **p<0.01 vs. respective control; $$p<0.01 vs. cortical neurons; ##p<0.01 vs. cerebellar neurons; &&p<0.01 vs. respective condition without NAC.

3.6. UCB-induced reduction of neurite outgrowth and branching mainly in immature neurons from hippocampus, is closely followed by those from cerebellar and cortical regions, and is prevented by NAC .NO showed to be involved in cell death, as well as in impairment of neurite ramification

and extension of immature cortical neurons after exposure to UCB, as we recently

demonstrated (personal communication Ana Rita Vaz, 2010; personal communication

Sandra L Silva, 2010, respectively). To investigate if oxidative stress affects neuritic

arborization, we assessed the neuronal network dynamics in cells non-treated or treated with

UCB in the presence or absence of NAC. We selected the longest time of incubation (24 h),

based on our previous reports showing UCB-induced neurite impairment in immature cortical

0.0

0.5

1.0

1.5

DJ-

1 (f

old

vs.c

ontro

l)


Control

UCB 50 μM

1h 4h 24hCerebellar neurons

Hippocampal neurons

1h 4h 24h

##$$**

* *

*

##$$**

A B

0.0

0.5

1.0

1.5

DJ-

1 (f

old

vs.c

ontro

l)


1h 4h 24hCerebellar neurons

Hippocampal neurons

1h 4h 24h

Control

UCB 50 μM

&& && &&

&&

- 22 KDaDJ-1 -

β-actin - - 42 KDa

UCB (50 μM) - - -+ + +Cortical neurons

Cerebellar neurons

Hippocampal neurons

- 22 KDaDJ-1 -

β-actin - - 42 KDa

UCB (50 μM) - - -+ + +Cortical neurons

Cerebellar neurons

Hippocampal neurons

C D

Without N-acetylcysteine With N-acetylcysteine

Chapter IV __________________________________________________________________________

126

neurons after 24 h treatment (Falcão et al., 2007) and because the decreased expression of

DJ-1 was found at 24 h incubation. As shown in Figure IV.6, UCB led to a decrease in

neurite extension and number of nodes in immature neurons from hippocampus and

cerebellum (p<0.01 vs. respective control) followed by those from cortex (p<0.05 vs.

respective control). In accordance with all the other results, neurite impairment by UCB was

higher in hippocampal neurons, both in total neurite output (42% reduction vs. 19% and 32%

in cortical and cerebellar neurons) and in number of branch points (38% reduction vs. 27%

and 29% in cortical and cerebellar neurons). Interestingly, pre-incubation with NAC

significantly prevented UCB-diminished neurite outgrowth (p<0.05), suggesting that

glutathione content may account to the resistance against UCB-induced neurite network

disruption. Therefore, different susceptibilities between brain regions may be partially due to

distinct levels of antioxidants.

4. Discussion In previous reports we demonstrated that UCB-induced neurotoxicity differs from nerve

cell type and maturation state, being cortical neurons more susceptible than cortical

astrocytes (Brito et al., 2008b, Falcão et al., 2006) and immature cells (3 DIVs) more

vulnerable than more differentiated (8 DIVs) ones (Falcão et al., 2005, Falcão et al., 2006).

Furthermore, oxidative stress and disruption of neuronal network dynamics were indicated as

pathological hallmarks in UCB-induced neurotoxicity (Brito et al., 2008a, Falcão et al., 2007,

Vaz et al., 2010, Fernandes et al., 2009). Based on these data, here we investigated for the

first time whether brain UCB specific pattern toxicity is determined by differential regional

susceptibility to UCB-induced oxidative stress and disruption of neurite arborization in

immature neurons, due to the increased vulnerability of premature babies. We also looked

for potential defence mechanisms as that of DJ-1 protein expression and glutathione content

as modulators of UCB-induced neurotoxicity.

In this study, we demonstrate that UCB leads to nitrosative and oxidative stress, as we

observed nNOS increased protein expression, production of nitrites and cGMP, as well as an

increase in the levels of ROS and of oxidized glutathione in immature neurons. Interestingly,

in spite of these biomarkers of oxidative stress are observed in immature neurons isolated

from all the three regions studied (cortex, cerebellum and hippocampus), they are mainly

detected in cells from hippocampus. For this differential vulnerability to UCB within brain

regions may account distinct responses at the cellular or biochemical processes, such as

antioxidant defences. In fact, total glutathione levels were markedly lower in immature

neurons from hippocampus, compared with those from cortex and cerebellum, as

demonstrated in Figure IV.3B.


127

Figure IV.6 - Unconjugated bilirubin (UCB)-induced reduction of neurite outgrowth and branching in immature neurons from hippocampus is reduced at cerebellar and cortical levels, and is partly prevented by N-acetyl cysteine (NAC). Primary neuron cultures from rat cortex, hippocampus and cerebellum, at 3 days in vitro, were incubated with UCB (50 µM), in the presence of human serum albumin (100 μM), for 24 h at 37ºC. In parallel experiments, cells were incubated with 100 μM NAC, a precursor of glutathione synthesis for 1 h, prior to UCB addition. Neurite extension and number of nodes were evaluated by immunocytochemistry with MAP-2 labeling (A) as indicated in Methods. Scale bar 40 μM. Total neurite output (B) and number of branch points (C) were quantified in HCA-Vision Neurite Analysis software. Results are mean (± SEM) from at least three independent experiments performed in duplicate. *p<0.05, **p<0.01 vs. respective control; $$p<0.01 vs. cortical neurons; &p<0.05, &&p<0.01 vs. respective condition without NAC.

0

50

100

150

200

250

300

Tota

l neu

rite

outp

ut (m

icro

ns)

Control

UCB 50 μM

B

Cortical neurons

Cerebellarneurons

Hippocampalneurons

Cortical neurons

Cerebellarneurons

Hippocampalneurons

NAC 100 μM

Control

UCB 50 μM

C

Cortical neurons

Cerebellarneurons

Hippocampalneurons

Cortical neurons

Cerebellarneurons

Hippocampalneurons

NAC 100 μM

A Control UCB 50 μM NAC 100 μM UCB 50 μM + NAC 100 μM

Cor

tical

neu

rons

Cer

ebel

larn

euro

nsH

ippo

cam

paln

euro

ns

0

2

4

6

8

10

12

14

Num

ber o

f bra

nch

poin

ts

&

&& &&

&&

&

*

** $$

***

* **

Chapter IV __________________________________________________________________________

128

Supporting this concept, it was reported that antioxidant enzymes, such as xantine

oxidase and catalase, have maximum activity in cortex, followed by cerebellum and

hippocampus in developing mouse brain exposed to lead (Prasanthi et al., 2010).

Furthermore, primary astroglial cultures isolated from cortex, striatum, or hippocampus

revealed distinct profiles of vulnerability when subjected to injury. While astrocytes from

striatum showed increased injury by oxygen and glucose deprivation, they were more

resistant to an oxidative insult resultant from exposure to H2O2 since they have higher levels

of antioxidant defences, such as glutathione levels and glutathione peroxidase and

superoxide dismutase activities (Xu et al., 2001). Interestingly, glutathione peroxidase activity

was considered determinant in the recovery of the immature mouse brain subjected to

traumatic brain injury (Tsuru-Aoyagi et al., 2009). Taking these into account, together with

the preventive effects of NAC on UCB-induced neurotoxicity, we hypothesize that

hippocampal vulnerability to UCB is, at least in part, due to the lower content of the

antioxidant molecule glutathione.

For the differential susceptibilities to UCB neurotoxicity in the different brain areas may

also account the UCB specific affinities, after crossing the blood brain barrier. Hippocampus

is one of the brain regions stained by UCB in severely jaundiced neonates who died with

kernicterus (Ahdab-Barmada and Moossy, 1984, Hansen, 2000). The selective bilirubin

deposition in cases of kernicterus seems to take into account the areas more vulnerable to

hypoxic ischemic injury, such as the pyramidal cell layer of the hippocampus (Perlman et al.

1997), which raises the question of whether hypoxic-ischemic injury is important to the

development of the lesions by kernicterus. In addition, ischemic injury is also usually

associated with an increased production of the VEGF, which was recently detected in

hippocampal neurons from a kernicteric patient (personal communication Alexandra Brito,

2010). It is known that hippocampus is particularly affected by hypoxic-ischemic insult in

animal models of both mature and immature brain (Guzzetta et al., 2000, Jiang et al., 2004).

To this feature may account the distribution of immature NMDA receptors, which

corresponds to regions that preferentially express nNOS (Black et al., 1995, Greenamyre et

al., 1987, Mitani et al., 1998). Interestingly, hippocampal neurons were the main responders

to UCB in terms of nNOS protein expression and activation (increased concentrations of

nitrates and cGMP). Inhibition of mitochondrial cytochrome c oxidase observed in immature

neurons exposed to UCB (Vaz et al., 2010) is surely one of the causes. Accordingly, UCB

was shown to promote cytochrome c release in isolated mitochondria from the brain and liver

of adult male Wistar rats (Rodrigues et al., 2002, Rodrigues et al., 2000). Recently, our group

demonstrated that .NO signalling mediates, at least in part, UCB-induced neuronal injury,

both in mature (Brito et al., 2010) and in immature neurons (personal communication Ana


129

Rita Vaz, 2010), and also that inhibition of nNOS prevented glutathione impairment (Brito et

al., 2010). Additionally, neuronal damage mediated by nNOS was noticed in hypoxia-

ischemia (Ferriero et al., 1996, Ferriero et al., 1995) and its inhibition confered tissue

protection and reduction of caspase-3 activation (Peeters-Scholte et al., 2002, Zhu et al.,

2004). All these findings are reinforced by data demonstrating that .NO and NOS mediate

apoptosis during inflammation in neuronal cells (Hemmer et al., 2001, Heneka et al., 1998,

Thomas et al., 2008), indicating that .NO is a player in the developing brain cytotoxicity.

Besides antioxidant defences, regional differences in the Ca2+-induced mitochondrial

permeability transition (Friberg et al., 1999) and DNA damage (Cardozo-Pelaez et al., 2000)

were reported between hippocampus, cortex, and striatum, which are also signs of increased

pro-oxidant activity. Accordingly, our results demonstrated that intracellular production of

ROS from those specific brain regions is a result of UCB interaction. Protein oxidation, lipid

peroxidation, alterations in glutathione stores, decreased NADPH concentration and

superoxide anion radical production are also additional features produced by UCB in cortical

neurons (Brito et al., 2008b, Brito et al., 2010, Vaz et al., 2010).

Here, we evidenced that UCB-induced oxidative and nitrosative stress occur in parallel

with necrotic and apoptotic cell death, as demonstrated by increased levels of PI uptake and

caspase-3 activation, thus participating in the mechanisms of UCB-induced neurotoxicity.

Accordingly, experimental obstructive jaundice was shown to be correlated with oxidative

stress in rats' brain (Chroni et al., 2006). This surely accounts for the preventive effect of

NAC on UCB-induced oxidative stress and necrotic cell death. This is not without precedent

since NAC treatment was shown to be neuroprotective in lead-induced lipid peroxidation and

in antioxidant enzyme activities deficiencies of rats’ brain (Nehru and Kanwar, 2004), as well

as during hypoxia in cultures of rats' hippocampal neurons (Jayalakshmi et al., 2005).

Furthermore, results from our group demonstrated that NAC treatment protects from UCB-

induced protein oxidation (Brito et al., 2008b).

We extended our studies to the evaluation of the expression of DJ-1, a protein involved

in Parkinson’s disease pathogenesis (Bonifati et al., 2003), with particular relevance in

neuroprotection against oxidative stress and cell death (Lev et al., 2009, Lev et al., 2008).

Our data indicate that DJ-1 protein expression is transiently up-regulated suggesting an

intervening role to diminish acute UCB-induced oxidative in hippocampal neurons. Recently

DJ-1 was indicated as a protective factor against UCB-induced oxidative stress in human

neuroblastoma SH-SY5Y cell line (Deganuto et al., 2010). DJ-1 was also shown to negatively

regulate N-methyl-d-aspartate receptor (NMDAR) function and suppression of this protein led

to NMDAR-induced cell death (Chang et al., 2010). This is of particular interest, since UCB-

Chapter IV __________________________________________________________________________

130

induced neurotoxicity is mediated by glutamate receptors in cultured rat brain neurons

(Grojean et al., 2000, Grojean et al., 2001, Brito et al., 2010). Accumulation of extracellular

glutamate, secreted by UCB-exposed neurons (Brito et al., 2010, Falcão et al., 2006), and

glial cells, namely by microglia and immature astrocytes (Fernandes et al., 2004, Falcão et

al., 2005, Gordo et al., 2006) is a dominant cell response. Nevertheless, persistent

hyperbilirubinemia seems to down-regulated DJ-1, notoriously in hippocampus, what can

contribute for the deleterious effects of the condition. Most important, this effect was reverted

by NAC.

It was reported by our group that UCB impairs of neurite extension and ramification, in

immature cortical neurons, from which cells do not recover along the differentiation (Falcão

et al., 2007). UCB also showed to interfere with the development of hippocampal neurons,

reducing dendritic and axonal elongation and branching, axonal growth cones and number of

dendritic spines and synapses (Fernandes et al., 2009). This UCB-elicited impairment in

neuritic outgrowth was demonstrated to be mediated by .NO and overstimulation of NMDA

receptors and, thus, prevented by the use of inhibitors, even during nerve cell maturation

(personal communication Sandra L Silva, 2010). Interestingly, here we demonstrate that

hippocampus is the most affected in neurite impairment by UCB, what raises the possibility

that neonatal jaundice could have an impact on the infant’s learning and memory.

Observations by Weir and Millar revealed that adverse effects for learning are observed in

severe cases of hyperbilirubinemia history (Weir and Millar, 1997).

In conclusion, the data obtained show that UCB, in conditions mimicking a moderate

hyperbilirubinemia in the early neonatal period, induces oxidative stress in different brain

regions, particularly in the hippocampus. Furthermore, these results provide specific features

that may explain this differential susceptibility along different brain areas to UCB exposure,

namely local differences in glutathione content and in DJ-1 expression, important molecules

involved in the regulation of oxidative damage. Moreover, here we suggest that UCB

neuronal exposure will differently affect neurite outgrowth and ramification in distinct cell

populations, pointing hippocampal neurons as preferential target to short and long-term

UCB-induced neuronal dysfunction.


131

5. References Ahdab-Barmada, M. and Moossy, J. (1984) The neuropathology of kernicterus in the


Black, S. M., Bedolli, M. A., Martinez, S., Bristow, J. D., Ferriero, D. M. and Soifer, S. J.

(1995) Expression of neuronal nitric oxide synthase corresponds to regions of selective

vulnerability to hypoxia-ischaemia in the developing rat brain. Neurobiol Dis, 2, 145-155.

Bonifati, V., Rizzu, P., van Baren, M. J. et al. (2003) Mutations in the DJ-1 gene associated

with autosomal recessive early-onset parkinsonism. Science, 299, 256-259.

Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-

254.

Brito, M. A., Brites, D. and Butterfield, D. A. (2004) A link between hyperbilirubinemia,

oxidative stress and injury to neocortical synaptosomes. Brain Res, 1026, 33-43.













Cardozo-Pelaez, F., Brooks, P. J., Stedeford, T., Song, S. and Sanchez-Ramos, J. (2000)

DNA damage, repair, and antioxidant systems in brain regions: a correlative study. Free

Radic Biol Med, 28, 779-785.

Chang, N., Li, L., Hu, R. et al. (2010) Differential regulation of NMDA receptor function by DJ-

1 and PINK1. Aging Cell.

Chroni, E., Patsoukis, N., Karageorgos, N., Konstantinou, D. and Georgiou, C. (2006) Brain

oxidative stress induced by obstructive jaundice in rats. J Neuropathol Exp Neurol, 65,

193-198.

Dalman, C. and Cullberg, J. (1999) Neonatal hyperbilirubinaemia--a vulnerability factor for

mental disorder? Acta Psychiatr Scand, 100, 469-471.

Chapter IV __________________________________________________________________________

132

Deganuto, M., Cesaratto, L., Bellarosa, C. et al. (2010) A proteomic approach to the bilirubin-

induced toxicity in neuronal cells reveals a protective function of DJ-1 protein. Proteomics,

10, 1645-1657.

Dennery, P. A., Seidman, D. S. and Stevenson, D. K. (2001) Neonatal hyperbilirubinemia. N

Engl J Med, 344, 581-590.



592.


671.









Falcão, A. S., Silva, R. F. M., Pancadas, S., Fernandes, A., Brito, M. A. and Brites, D. (2007)

Apoptosis and impairment of neurite network by short exposure of immature rat cortical

neurons to unconjugated bilirubin increase with cell differentiation and are additionally

enhanced by an inflammatory stimulus. J Neurosci Res, 85, 1229-1239.

Fernandes, A., Falcão, A. S., Abranches, E., Bekman, E., Henrique, D., Lanier, L. M. and

Brites, D. (2009) Bilirubin as a determinant for altered neurogenesis, neuritogenesis, and

synaptogenesis. Dev Neurobiol, 69, 568-582.











133




Fink, S. L. and Cookson, B. T. (2005) Apoptosis, pyroptosis, and necrosis: mechanistic

description of dead and dying eukaryotic cells. Infect Immun, 73, 1907-1916.

Friberg, H., Connern, C., Halestrap, A. P. and Wieloch, T. (1999) Differences in the activation

of the mitochondrial permeability transition among brain regions in the rat correlate with

selective vulnerability. J Neurochem, 72, 2488-2497.

Gkoltsiou, K., Tzoufi, M., Counsell, S., Rutherford, M. and Cowan, F. (2008) Serial brain MRI

and ultrasound findings: relation to gestational age, bilirubin level, neonatal neurologic

status and neurodevelopmental outcome in infants at risk of kernicterus. Early Hum Dev,

84, 829-838.

Gomes, A., Fernandes, E. and Lima, J. L. (2005) Fluorescence probes used for detection of

reactive oxygen species. J Biochem Biophys Methods, 65, 45-80.



Greenamyre, T., Penney, J. B., Young, A. B., Hudson, C., Silverstein, F. S. and Johnston, M.

V. (1987) Evidence for transient perinatal glutamatergic innervation of globus pallidus. J

Neurosci, 7, 1022-1030.

Grojean, S., Koziel, V., Vert, P. and Daval, J. L. (2000) Bilirubin induces apoptosis via

activation of NMDA receptors in developing rat brain neurons. Exp Neurol, 166, 334-341.

Grojean, S., Lievre, V., Koziel, V., Vert, P. and Daval, J. L. (2001) Bilirubin exerts additional

toxic effects in hypoxic cultured neurons from the developing rat brain by the recruitment

of glutamate neurotoxicity. Pediatr Res, 49, 507-513.

Guzzetta, F., Deodato, F. and Rando, T. (2000) Brain ischemic lesions of the newborn.

Childs Nerv Syst, 16, 633-637.




29, 765-778, viii.






Chapter IV __________________________________________________________________________

134



Jayalakshmi, K., Sairam, M., Singh, S. B., Sharma, S. K., Ilavazhagan, G. and Banerjee, P.

K. (2005) Neuroprotective effect of N-acetyl cysteine on hypoxia-induced oxidative stress

in primary hippocampal culture. Brain Res, 1046, 97-104.

Jiang, X., Mu, D., Manabat, C., Koshy, A. A., Christen, S., Tauber, M. G., Vexler, Z. S. and

Ferriero, D. M. (2004) Differential vulnerability of immature murine neurons to oxygen-

glucose deprivation. Exp Neurol, 190, 224-232.

Jin, J., Meredith, G. E., Chen, L., Zhou, Y., Xu, J., Shie, F. S., Lockhart, P. and Zhang, J.

(2005) Quantitative proteomic analysis of mitochondrial proteins: relevance to Lewy body

formation and Parkinson's disease. Brain Res Mol Brain Res, 134, 119-138.

Karageorgos, N., Patsoukis, N., Chroni, E., Konstantinou, D., Assimakopoulos, S. F. and

Georgiou, C. (2006) Effect of N-acetylcysteine, allopurinol and vitamin E on jaundice-

induced brain oxidative stress in rats. Brain Res, 1111, 203-212.

Knowles, R. G., Palacios, M., Palmer, R. M. and Moncada, S. (1989) Formation of nitric

oxide from L-arginine in the central nervous system: a transduction mechanism for

stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci U S A, 86, 5159-5162.

Lev, N., Ickowicz, D., Barhum, Y., Lev, S., Melamed, E. and Offen, D. (2009) DJ-1 protects

against dopamine toxicity. J Neural Transm, 116, 151-160.

Lev, N., Ickowicz, D., Melamed, E. and Offen, D. (2008) Oxidative insults induce DJ-1

upregulation and redistribution: implications for neuroprotection. Neurotoxicology, 29, 397-

405.

Mattson, M. P. and Liu, D. (2002) Energetics and oxidative stress in synaptic plasticity and

neurodegenerative disorders. Neuromolecular Med, 2, 215-231.

Mitani, A., Watanabe, M. and Kataoka, K. (1998) Functional change of NMDA receptors

related to enhancement of susceptibility to neurotoxicity in the developing pontine

nucleus. J Neurosci, 18, 7941-7952.

Nehru, B. and Kanwar, S. S. (2004) N-acetylcysteine exposure on lead-induced lipid

peroxidative damage and oxidative defence system in brain regions of rats. Biol Trace

Elem Res, 101, 257-264.

Ocal, K., Avlan, D., Cinel, I., Unlu, A., Ozturk, C., Yaylak, F., Dirlik, M., Camdeviren, H. and

Aydin, S. (2004) The effect of N-acetylcysteine on oxidative stress in intestine and

bacterial translocation after thermal injury. Burns, 30, 778-784.




135

Ostrow, J. D., Pascolo, L. and Tiribelli, C. (2003) Reassessment of the unbound

concentrations of unconjugated bilirubin in relation to neurotoxicity in vitro. Pediatr Res,

54, 926.

Özkan, H., Akkoc, N., Aydin, A., Kavukcu, Olgun, N., Irken, G., Akyol, F. and Cevik, N. T.

(1995) Relationship between serum unconjugated bilirubin levels and the

autofluorescence of white blood cells in neonatal jaundice. Biol Neonate, 68, 100-103.



2310.

Perlman, J. M., Rogers, B. B. and Burns, D. (1997) Kernicteric findings at autopsy in two sick

near term infants. Pediatrics, 99, 612-615.




Rodrigues, C. M. P., Solá, S. and Brites, D. (2002) Bilirubin induces apoptosis via the




Mol Med, 6, 936-946.



Shendelman, S., Jonason, A., Martinat, C., Leete, T. and Abeliovich, A. (2004) DJ-1 is a

redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation.

PLoS Biol, 2, e362.



Soorani-Lunsing, I., Woltil, H. A. and Hadders-Algra, M. (2001) Are moderate degrees of

hyperbilirubinemia in healthy term neonates really safe for the brain? Pediatr Res, 50,

701-705.



Sunico, C. R., Gonzalez-Forero, D., Dominguez, G., Garcia-Verdugo, J. M. and Moreno-

Lopez, B. (2010) Nitric oxide induces pathological synapse loss by a protein kinase G-,

Rho kinase-dependent mechanism preceded by myosin light chain phosphorylation. J

Neurosci, 30, 973-984.

Chapter IV __________________________________________________________________________

136




105, 2039-2052.



Vaz, A. R., Delgado-Esteban, M., Brito, M. A., Bolaños, J. P., Brites, D. and Almeida, A.

(2010) Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in

immature cortical neurons: assessment of the protective effects of glycoursodeoxycholic

acid. J Neurochem, 112, 56-65.



Weir, C. and Millar, W. S. (1997) The effects of neonatal jaundice and respiratory

complications on learning and habituation in 5- to 11-month-old infants. J Child Psychol

Psychiatry, 38, 199-206.

Weisiger, R. A., Ostrow, J. D., Koehler, R. K., Webster, C. C., Mukerjee, P., Pascolo, L. and

Tiribelli, C. (2001) Affinity of human serum albumin for bilirubin varies with albumin

concentration and buffer composition: results of a novel ultrafiltration method. J Biol

Chem, 276, 29953-29960.



Zachwieja, J., Zaniew, M., Bobkowski, W., Stefaniak, E., Warzywoda, A., Ostalska-Nowicka,

D., Dobrowolska-Zachwieja, A., Lewandowska-Stachowiak, M. and Siwinska, A. (2005)

Beneficial in vitro effect of N-acetyl-cysteine on oxidative stress and apoptosis. Pediatr

Nephrol, 20, 725-731.




Chapter V

V. Final considerations

Final considerations __________________________________________________________________________

139

1. Concluding remarks and perspectives The present thesis was designed to bring new insights into the cellular mechanisms

underlying neonatal hyperbilirubinemia neurotoxicity, and particular attention was given to

the role of oxidative stress in immature neurons exposed to unconjugated bilirubin (UCB). It

is widely accepted that hyperbilirubinemia in preterm infants is more prevalent, more severe

and represents an increased risk for the development of neurologic dysfunction in later life

(Stevenson et al., 2001, Watchko, 2006). Therefore, it is important to better understand the

essential mechanisms that contributes to the increased vulnerability of prematures to UCB

encephalopathy.

Cultured neuronal cells are powerful experimental models that we have adopted to

evaluate the deleterious effects of UCB. We have decided to use rat primary cultures of

neurons at 3 days in vitro (DIV), to mimic a condition of prematurity. This model was chosen

based on previous results demonstrating that immature cells are more susceptible to UCB-

induced neurotoxic effects than more differentiated ones (Falcão et al., 2005, Falcão et al.,

2006). In addition, this work focused on primary cultures of neurons, since they showed to be

more prone to oxidative injury and mitochondrial dysfunction and consequent cell death than

astrocytes, for which differences in glutathione content appear to have an important role

(Almeida et al., 2002, Almeida et al., 1998, Bolaños et al., 1996, Brito et al., 2008b).

Using this model, in the first experimental part of this thesis (chapter II), it is

demonstrated that UCB neuronal exposure rapidly inhibits cytochrome c oxidase (complex

IV) activity and ascorbate-driven oxygen consumption in 3 DIV rat cortical neurons. This

impairment of mitochondrial respiration was accompanied by a bioenergetic crisis, as judged

by the collapse of the inner-mitochondrial membrane potential, increased glycolytic activity,

and adenosine triphosphate (ATP) release. In addition, it was observed a disruption of

oxidized status of the cells, with increased superoxide anion radical production, disruption of

glutathione metabolism and impairment of reduced nicotinamide adenine dinucleotide

phosphate (NADPH) production. These events coursed in parallel with apoptotic cell death,

as determined by increased activities of mitochondrial-dependent caspases-9 and -3, as well

as increased annexin V+ neurons and condensed or fragmented nuclei. The mitochondrial

dysfunction observed by UCB may involve nitric oxide (.NO), which is capable of rapidly and

reversibly inhibit the mitochondrial respiratory chain (Bolaños et al., 1994, Brown and

Cooper, 1994, Cleeter et al., 1994). In fact, recent studies reported that neuronal oxidative

disruption by UCB is counteracted by inhibition of neuronal NO synthase (nNOS) (Brito et al.,

2008a, Mancuso et al., 2008). In the present work, the mitochondrial impairment by UCB

accompanied by the up-regulation of glycolysis suggests an attempt of immature neuronal

cells to support the bioenergetic crisis, as previously demonstrated for astrocytes exposed to

Chapter V __________________________________________________________________________

140

toxic stimulus (Bolaños et al., 2004). Nevertheless, excess of neuronal released ATP

determines .NO production and may be associated with neuronal apoptosis (Figueroa et al.,

2006, López et al., 2006). Finally, since NADPH levels are necessary to restore reduced

glutathione (Dringen, 2000), UCB-induced up-regulation of glycolysis may lead to inhibition of

the pentose-phosphate pathway (PPP), causing decrease of NADPH levels and,

consequently, glutathione oxidation, a notion recently reported for neuronal cultures

(Herrero-Mendez et al., 2009). In this study, neuroprotective effects of glycoursodeoxycholic

acid (GUDCA) were tested, since this bile acid was recently demonstrated to inhibit neuronal

cell death and oxidative stress (Brito et al., 2008a), as well as astrocytic inflammatory

response and consequent apoptotic and necrotic cell death (Fernandes et al., 2007b). In

summary, treatment of neurons with GUDCA prior to exposure to UCB prevented inhibition of

cytrochrome c oxidase activity, preserved cellular redox status and maintained cellular

viability. Taken together, these data suggest that cytochrome c oxidase inhibition is involved

in the neurotoxicity associated with UCB-induced neurological dysfunction and strongly

indicates the possible therapeutic potential of GUDCA in the treatment of neonatal jaundice.

In the second part of the work presented (chapter III), it is discussed the role of

neuroinflammation, a risk factor that is often associated with neonatal hyperbilirubinemia and

that is responsible for the alteration of blood brain barrier permeability through the release of

great amounts of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α+,

interleukin-1β (IL-1β) and interleukin-6 (IL-6) (Goldenberg and Andrews, 1996). In fact,

infection increases the risk for UCB encephalopathy (Dawodu et al., 1984) and presence of

inflammatory features, namely fever episodes and brain edema, were already described

during or following moderate to severe hyperbilirubinemia (Kaplan and Hammerman, 2005).

Interestingly, immature neuronal and astroglial cells showed higher levels of released

glutamate and TNF-α induced by exposure to UCB than more differentiated ones (Falcão et

al., 2006). Moreover, LPS showed to exacerbate the release of TNF-α and IL-1β by

immature astrocytes (Falcão et al., 2005), which provides a basis for the increased risk of

hyperbilirubinemia in the presence of sepsis. The results presented here reveal neuronal

dysfunction in 3 DIV rat cortical neurons by exposure to UCB, resulting from activation of

both the intrinsic and extrinsic pathways of apoptosis and loss of cellular functionality. Most

important, the results provide supportive evidence for the commonly indicated higher risk of

UCB brain damage in a condition of inflammation, since the association of pro-inflammatory

cytokines, TNF-α+IL-1β, to the condition of hyperbilirubinemia significantly increased the

cytotoxic potential of UCB through the same cascade of mediators. In this study, .NO

signalling and JNK1/2 activation are pointed to be key players in neurotoxicity induced by

hyperbilirubinemia and associated inflammation. In fact, several studies highlighted the


141

association between inflammation and the generation of reactive oxygen species and/or

reactive nitrogen species (ROS/RNS) (Bian and Murad, 2001, Brito et al., 2007, Sener et al.,

2005) and one of them, .NO, is important in the pathogenesis of inflammation (Korhonen et

al., 2005). .NO and induction of NOS are involved in apoptosis induced by inflammatory

mediators in neuronal cells (Hemmer et al., 2001, Heneka et al., 1998, Thomas et al., 2008).

Interestingly, in recent studies with mature neurons (rat primary cultures used at 8 DIV), UCB

exposure increased the expression of nNOS and production of .NO, cyclic guanosine 3',5'-

monophosphate (cGMP) and ROS, along with protein oxidation and depletion of glutathione

(Brito et al., 2008b, Brito et al., 2010). Furthermore, stress-activated protein kinases, such as

c-Jun N-terminal kinases 1/2 (JNK1/2), become activated in response to toxic stimulus, such

as the RNS (Luo et al., 1998, Marques et al., 2003), and the pro-inflammatory cytokines

TNF-α and IL-1, pointing these kinases as strong effectors of neuronal apoptosis (Mielke and

Herdegen, 2000, Tibbles and Woodgett, 1999). More recently, JNK 1/2 showed to directly

mediate the UCB-stimulation of TNF-α by astrocytes (Fernandes et al., 2007a). This feature

may be relevant if we consider that astrocytic activation has also been reported in several

neurodegenerative disorders and that this transition may be accompanied by dysfunction of

astrocytes leading to incorrect glia-to-neuron cross-talk (Rossi and Volterra, 2009). In

conclusion, these results provide evidence for the commonly indicated higher risk of UCB

brain damage in an inflammatory-associated condition.

In the third part of this work (chapter IV), it is discussed the different regional

vulnerability to UCB-induced neurotoxicity. For this purpose, rat primary neurons were

isolated from cortex, hippocampus and cerebellum and cultured for 3 DIV. Hippocampal and

cerebellar neurons were chosen based on preferential deposition pattern in the brain in

kernicteric conditions (Ahdab-Barmada and Moossy, 1984, Hansen, 2000). Here it is

demonstrated that neurons from hippocampus are more susceptible to UCB-induced

oxidative and nitrosative stress than those from cerebellum and cortex. For this differential

vulnerability to UCB within brain regions may account distinct antioxidant defences, since the

lowest levels of total glutathione were found in immature neurons from hippocampus. In

agreement with this concept, antioxidant enzymes, such as xantine oxidase, catalase and

glutathione peroxidase, showed minimum activity in hippocampus in different models, such

as developing mouse brain and astrocytic cultures (Prasanthi et al., 2010, Tsuru-Aoyagi et

al., 2009, Xu et al., 2001). Considering these facts, together with the results achieved with

the precursor of glutathione, N-acetylcysteine (NAC), where there was a protection from UCB

neurotoxicity, it is conceivable that hippocampal preferential vulnerability to UCB is, at least

in part, due to the lower glutathione content. Another important finding is the increased

vulnerability of hippocampal neurons to UCB-induced neurite arborization impairment.

Chapter V __________________________________________________________________________

142

Interestingly, in a very recent study with the same cellular model, inhibition of nNOS

abrogated the deleterious effects produced by UCB in network dynamics (personal

communication Sandra L Silva, 2010), result that were maintained along cell maturation,

indicating that short and long-term UCB toxic effects are prevented through inhibition of .NO

release. Since hippocampus is crucial for the linkage of short-term memory to the learning

process and the storage of spatial information (de Hoz et al., 2003, O'Neill et al., 2010,

Scoville and Milner, 1957), disassembly of neuritic development in neonatal

hyperbilirubinemia may account for a reduced learning memory ability and memory loss.

Cytoskeleton disassembly, loss of dendrites and axons and impairment of

neurotransmission are responsible for synaptic connectivity disruption, which are contributors

to the development of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s

diseases (Benitez-King et al., 2010, Evans et al., 2008). Since oxidative stress is a hallmark

of these pathologies (Halliwell, 2006, Lin et al., 2005), a better understanding of the neuronal

models comprising both oxidative stress and neuronal network dynamics is extremely

important in the comprehension of neurodegenerative diseases pathogenesis. In this study it

was also evaluated the expression of DJ-1, a protein involved in Parkinson’s disease

pathogenesis (Bonifati et al., 2003). However, in our experimental model, the effort to

prevent oxidative stress by increasing DJ-1 levels fails after long periods of UCB neuronal

exposure in neurons from all the three regions. Since down-regulation of DJ-1 is suppressed

in the presence of NAC, we consider that UCB-induced oxidative stress participates in DJ-1

regulation. In conclusion, the data obtained in the present study show that the condition of

neonatal hyperbilirubinemia induces oxidative stress in different brain regions, particularly in

the hippocampus. Furthermore, these results provide specific features that may explain the

differential susceptibility throughout different brain areas to UCB exposure, such as local

differences in glutathione content and in DJ-1 expression, important molecules involved in

the regulation of oxidative damage.

The major findings of this thesis are summarized in Figure V.1. Although we were able to

answer the questions that constitute the starting point of this work, others were raised and

remain to be clarified.


143

Figure V.1 – Integrative schematic representation of the major findings achieved in the present work, using immature neurons (3 DIV) isolated from brain cortex of fetuses from 16 days (E16) pregnant Wistar rats as experimental model. In the first part of this work (A), 3 DIV cortical neurons (Cx) exposed to unconjugated bilirubin (UCB), in conditions mimicking neonatal hyperbilirubinemia in the prematures, suffered oxidative stress, mitochondrial dysfunction associated with bioenergetic crisis and cell death, effects that were prevented in the presence of glycoursodeoxycholic acid (GUDCA). In the second part (B), it is concluded that UCB-induced nitrosative stress, c-Jun N-terminal kinases 1 and 2 (JNK1/2) and cell death are intensified by pro-inflammatory cytokines as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), used to mimic infection, in 3 DIV cortical neurons. In addition, both L-NAME (nitric oxide synthase inhibitor) and SP600125 (JNK1/2 inhibitor) reversed the effects produced by UCB either alone, or in association to pro-inflammatory cytokines. In the third part (C), neurons were isolated not only from cortex but also from cerebellum (Cb) and hippocampus (Hc). Hc neurons were the most susceptible to UCB-induced oxidative and nitrosative stress, as well as to UCB-induced neuritic impairment and cell death. N-acetylcysteine (NAC), a precursor of glutathione synthesis, was able to counteract the UCB-induced neurotoxicity.

Since hippocampal neurons were shown to be more vulnerable to UCB-induced

neurotoxicity than those from cortex and cerebellum, and that antioxidant defences will

appear to account for this differential vulnerability, it would be interesting to study whether

treatment of neuronal cultures with molecules that showed here to have antioxidant capacity,

such as GUDCA and NAC, are able to prevent from long term oxidative stress and energy

dysfunction in immature cultured neurons exposed to UCB, either alone or in association with

pro-inflammatory cytokines. This will contribute to elucidate the molecular events underlying

the relation between neonatal hyperbilirubinemia, alone or with associated inflammation, and

the development of neurological disorders in latter life. It would also be important to

determine if glutathione is the only molecule presented in lower levels in neurons from

hippocampus or if other antioxidant systems, like superoxide dismutase and catalase are

also compromised in these cells.

E16 rat brain3 DIV neurons

CxHc Cb

UCBOxidative stressMitochondrial dysfunction

Bioenergetic crisisCell death

(A)

UCB Nitrosative stressJNK1/2 signalling

Cell deathUCB + TNF-α + IL-1β

(B)

UCBOxidative/Nitrosative

stressNeuritic impairment

Cell death

(C)

GUDCA

L-NAME; SP600125

NAC

Chapter V __________________________________________________________________________

144

Other interesting issue that deserves to be addressed is whether activated astrocytes

and microglia exert a preventive or toxic effect on immature neuronal cells. Neuronal-glial

interactions constitute a major feature in the maintenance of neuronal homeostasis regarding

vascular, ionic, redox and metabolic function in the brain. In fact, astrocytes provide neurons

with energy and substrates for neurotransmission, as well as glutathione precursors (Allen

and Barres, 2005, Dringen et al., 2000). In addition, microglia is one of the brain’s major

sources of ROS and RNS and elevated concentrations of .NO (Bishop and Anderson, 2005),

and may be implicated in neurodegenerative diseases and neural injury as previously

discussed. Interestingly it has been demonstrated that UCB is able to activate microglia by

the acquisition of a phagocytic and inflammatory phenotype, which includes the release of

pro-inflammatory cytokines and release of glutamate (Gordo et al., 2006, Silva et al., 2010).

Interestingly, studies performed in our laboratory (personal communication Sandra L Silva,

2010) suggest that microglia exposure to UCB increases .NO production, effect that seems to

be further augmented when microglia is incubated with UCB-treated neuron conditioned

medium. In addition, stimulation of hippocampal slice cultures by UCB was recently reported

to increase nitrites release into the culture medium, as well as intracellular production of

glutamate, which is completed abrogated in microglia-depleted slice cultures. Since .NO

signalling by UCB is one important finding of the studies discussed in this thesis, these

observations suggest that microglial reactivity may be important for UCB-induced

neurotoxicity. Therefore, it would be interesting to evaluate whether microglia plays a role in

mitochondrial dysfunction and consequent cell death observed in chapter II, by using other

experimental models, such as neuron-microglia co-cultures or in models with depleted or

non-depleted microglia slices.

These advances may substantiate target-driven approaches to the prevention and

treatment of UCB-induced neurological damage, and provide fruitful opportunities for future

investigations.


145

2. References

Ahdab-Barmada, M. and Moossy, J. (1984) The neuropathology of kernicterus in the


Allen, N. J. and Barres, B. A. (2005) Signaling between glia and neurons: focus on synaptic

plasticity. Curr Opin Neurobiol, 15, 542-548.

Almeida, A., Delgado-Esteban, M., Bolanos, J. P. and Medina, J. M. (2002) Oxygen and

glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones

but not in astrocytes in primary culture. J Neurochem, 81, 207-217.

Almeida, A., Heales, S. J., Bolanos, J. P. and Medina, J. M. (1998) Glutamate neurotoxicity

is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione

depletion. Brain Res, 790, 209-216.

Benitez-King, G., Ortiz-Lopez, L., Jimenez-Rubio, G. and Ramirez-Rodriguez, G. (2010)

Haloperidol causes cytoskeletal collapse in N1E-115 cells through tau

hyperphosphorylation induced by oxidative stress: Implications for neurodevelopment. Eur

J Pharmacol, 644, 24-31.



Bishop, A. and Anderson, J. E. (2005) NO signaling in the CNS: from the physiological to the

pathological. Toxicology, 208, 193-205.

Bolaños, J. P., Cidad, P., García-Nogales, P., Delgado-Esteban, M., Fernández, E. and

Almeida, A. (2004) Regulation of glucose metabolism by nitrosative stress in neural cells.

Mol Aspects Med, 25, 61-73.

Bolaños, J. P., Heales, S. J., Peuchen, S., Barker, J. E., Land, J. M. and Clark, J. B. (1996)

Nitric oxide-mediated mitochondrial damage: a potential neuroprotective role for

glutathione. Free Radic Biol Med, 21, 995-1001.



Neurochem, 63, 910-916.

Bonifati, V., Rizzu, P., van Baren, M. J. et al. (2003) Mutations in the DJ-1 gene associated

with autosomal recessive early-onset parkinsonism. Science, 299, 256-259.




Chapter V __________________________________________________________________________

146












Lett, 356, 295-298.




345, 50-54.



Geogr Med, 36, 127-132.

de Hoz, L., Knox, J. and Morris, R. G. (2003) Longitudinal axis of the hippocampus: both

septal and temporal poles of the hippocampus support water maze spatial learning

depending on the training protocol. Hippocampus, 13, 587-603.


671.

Dringen, R., Gutterer, J. M. and Hirrlinger, J. (2000) Glutathione metabolism in brain

metabolic interaction between astrocytes and neurons in the defence against reactive

oxygen species. Eur J Biochem, 267, 4912-4916.

Evans, N. A., Facci, L., Owen, D. E., Soden, P. E., Burbidge, S. A., Prinjha, R. K.,

Richardson, J. C. and Skaper, S. D. (2008) Abeta(1-42) reduces synapse number and

inhibits neurite outgrowth in primary cortical and hippocampal neurons: a quantitative

analysis. J Neurosci Methods, 175, 96-103.





147




Fernandes, A., Falcão, A. S., Silva, R. F. M., Brito, M. A. and Brites, D. (2007a) MAPKs are



Fernandes, A., Vaz, A. R., Falcão, A. S., Silva, R. F. M., Brito, M. A. and Brites, D. (2007b)



Figueroa, S., Oset-Gasque, M. J., Arce, C., Martinez-Honduvilla, C. J. and González, M. P.

(2006) Mitochondrial involvement in nitric oxide-induced cellular death in cortical neurons

in culture. J Neurosci Res, 83, 441-449.

Goldenberg, R. L. and Andrews, W. W. (1996) Intrauterine infection and why preterm

prevention programs have failed. Am J Public Health, 86, 781-783.



Halliwell, B. (2006) Oxidative stress and neurodegeneration: where are we now? J

Neurochem, 97, 1634-1658.















Acta, 356, 9-21.



Chapter V __________________________________________________________________________

148

Lin, S., Wei, X., Bales, K. R., Paul, A. B., Ma, Z., Yan, G., Paul, S. M. and Du, Y. (2005)

Minocycline blocks bilirubin neurotoxicity and prevents hyperbilirubinemia-induced

cerebellar hypoplasia in the Gunn rat. Eur J Neurosci, 22, 21-27.

López, E., Arce, C., Oset-Gasque, M. J., Canadas, S. and González, M. P. (2006) Cadmium

induces reactive oxygen species generation and lipid peroxidation in cortical neurons in

culture. Free Radic Biol Med, 40, 940-951.


apoptosis through an oxidation-involved SAPK/JNK activation pathway. J Biol Chem, 273, 3756-3764.







Biol Chem, 278, 28294-28302.



O'Neill, J., Pleydell-Bouverie, B., Dupret, D. and Csicsvari, J. (2010) Play it again:

reactivation of waking experience and memory. Trends Neurosci, 33, 220-229.




Rossi, D. and Volterra, A. (2009) Astrocytic dysfunction: insights on the role in

neurodegeneration. Brain Res Bull, 80, 224-232.

Scoville, W. B. and Milner, B. (1957) Loss of recent memory after bilateral hippocampal

lesions. J Neurol Neurosurg Psychiatry, 20, 11-21.



Today, 35, 52-59.



phagocytosis to inflammation. Neurobiol Dis.




149



neuroinflammation-relevance in a mouse model of Parkinson's disease. J Neurochem,

105, 2039-2052.


Mol Life Sci, 55, 1230-1254.







UNIVERSIDADE DE LISBOArepositorio.ul.pt/bitstream/10451/2476/1/ulsd059472_Td_Ana_Vaz.pdf · À...

Documents

Transcript of UNIVERSIDADE DE LISBOArepositorio.ul.pt/bitstream/10451/2476/1/ulsd059472_Td_Ana_Vaz.pdf · À...