The Neuroprotective Effect of BDNF on Oxidative DNA Damage...

UNIVERSIDADE DE LISBOA

FACULDADE DE MEDICINA

The Neuroprotective Effect of BDNF on Oxidative DNA Damage in Rat Cortical Neurons: Evaluation by Comet Assay

Rúben Balau Delgado Gonçalves

Mestrado em Neurociências

Lisboa, 2012

UNIVERSIDADE DE LISBOA

FACULDADE DE MEDICINA

The Neuroprotective Effect of BDNF on Oxidative DNA Damage in Rat Cortical Neurons: Evaluation by Comet Assay

Rúben Balau Delgado Gonçalves

Orientador: Professora Doutora Ana Sebastião, FMUL Co-Orientador: Professor Doutor João Ferreira, FMUL

Todas as afirmações contidas neste trabalho são da exclusiva responsabilidade do candidato, não cabendo à Faculdade de Medicina da Universidade de Lisboa qualquer responsabilidade.

Mestrado em Neurociências

Lisboa, 2012

Esta dissertação foi aprovada pelo Conselho Científico da Faculdade de Medicina da Universidade de Lisboa em reunião de 19 de Fevereiro de 2013

Index

Abstract ................................................................................................................................... i

Resumo .................................................................................................................................. ii

Abbreviations List .................................................................................................................. iii

Figures Index .......................................................................................................................viii

Agradecimentos .................................................................................................................... xi

1. Introduction......................................................................................................................... 1

1.1 - BDNF and Other Neurotrophins .......................................................................... 1

1.2 - Oxidative Stress, ROS and DNA Damage ......................................................... 10

1.3 - Antioxidant Defences ........................................................................................ 15

1.4 - Ageing .............................................................................................................. 17

1.5 - Neurodegenerative Disorders and Oxidative Stress in Neurons ........................ 19

1.6 - Alzheimer’s Disease and Aβ Peptide ................................................................ 20

2. Objectives ........................................................................................................................ 25

3. Methodology ..................................................................................................................... 26

3.1 - Primary Cell Culture .......................................................................................... 26

3.1.1 - Culture Maintenance ................................................................................. 27

3.2 - Immunocytochemistry ....................................................................................... 27

3.3 - Cell Viability ..................................................................................................... 28

3.4 - Comet Assay .................................................................................................... 29

3.4.1 General Considerations ............................................................................... 29

3.4.2 Experimental Design.................................................................................... 33

3.4.3 Technique Description ................................................................................. 37

3.4.3.1 Electrophoretic Force (V/cm) Calculation .............................................. 38

3.4.3.2 Standard Cells ...................................................................................... 39

4. Results and Discussion .................................................................................................... 40

4.1 - The Oxidative Comet Assay: Implementation and Optimization ........................ 40

4.2 - Cell Culture Characterization: Are the cultured neurons viable at the times of

experiment? What percentage of glial cells is present in culture?.............................. 49

4.3 - Do neurons accumulate DNA damage, either structural or oxidative, as they

mature in culture? ..................................................................................................... 55

4.4 - Does Aβ25-35 peptide induce structural and/or oxidative DNA damage at sub-

lethal concentrations? ............................................................................................... 59

4.5 - Does BDNF protect against H2O2 induced DNA damage? ................................ 63

5. Conclusions ...................................................................................................................... 68

6. Future Work...................................................................................................................... 69

7. References ....................................................................................................................... 71

8. Annexes ........................................................................................................................... 76

Annex I – Comet Assay Protocol ............................................................................... 76

Annex II – Standard Cells Protocol............................................................................ 95

i

Abstract

Alzheimer’s disease is an increasing prevalent disease nowadays although its

underlying mechanisms are not fully understood. In addition to other factors, it is

known that the presence of amyloid-β peptide in the senile plaques of patients with

Alzheimer disease, oxidative stress and neurotrophic deprivation are involved in the

onset of the disease.

In vitro studies refer that incubation with BDNF prevented Aβ-induced apoptosis

and also increased the activity of antioxidant enzymes. However, most studies only

refer oxidative stress and the loss of cell viability and do not focus in oxidative

damage particularly to the nuclear DNA.

In this study, the structural and oxidative DNA damage of primarily cultured rat

cortical neurons was measured by Comet Assay. The assay was performed after

incubation with the Aβ25-35 peptide at sub-lethal concentrations for a 24 hour period

and after a 10 minutes exposure to 10 µM H2O2 in the presence or absence of BDNF.

Although no DNA damage was observed after incubation with the Aβ25-35 peptide,

the H2O2 incubation induced both structural and oxidative damage to the nuclear

DNA, while a 48 hour pre-incubation with BDNF decreased the induced oxidative

damage. This study thus provided a useful insight into BDNF’s protective effects on

DNA damage. However, further studies are necessary to evaluate if the Aβ25-35

peptide induces DNA damage at higher concentrations.

Keywords: Cortical neurons, BDNF, Aβ25-35 peptide, H2O2, Comet Assay, DNA

damage.

ii

Resumo

A doença de Alzheimer é cada vez mais prevalente actualmente devido ao

envelhecimento da população mundial, no entanto os mecanismos biológicos que

levam ao seu aparecimento ainda não são conhecidos. Entre outros factores, sabe-

se que a presença de agregados do péptido β-amilóide assim como o stress

oxidativo e deprivação neurotrófica estão envolvidos no desenvolvimento da doença.

Estudos in vitro demonstraram que a incubação com BDNF evita a apoptose de

neurónios induzida pelo péptido β-amilóide assim como aumenta a actividade de

enzimas antioxidantes. No entanto, a maioria dos estudos foca-se na presença de

stress oxidativo e na perda de viabilidade celular, não estudando os níveis de dano

ao DNA nuclear, especialmente de natureza oxidativa.

Neste estudo, o dano estrutural e oxidativo ao DNA nuclear de culturas primárias

de neurónios corticais de rato foi medido utilizando a técnica de Comet Assay. A

técnica foi realizada após incubação com o péptido Aβ25-35 em concentrações sub-

letais por um período de 24 horas e após exposição a 10 µM de H2O2 na presença

ou ausência de BDNF.

Apesar de nenhum dano ao DNA ter sido observado após incubação com o

péptido Aβ25-35, a incubação com H2O2 induziu dano estrutural e oxidativo ao DNA

nuclear sendo que uma pré-incubação com BDNF reduziu os níveis medidos de

dano oxidativo. O presente estudo permitiu então determinar que o BDNF tem um

efeito protector em condições de dano ao DNA. No entanto, são necessários mais

estudos para avaliar se o péptido Aβ25-35 induz dano ao DNA quando presente em

concentrações mais elevadas.

Palavras-Chave: Neurónios Corticais, BDNF, Péptido Aβ25-35, H2O2, Ensaio Comet,

Dano ao DNA.

iii

Abbreviations List

8-oxoG - 8-oxoGuanine

AD - Alzheimer’s Disease

Akt – also known as PKB (Protein Kinase B)

ANOVA - Analysis of Variance

AP sites - Apurinic/Apyrimidinic sites

APP - β-Amyloid Precursor Protein

araC - Cytosine Arabinoside

ATP – Adenosine Tri-Phosphate

Aβ peptide - Amyloid-β peptide

Aβ1-40 - Amyloid-β peptide 1-40



BAD - Bcl-2-associated death promoter (BAD)

Bax - Bcl-2–associated X protein

Bcl2 - B-cell lymphoma 2

Bcl-xL- B-cell lymphoma-extra large

BDNF - Brain-Derived Neurotrophic Factor

BER - Base Excision Repair

BSA - Bovine Serum Albumin

iv

CDK2 - Cyclin-dependent kinase 2

CDK3 - Cyclin-dependent kinase 3

DAG - Diacylglycerol

DAPI - 4’, 6’ – Diamidino-2-Phenylindole

DIV - Days In Vitro

DMEM - Dulbecco's Modified Eagle Medium

DMSO - Dimethyl sulfoxide

DSBs - Double-Strand DNA Breaks

EDTA - Ethylenediaminetetraacetic acid

EndoIII - Endonuclease III

ERK - Extracellular Signal-Regulated Kinase

EtBr - Ethidium bromide

Fapy - Formamidopyrimidines

FBS - Fetal Bovine Serum

FISH - Fluorescent in situ hybridization

FKHRL-1 - Forkhead Transcription Factor

Fpg - Formamidopyrimidine DNA Glycosylase

Gab1 - GRB2-associated-binding protein 1

GC-MS - Gas Chromatography-Mass Spectrometry

GDP - Guanosine Di-Phosphate

GFAP - Glial Fibrillary Acidic Protein

v

GPx - Glutathione Peroxidase

GR - Glutathione Reductase

Grb2 - Growth factor receptor-bound protein 2

GSH - Glutathione

GSSG - Glutathione Disulfide

GTP - Guanosine Tri-Phosphate

H2O2 – Hydrogen Peroxide

HBSS - Hanks’ Balanced Salt Solution

HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC - High-Pressure Liquid Chromatography

IP3 - Inositol 1,4,5-Trisphosphate

IκB - Inhibitor of κB

KSR1 - Kinase Suppressor of Ras 1

LMPA - Low Melting Point Agarose

LY-294002 - 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (inhibitor of PI-3

kinase)

MAP2 - Microtubule Associated Protein 2

MAPK - Mitogen-Activated Protein Kinase

MEK - MAPK-ERK Kinase

Mn-SOD – Manganese Superoxide Dismutase

MPTP - 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

vi

NADPH – reduced form of Nicotinamide Adenine Dinucleotide Phosphate

NF-κB - Nuclear factor kappa-light-chain-enhancer of activated B cells

NGF - Nerve Growth Factor

NMA - Normal Melting Agarose

NMDA - N-Methyl-D-aspartate

NT-3 - Neurotrophin-3

NT-4/5 - Neurotrophin-4/5



1O2 - Singlet Oxygen

O2•– - Superoxide Anion

Ogg1 - 8-oxo-Guanine-DNA Glycosylase

OH• - Hydroxyl Radical

OHdG - 8-hydroxy-2-deoxyguanosine

PBS - Phosphate Buffer Saline

PD - Parkinson’s Disease

PDL - Poly-D Lysine

Pen/Strep - Penicillin/Streptomycin

PI3-K - Phosphatidylinositol 3 - Kinase

PKC - Protein Kinase C

PLC-γ1 - Phospholipase C – γ1

vii

pRb - Retinoblastoma Protein

PTB - Phosphotyrosine-Binding

PtdIns (4,5)P2 - Phosphatidylinositol 4,5-Bisphosphate

RNAi - Interference RNA

ROS - Reactive Oxygen Species

SCGE - Single Cell Gel Electrophoresis

SH2 - Src Homology 2

SOD - Superoxide Dismutase

SOS - Son Of Sevenless

SSBs - Single-Strand DNA Breaks

TG - Thymine Glycol

TNF - Tumour Necrosis Factor

Trk - Tropomyosin-Related Kinase

U-0126 - 1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (inhibitor of

MAPK)

U-73122 - 1-[6-[[(17b)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-

2,5-dione (inhibitor of Phospholipase C)

viii

Figures Index

Figure 1 – Neurotrophin interaction with Trk and p75NTR receptors. ............................ 2

Figure 2 – Neurotrophin signalling through Trk receptor activation. ............................ 4

Figure 3 – BDNF induced action in several signalling pathways after exposure to

NMDA or H2O2. ............................................................................................................ 8

Figure 4 – Hydroxyl radical (OH•) production in mitochondria through Fenton’s

reaction. .............................................................................................................. 11

Figure 5 – Stochastic causes of ageing. .................................................................... 18

Figure 6 – APP cleavage by secretase enzymes. ..................................................... 21

Figure 7 – Diagrams explaining the comet assay technique and mechanisms. ......... 31

Figure 8 – Representation of the analysis process used by comet analysis software

............................................................................................................................ 32

Figure 9 – Schematic representation of the comet assay timeline. ............................ 33

Figure 10 – Schematic representation of the comet assay timeline with a 24 hour

exposure to Aβ25-35. ............................................................................................. 34


exposure to Aβ25-35 and a 48 hour incubation with BDNF.................................... 35


incubation with BDNF and exposed to H202. ....................................................... 36

Figure 13 – Comet assay using the THP-1 cell line after incubation with etoposide for

one hour at several concentrations. .................................................................... 42

ix

Figure 14 – Graphic representations of the comet assay tail length data using the

THP-1 cell line after incubation with etoposide for one hour at several

concentrations. .................................................................................................... 43

Figure 15 – Graphic representations of the comet assay percentage of DNA in tail

data using the THP-1 cell line after incubation with etoposide for one hour at

several concentrations ........................................................................................ 44

Figure 16 – Image of comets obtained from primary cultured cortical neurons

exposed to H2O2. ................................................................................................. 46

Figure 17 – Graphic representations of the standard cells tail length and percentage

of DNA in tail data mean values. ......................................................................... 47

Figure 18 – Graphics of the fluorescence and absorbance data obtained at various

DIVs. ................................................................................................................... 50

Figure 19 – Overview of a neuronal culture ............................................................... 51

Figure 20 – Detail of an astrocyte at the centre of the image. ................................... 51

Figure 21 – Graphic representation of structural and oxidative DNA damage as the

neuronal culture matures. .................................................................................... 55


neuronal culture matures. .................................................................................... 56

Figure 23 – Graphic representation of structural and oxidative DNA damage caused

in the presence of the Aβ25-35 and in the presence or absence of the B27

supplement. ......................................................................................................... 57


in the presence of the Aβ25-35 and in the presence or absence of the B27

supplement. ......................................................................................................... 58


by sub-lethal concentrations of Aβ25-35 ................................................................ 61

x


by sub-lethal concentrations of Aβ25-35 ................................................................ 62

Figure 27 – Graphic representation of structural and oxidative DNA damage after

exposure to H2O2 with or without a pre-incubation with BDNF ............................ 64


exposure to H2O2 in the presence or absence of BDNF. ..................................... 65

Figure A – Outline of the slide cleaning and coating first steps ................................. 81

Figure B – Outline of the slide cleaning and coating last steps. ................................. 81

Figure C – Outline of the agarose embedment and cell lysis steps ........................... 84

Figure D – Outline of the staining steps. .................................................................... 90

xi

Agradecimentos

Quero agradecer à minha mãe e avó que me apoiaram imenso neste período da

minha vida. Por toda a paciência, carinho e amor, sem os quais não me teria sido

possível superar mais esta etapa.

Agradeço igualmente ao meu pai, pelos conselhos práticos e pela perspectiva que

proporcionou sobre o que verdadeiramente importa na vida profissional.

Estou profundamente grato à minha namorada pelo apoio, carinho e amor. Por

sempre me conseguir animar, mesmo nos dias de maior frustração e por me ter

aturado e ajudado a concentrar durante o processo de escrita, sem cujo apoio esta

dissertação não existiria.

Quero agradecer ao Prof. Doutor Alexandre Ribeiro, por me ter acolhido no

laboratório e me ter proporcionado esta oportunidade.

A todos os colegas de laboratório da UFN, e em especial ao Jorge Valadas, André

Santos e Pedro Pereira (UBCR), pelo apoio e conselhos práticos, o meu obrigado.

Um agradecimento especial à minha orientadora, Prof. Doutora Ana Sebastião,

pela liberdade e possibilidade de desenvolver trabalho com uma técnica não

previamente utilizada no laboratório, pela acessibilidade e disponibilidade e pelos

conselhos e orientação sempre pertinentes.

Agradeço igualmente ao Prof. Doutor João Ferreira, meu co-orientador, pelo rigor

e conselhos experimentais aquando da montagem da técnica.

Um agradecimento final ao Prof. Doutor Andrew Collins (U. Oslo) pela enzima FPG

e pelas sugestões referentes ao protocolo de comet sem as quais não teria sido

possível realizar o ensaio em neurónios.

1

1. Introduction

1.1 BDNF and Other Neurotrophins

Neurotrophins are a highly conserved family of secreted proteins that regulate brain

functions, particularly development, differentiation, survival and plasticity [23,55]. The

neurotrophin family includes the nerve growth factor (NGF), neurotrophin-3 (NT-3),

neurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6), neurotrophin-7 (NT-7) and the

brain-derived neurotrophic factor (BDNF) [20, 50]. NT-6 and NT-7 were isolated from

fish and have no mammalian or bird orthologous but appear to interact with the same

receptors as the related proteins in mammals [44]. The effect of neurotrophins is

exerted through the binding to two different classes of cell surface receptors, the

tropomyosin-related kinase (Trk) receptor family composed by TrkA, TrkB and TrkC

receptors and the neurotrophin receptor p75NTR, which is a member of the tumour

necrosis factor (TNF) receptor family. While the p75NTR receptor allows the binding of

all mature neurotrophins, the Trk receptor family exhibits ligand specificity to each

receptor, namely NGF preferentially binds to TrkA, BDNF and NT-4 to TrkB and NT-3

to TrkC [25]. The p75NTR receptor is known to regulate the Trk receptors response to

neurotrophins. In the presence of this receptor, NT-3 has a diminished affinity to TrkA

and together with NT-4 is also much less effective at activating TrkB. The TrkA and

TrkB receptors ligand specificity is then enhanced by the p75NTR receptor which leads

to a higher affinity of these receptors to their primary ligands, NGF and BDNF,

respectively [23].

The BDNF protein is processed from a pro-protein to a mature form and it is the

mature form that binds specifically to the TrkB receptor leading to cell survival or

2

differentiation. Pro-BDNF and other proneurotrophins, on the other hand, can bind

with high affinity to the p75NTR receptor, activating signalling pathways that often

result in apoptosis [44].

As can be seen in Figure 1, the Trk receptors are highly conserved and present an

extracellular domain composed by a cluster rich in cysteine residues. These are

Figure 1 – Neurotrophin interaction with Trk and p75NTR receptors. Conserved protein

domains are present in Trk receptors (TrkA, TrkB and TrkC). NGF, NT-3, NT-4 and BDNF

neurotrophins have an equal low affinity to the p75NTR receptor, but their pro-protein form

binds with high affinity to this receptor. NGF binds preferentially to the TrkA receptor. BDNF

and NT-4 specifically bind to the TrkB receptor. NT-3 favourably binds to TrkC but in certain

situations, it also has a low affinity to the other Trk receptors. CR1-CR4: cysteine-rich motifs;

C1/C2: cysteine-rich clusters; LRR1–3: leucine-rich repeats: Ig1/Ig2: immunoglobulin-like

domains [44].

3

followed by three leucine-rich repeats, another cysteine-rich cluster and two Ig-like

domains. The transmembrane region of these receptors contains a terminal tyrosine

kinase domain that is surrounded by many tyrosine residues which allows the binding

of cytoplasmic adaptors and enzymes in a phosphorylation dependent way [50].

BDNF, as a member of the family of neurotrophins, then influences cell survival,

differentiation and death [5] by the activation of its high-affinity receptor, the tyrosine

kinase receptor B (TrkB) [20]. So far, three different TrkB receptors have been

described with different signalling capabilities: TrkB.FL, a full-length catalytic receptor

and two truncated isoforms of the latter, TrkB.T1 and TrkB.T2. The TrkB.FL is a

transmembrane tyrosine kinase receptor with a conserved intracellular domain that

interacts with several signalling pathways including the Ras/MAPK, PI3K and PLC-γ1

pathways. The TrkB.T1 and TrkB.T2 receptors allow a signal transduction but lack an

intracellular tyrosine kinase activity. The gene expression of BDNF and TrkB varies,

depending on the development stage, age and cognitive performance. During

development, the TrKB.FL protein levels increase and there is a reduction of the

expression of this receptor in the hippocampus of aged rats. A decreasing expression

of BDNF and its receptors in dendrites could then partially explain memory

impairment in aged animals [55].

The activation of the Trk receptors is stimulated by neurotrophin binding that

induces dimerization of these membrane proteins and ultimately leads to the

transphosphorylation of their tyrosine kinase terminal domain. Of note that this

activation only occurs if the neurotrophin bound to the receptor is in its mature form

and not in the pro-form. The several tyrosine residues present in the terminal domain

4

of the Trk receptors are also subject to additional phosphorylation by the tyrosine

kinase receptor domain. The phosphorylated tyrosines then allow the recruitment of

several adaptor proteins and enzymes that propagate the signal transduction. The

proteins that bind to these phosphotyrosines and surrounding amino acid residues

usually contain phosphotyrosine-binding (PTB) or Src homology 2 (SH2) domains [50].

One of the major Trk receptor activating pathways is the Ras pathway which is

required for normal differentiation of neurons as well as promoting neuronal survival.

The adaptor protein Shc is recruited by a phosphotyrosine residue in the Trk receptor

by interacting with its PTB domain. The Trk receptor then mediates the

Figure 2 – Neurotrophin signalling through Trk receptor activation. The main activated

signalling pathways are depicted. Neurotrophin (NT) binding to the Trk receptor leads to its

dimerization and consequently to autophosphorylation. The Ras/Raf/MEK pathway promotes

the expression of prodifferentiation genes whereas the PI3-K/Akt pathway leads to the

expression of prosurvival genes. Adapted from Skaper (2012) [50].

5

phosphorylation of a tyrosine residue in Shc which leads to the recruitment of another

adaptor protein, Grb2. Grb2 in turn bounds to the Ras exchange factor SOS (Son Of

Sevenless) which activates Ras by replacing GDP with GTP [44]. The activated Ras

protein then interacts with the serine-threonine kinase Raf which sequentially leads to

activation of MEK (MAP kinase- ERK (Extracellular signal-regulated) kinase) that

further activates MAPK (mitogen-activated protein kinase). The translocation of

activated MAPK to the nucleus leads to the phosphorylation of transcription factors

that promote neurons to differentiate [50] (see Figure 2). There is a feedback in the

MAPK signalling cascade that attenuates and terminates signal responses through

the phosphorylation of intermediates and activation of phosphatases. For example,

ERK mediates SOS phosphorylation resulting in the dissociation of the SOS-Grb2

complex [44].

Activation of the PI3-K (phosphatidylinositol 3 - kinase) pathway can occur in a

Ras-dependent way or in a Ras-independent way which involves the Gab1 factor.

Both activating pathways lead to the promotion of neuronal survival and growth [50].

As seen in Figure 2, the PI3-kinase Ras-independent activation is initiated by the

recruitment of Gab1 by the phosphorylated form of Grb2 which subsequently leads to

the binding and activation of PI3-K. Activated PI3-K generates P3-phosphorylated

phosphoinosides, neuron survival essential lipids that are substract of many

phosphoinositide-dependent kinases that together with PI3-K activate the Akt protein

kinase. Through phosphorylation, Akt protein then controls the activity of several

proteins that are important in promoting neuronal survival. These proteins include

BAD and Bcl2-family members that are substrates that directly regulate the caspase

6

signal transduction pathway by binding to Bcl-xL, thus preventing this factor from

inhibiting the pro-apoptotic activity of the Bax protein. Phosphorylation of BAD leads

to its sequestering by the 14-3-3 proteins which prevents its pro-apoptotic actions.

Akt also regulates the activity of the forkhead transcription factor (FKHRL-1)

transcription factor by phosphorylation leading to the cytoplasmic sequestering of its

phosphorylated form by 14-3-3 proteins. This, in turn, prevents FKHRL-1 from

activating the transcription of several genes, which had the purpose of promoting

apoptosis. Furthermore, the degradation of IκB is mediated by phosphorylation

induced by the Akt protein, which leads to the liberation of the NF-κB factor that

promotes the transcription of genes involved in the sensory promotion of neuronal

survival [44].

PLC-γ1 (phospholipase C – γ1) activation results in the activation of two pathways:

the Ca2+-regulated pathway and the protein kinase (PKC)-regulated pathway. Both

ways lead to the promotion of synaptic plasticity [50]. The phosphorylated tyrosine

residues in the Trk receptor recruit PLC-γ, activating it through Trk-mediated

phosphorylation which then leads to the hydrolysis of PtdIns (4,5)P2, generating IP3

and DAG (see Figure 2). The presence of IP3 induces the release of Ca2+ from

cytoplasmic stores while DAG stimulates the DAG-regulated isoform of PKC. These

two signalling molecules can potentially activate many intracellular enzymes,

including the majority of PKC isoforms, Ca2+ - calmodulin-dependent protein kinases

and other Ca2+ - calmodulin-regulated targets [44].

The activation of the MAPK signalling cascade and increased levels of ERK1/2,

has been shown to occur both by pro-survival stimulus such as BDNF and toxic

7

stimuli including oxidative stress and are, as such, not predictive of the effect upon

neuronal survival [15,52]. This effect has, however, been found to be dependent upon

ERK1/2 localization, possibly explaining a biphasic effect of MAPK activation

following oxidative injury in which ERK1/2 has a protective effect upon the DNA

through the glutathione metabolism in early stages and later, following long exposure

to oxidative agents, contributes to cellular toxicity and death [15,29]. These findings are

consubstantiated by Hetman et al (2007) that reports a BDNF dependent activation

of MAPK involving co-localization of ERK1/2 with Kinase suppressor of Ras 1 (KSR1)

in membrane vesicles resembling the Golgi. Despite this, ERK1/2 also suffers a initial

rapid nuclear translocation though to be necessary for cell cycle re-entry [15,54].

A recent study by N. Boutahar et al (2010) has concluded that BDNF protects

cortical neurons from oxidative stress through the Ras/MAPK pathway and proteins

E2F1 and Rb (Retinoblastoma protein). As post-mitotic cells, neurons pause in the

G0 phase of the cell cycle. To re-enter it, cell cycle proteins must be activated so that

neurons can exit G0 and enter G1 phase. However, protein activation may also

potentiate a cell death mechanism in which the re-entry in the cell cycle ends in

mitotic aberration and ultimately cell death. Proteins Rb and from the E2F protein

family are included in the group of factors that regulate the cell cycle progression. In

quiescent cells, protein Rb is mainly hypo-phosphorylated and sequesters

transcriptions factors from the E2F family, hence inhibiting the cell from re-entering

the cell cycle. In contrast, the hyper-phosphorylation of Rb leads to its dissociation

from the E2F1 transcription factor, allowing the activation of E2F1-responsive genes,

S phase progression and neuronal death [5].

8

The image in Figure 3 depicts the action of BDNF in signalling pathways after

exposure to NMDA or H2O2. The H2O2 exposure induced ERK activation and cell

death which was prevented by BDNF [5]. These results, together with the fact that the

PI3-K inhibitor tested in the paper had no effect on ERK activation, lead to the

conclusion that the PI3-K pathway was probably not involved in the activation of the

Ras/MAPK pathway when the cortical neurons were exposed to H2O2 [5]. Cortical

neurons treated with NMDA or H2O2 had an increase in phosphorylation of protein Rb

and E2F1 expression and this effect was completely abolished when these

neurotoxins were combined with BDNF, leading to the previously reported conclusion

that neuroprotection induced by BDNF also involves proteins Rb and the E2F1

transcription factor [5].

Figure 3 – BDNF induced action in several signalling pathways after exposure to NMDA

or H2O2. The PI3-K pathway was activated by BDNF. On the other hand, BDNF stopped both

the NMDA and H2O2 activated MEK pathway and the activation of proteins Rb and E2F1

induced by both compounds. NMDA induced expression of ER stress was not modified by

BDNF [5].

9

The conclusion that BDNF prevents ERK activation induced by toxic agents while

capable to induce ERK activation on its own is of particular interest. One must

consider that these kinases can be activated by several extracellular signals

transducing them to various effector mechanisms [54]. This includes the ability to

mediate suppression of apoptosis by neurotrophins [54] but ERK kinases also play a

major role in promoting cell cycle progression and particularly ERK1 and ERK2 are

involved in DNA damage response [63]. In cortical neurons, the activation of ERK1/2

by BDNF suppresses apoptosis induced by DNA damaging agents [54]. Although the

way DNA damage induces ERK activation is still poorly understood, the general

consensus is that MEK mediates ERK activation in DNA damage responses. The use

of MEK inhibitors lead to the inhibition of ERK activation induced by several

genotoxic agents but whether DNA damage activates MEK through Raf remains to

be elucidated. Of note that the impact of the MEK/ERK pathway on checkpoint

activation in DNA damage response is dependent of the cell type [63].

BDNF is known to protect neurons from excitotoxicity through a signalling pathway

that activates NF-κB, a transcription factor that not only induces the expression of

antioxidant enzymes such as Mn-SOD but also induces the expression of Bcl-2, an

anti-apoptotic protein [31]. Regarding the increase in the activity of antioxidant

enzymes, previous studies have indicated that BDNF increased the GPx (glutathione

peroxidase) and GR (glutathione reductase) activities. Neurotrophic factors can

protect neurons against oxidative insults and it has indeed been reported the ability

of BDNF to protect mesencephalic neurons against 1-methyl-4-phenyl-1,2,3,6-

10

tetrahydropyridine (MPTP) toxicity and hippocampal neurons against the

accumulation of ROS (reactive oxygen species) induced by glutamate exposure [32].

1.2 Oxidative Stress, ROS and DNA Damage

Oxidative stress is a state of imbalance in which cells might suffer oxidative

damage. The equilibrium between the biochemical processes that lead to the

production of reactive oxygen species (ROS) and the ones that dispose of such

oxidative compounds is thus impaired [53].

The brain is a relative small organ but its oxygen consumption can be as high as

20% of the body’s total basal oxygen consumption due to its high demand on the

ATP molecule. These high levels of oxygen intake then lead to high levels of ROS

and as such, neurons are exposed to a more oxidative environment than any other

cell type [53].

Reactive oxygen species (ROS) are a group of highly reactive molecules derived

from oxygen. These free radicals contain one or more unpaired electrons which allow

them to act as oxidizing agents. These molecules are more reactive than their

corresponding non-radicals and their presence leads to oxidative aggressions

towards any cellular biomolecules [33].

There is a continuous formation of ROS derived from the normal cellular

metabolism and as the result of some extracellular processes. Peroxisome

metabolism, enzymatic synthesis of nitric oxide, phagocytic leukocytes, heat,

11

ultraviolet (UV) light, therapeutic drugs, ionizing radiation and redox-cycling

compounds are some of the pathways and processes that produce ROS [3]. These

comprise a group composed mainly by hydrogen peroxide (H2O2), superoxide anion

(O2•–), singlet oxygen (1O2) and hydroxyl radical (OH•) [3,33].

In mitochondria, oxygen is mostly converted to water. However, during the

oxidative metabolism almost 2% of this oxygen can be converted into ROS and one

of the reactive species produced is the superoxide anion (O2•–). The superoxide

anion is relatively weak in aqueous media but can be converted to H2O2 either

spontaneously or catalysed by superoxide dismutase (SOD). The H2O2 can in turn be

completely reduced to water or partially reduced to hydroxyl radical (OH•), a very

powerful oxidizing agent. Although it is not as reactive as other ROS, H2O2 plays an

important part in the cellular oxidative damage and carcinogenesis processes as it is

a particularly stable molecule and diffuses easily across biological membranes. Thus,

H2O2 allows other cellular compartments to suffer oxidative damage, further

increasing the cellular injury, particularly if it is converted to the highly reactive

hydroxyl radical (OH•) [33].

Fe2+ + H2O2 � Fe3+ + OH- + OH• Equation 1

Fe3+ + O2•– � Fe2+ + O2 Equation 2

Figure 4 – Hydroxyl radical (OH•) production in mitochondria through Fenton’s

reaction. Equation 1 resumes Fenton reaction. Equation 2 resumes the reduction reaction of

Fe3+ ions. Adapted from Thomas et al (2009) [56].

12

During oxidative stress, the mitochondria can thus produce OH• through the Fenton

reaction. This reaction is depicted in Figure 4 as Equation 1 where low molecular

ions and iron ligands present in mitochondria react with H2O2 to form Fe3+ and OH-

ions as well as the hydroxyl radical (OH•). Fe3+ ions can then interact with another

ROS, the superoxide anion (O2•–) and reduce iron to Fe2+ ions resulting in molecular

oxygen (O2) being produced (Equation 2). The reaction in Equation 2 thus provide for

more Fe2+ ions that can be reused through Fenton’s reaction leading to a OH• influx

that can cause significant biological damage [56].

Despite the cells tendency to eliminate ROS, it is important to refer that these

molecules also play significant roles in the regulation of numerous physiological

processes such as platelet adhesion, neurotransmission and vascular permeability.

The free radical nitric oxide acts as a second messenger in these processes and it is

a highly diffusible molecule derived from L-arginine. In itself, nitric oxide is not highly

reactive to macromolecules, but it can react with the superoxide anion (O2•–) and

produce the strong oxidant peroxynitrate (ONOO–) [33].

The level of ROS in cells is thus inevitably increased as a consequence of the

metabolic stress. If the amount of ROS present in a cell is higher than their ability to

dispose of such reactive molecules, oxidative stress can occur and lead to several

damaging effects namely modifications of proteins, lipids and DNA which in turn lead

to mitochondria and cellular dysfunction [6].

Although ROS can cause damage to any cellular biomolecule, these can usually be

replaced and if their turnover is high, damage may not even accumulate. The DNA

13

molecule however, is the prime information molecule of the cell and nuclear DNA in

particular must last throughout the lifetime of the cell. As such, damage in the vital

molecule seriously threatens the cell function and must be repaired immediately [14].

Any modification to DNA changes its coding properties, the transcription and

replication processes and is thus considered as DNA damage. The lesions that the

DNA molecule can endure include apurinic/apyrimidinic (AP) sites, adducts, single-

strand DNA breaks (SSBs), double-strand DNA breaks (DSBs), crosslinks between

proteins and DNA and also mismatches by insertion or deletion of nucleotides [30].

AP sites are frequent DNA lesions in which the DNA bases are freed from the

deoxyribose backbone. They can be formed both spontaneously and as

intermediates during the process of repairing oxidized, deaminated or alkylated

bases. These AP sites are also one of the major types of damage produced by ROS

in DNA molecules. Approximately 50,000 to 200,000 AP sites can be found in a

mammalian cell induced by ROS action and in fact brain cells comprise most of these

AP sites [30].

DNA damage caused by ROS also includes common lesions such as 8-oxoguanine

(8-oxoG) and thymine glycol (TG). The levels of these lesions are increased in cells

treated with UV light, ionizing irradiation or chemical mutagens that generate oxygen

radicals. 8-oxoG lesions adopt a syn conformation and base pairs with adenine

leading to transversion mutations that can potentially play a role in the process of

ageing and the development of cancer. On the other hand, TG lesions strongly block

DNA replication and transcription and have to be efficiently removed and repaired to

14

maintain a genetic stability. The main DNA repair system that removes these types of

lesions is the base excision repair (BER) system [3].

These 8-oxoG lesions are included in the group of the most frequently studied

oxidized base lesions that also comprise the 8-hydroxy-2-deoxyguanosine (OHdG)

and 5-hydroxyuracil lesions. OHdG lesions can be generated from hydroxyl radicals

(OH•) which arise trough the hemolytic cleavage of H2O2 through the Fenton reaction

or by the decomposition of peroxynitrite (ONOO-), formed by the combination of

superoxide with nitric oxide. OHdG lesions accumulate in specific DNA sequence

sites and it is also postulated that in the genome of the human brain, DNA damage

occurs preferentially in some promotor regions. As previously mentioned, 8-oxoG

and OHdG DNA lesions are mutagenic as they mispair with adenine during the

replication and transcription processes. The accumulation of OHdG lesions in

preapoptotic neurons during retrograde degeneration and in prenecrotic neurons

during ischemic neurodegeneration have also been detected in well-characterized

animal models. 5-hydroxyuracil arises from cytosine oxidation leading to unstable

cytosine glycol which undergoes deamination. This lesion is also premutagenic as it

gives rise to C to T transitions resulting in base transversions [30].

Cells have developed several mechanisms to identify and repair DNA lesions,

which include the use of several DNA repair enzymes such as Endonuclease III

(EndoIII), formamidopyrimidine DNA glycosylase (Fpg) and 8-oxo-guanine-DNA

glycosylase (Ogg1) [13]. Of note that Ogg1 protein in eukaryotes is a DNA glycosylase

that removes 8-oxoG and other oxidized guanine bases from nuclear and

mitochondrial DNA and that is a function analogue of the Fpg protein in bacteria [39].

15

The DNA glycosylase Fpg has AP-lyase activity and specifically recognizes several

oxidized DNA bases including 8-oxoG, 8-oxo-adenine, formamidopyrimidine (fapy)-

guanine, fapy-adenine, 5-hydroxycytosine and 5-hdroxyuracil [64].

1.3 Antioxidant Defences

Antioxidants convert the oxidative compounds to less reactive species and

constitute the first line of defence against the ROS induced cellular damage. As the

ratio of steady-state concentration of oxidants to antioxidants increases, so does

oxidative stress. Cellular response to this type of stress includes DNA repair, cell

cycle arrest and apoptosis [33].

Cellular antioxidants include molecules such as glutathione, α-tocopherol (vitamin

E), carotenoids and ascorbic acid and antioxidant enzymes such as catalase and

glutathione peroxidase. If the levels of reactive oxygen species surpass the capacity

of these cellular antioxidants, then the cell is under the effect of oxidative stress [53].

Neurons, as any other cell type, contain specific enzymes to eliminate ROS from

the cytoplasm. As mentioned above, this enzyme group includes catalase,

glutathione peroxidase (GPx) and superoxide dismutase (SOD) [19].

There are several types of superoxide dismutase (SOD) enzymes depending on

the ion they contain. Copper-zinc-SODs are stable enzymes which are present in the

cytosol, particularly in lysosomes and the nucleus. Manganese-SODs (MnSODs) are

present in yeast and animal mitochondrias while iron-SODs have yet to be

discovered in animal cells [10]. SOD is an enzyme that converts superoxide anion

16

(O2•–) into hydrogen peroxide which, as previously mentioned can readily diffuse

through neural membranes [19].

Catalase activity is mostly located in peroxisomes in all cell types but it is

particularly concentrated in hepatocytes. The catalase enzyme reduces H2O2 to O2

and H2O, particularly when this reactive species is present in high concentrations. In

fact, catalase requires two H2O2 molecules to carry out this reduction reaction and as

such, when the H2O2 concentration is low, its enzymatic activity is reduced. For the

same reason, catalase activity is augmented as the H2O2 concentration increases

and it is very difficult to saturate the catalase enzyme [10].

The GPx enzyme is distributed throughout animal cells and its levels are higher in

the kidney, liver and whole blood. Its substract is the reduced form of glutathione

(GSH) which functions as an electron donor in the removal of H2O2 from the cell. GPx

is considered to be the main peroxide removing enzyme in human cells, presenting a

high specificity to GSH but not to H2O2, as it also reacts with other peroxides [10].

Thus, GPx is reduced, leading to the oxidation of GSH to GSSG, its disulfide redox

partner, ultimately leading to the removal of H2O2 from the cell. The glutathione

reductase (GR) enzyme is then responsible to recycle GSH from its oxidized form

(GSSG) by NADPH oxidation. The glutathione conversion GSH/GSSG is commonly

used as a biomarker of oxidative stress in biological systems. A decrease in

GSH/GSSG ratio could induce mitochondrial membrane structural damage, activity

changes in mitochondrial enzymes and membrane potential leading to mitochondrial

dysfunction and possibly affecting excitability in neurons [61].

17

1.4 Ageing

All organisms are subject to the ageing of its cells and tissues and eventually

death. The study of ageing is a complex area of research and is the result of various

interconnected cellular processes. Many theories have been proposed to explain

ageing, however many of them are not mutually exclusive – see Figure 5.

Two major groups exist within the theories of aging: the genetic and the stochastic

theories. The first are based on the finite number of population doublings of mitotic

cells mediated by the telomeres, while the latter proposes ageing as a consequence

of the accumulation of cellular damage owing to environmental exposure [43].

The somatic mutation theory was formulated by Medawar in 1952, soon after the

discovery of the DNA structure. This theory proposes that the gradual accumulation

of genomic DNA alterations is in the basis for ageing. Mutation in somatic cells that

occur before the reproductive age are select against, however, those that occur

afterwards are not as much as they do not impair the ability to produce descendants.

The rate of somatic mutations would then increase with age correlating to the

increase in mis-repaired DNA damage. These mutations could lead to the production

of abnormal proteins but there is no direct evidence of an age-dependent protein mis-

synthesis, although the rate of protein synthesis is known to be altered with aging

[43,59].

18

Although the somatic mutation theory has not been disproven and it is quite robust,

there is another theory which can better explain the source of cellular damage in

aging. This theory is termed oxidative theory of ageing which refers that aging occurs

as a consequence of the action of free radicals, namely ROS, produced by

mitochondria during the cellular respiration. ROS would then inflict damage upon

various cell organelles and biomolecules leading impaired functions. Most importantly

however, they would generate damage in the mitochondrial DNA leading to positive

feedback in ROS production [43,59].

Figure 5 – Stochastic causes of ageing. Illustration of various factors involved in cellular

ageing [43].

19

1.5 Neurodegenerative Disorders and Oxidative Stress in Neurons

As neurons consume substantial amounts of oxygen, have a low glutathione

content and a high proportion of polyunsaturated fatty acids, they are particularly

vulnerable to oxidative stress. Being postmitotic cells also implies that they cannot be

replaced if irreversible damage occurs. Accumulation of nuclear DNA damage in

neurons has been suggested as one of the main forms of brain aging and

neurodegeneration [6].

Neurodegenerative diseases onset and progression is thought to be dependent in

genetic factors, oxidative stress and the complex interactions between the individual

genetic background and environmental factors. The risks associated with each of

these factors are still poorly understood as well as the relation between neuronal

death and the diseases clinical expression. Neurodegenerative diseases are

characterized by site specific premature and slow death of determined neuronal

population. For example, in Alzheimer’s disease (AD), degeneration of neurons

occurs mainly in the nucleus basalis while, in Parkinson’s disease (PD), the affected

neurons are in the substantia nigra. It is also known that the neuronal populations

affected by neurodegeneration are usually synaptically interconnected, although the

mechanisms associated with the specificity that leads these neurons to cell death is

still not elucidated. Increasing evidence has been suggesting that this specificity may

be involved with alterations in the energy status of degenerative neurons, ubiquitin-

proteasome system defects, presence of aggregates of abnormal proteins (like β-

amyloid and tau proteins in AD), trophic factor deficiency, alterations in cytokine

levels and disruption of both ionic gradient and signal transduction processes [19].

20

1.6 Alzheimer’s Disease and Aβ Peptide

One of the most common neurodegenerative diseases is Alzheimer’s disease (AD),

characterized by mild cognitive impairments at onset and multiple cortical function

deficiencies in the later stages of the disease. Numerous senile plaques and

neurofibrillary tangles accompanied by neuronal death can be observed in the

dementia stages. These plaques are mostly composed of amyloid β peptide (Aβ

peptide). The Aβ peptide is a fragment of the β-amyloid precursor protein or APP,

containing 40 to 42 amino acid residues [2]. APP is a ubiquitously expressed

transmembrane glycoprotein and its secreted form, sAPP, is considered as a cell

survival signal with extensive influence on neuronal development. sAPP is released

in an activity-dependent manner promoting neurite outgrowth and preventing cell

death in hippocampal neurons [20].

As seen in Figure 6, the APP can be cleaved by three different secretase enzymes.

The α-secretase leads to the release of sAPP by cleaving APP in the centre of the β

amyloid domain whereas the β-secretase and γ-secretase action lead to the release

of the Aβ peptide. Following its release, the Aβ peptide can thus form aggregates.

Mutations in the APP gene inhibit the action of the α-secretase enzyme,

consequently allowing the β-secretase cleavage. Mutations in components of the γ-

secretase complex, the presenilin-1 and presenelin-2 (PSEN-1 and PSEN-2) genes,

also increase cleavage by the γ-secretase enzyme. The Aβ peptide production is

thus excessive and it is suggested that the soluble oligomers can impair synaptic

function between neurons. On the other hand, the Aβ aggregates may trigger a local

inflammatory response [40].

21

The APP cleavage by secretase enzymes can thus give rise to two main forms of

the Aβ peptide: Aβ1-40 which is the more soluble form of the Aβ peptide and Aβ1-42

which is the primary component of senile plaques and more neurotoxic than Aβ1-40 [8].

On the other hand, the neurofibrillary tangles are composed of paired helical

filaments aggregated, which in turn are formed by the hyper-phosphorylated form of

tau protein [38].

The amyloid β peptide is derived from APP through an initial β-secretase cleavage

which is followed by an intramembraneous cut by γ-secretase. An early onset of AD

has been associated with an autosomal dominant mutation in APP that results in

increased formation of the Aβ peptide. It is also known that, in primary neuronal

cultures, β-amyloid induces cell death. The tau protein is a microtubule-associated

Figure 6 – APP cleavage by secretase enzymes. The α-secretase cleaves APP in the

middle of the amyloid β domain leading to the release of the normal secreted form of APP.

When APP is cleaved by the β-secretase and γ-secretase it leads to the release of the Aβ

peptide which can thus form aggregates [40].

22

protein that contributes to the stability of these cell structures. The hyper-

phosphorylation of tau leads to the formation of neurofibrillary tangles, which

destabilizes the microtubule network, while its hypo-phosphorylated state has a high

microtubule affinity. This change in function of the tau proteins has been linked to the

disruption of neuronal function and alterations in the distribution of several organelles

including mitochondria [38].

Oxidative stress, inflammation and neurotoxicity are some of the results of Aβ

peptide accumulation in neurons. These processes can then lead to the deterioration

of the neurotransmission systems and ultimately to apoptosis [2]. In AD, the formation

and resulting effects of the senile plaques and neurofibrillary tangles has been

associated with oxidative stress and mitochondrial defects. The oxidation of proteins

often leads to altered protein solubility and hence, an increase in the formation of

protein aggregates. The cytotoxicity induced by multimeric Aβ peptide aggregates

can be explained by ROS production of copper ions which seem to be essential, in

vitro, for the Aβ1-42 inhibition of cytochrome C oxidase terminal complex. Tau protein

is also influenced by oxidative stress as its phosphorylation and aggregation

processes can be modulated by oxidative signals. Moreover, in primary hippocampal

neurons, tau phosphorylation is modified by iron-induced oxidative stress. In primary

neuronal cultures, oxidation by H2O2 induced the dephosphorylation of the tau

protein, but this is thought to have occurred as the concentration of H2O2 used was

high and it might have increased the over-oxidation of fatty acids that is known to

inhibit the polymerization of tau. In fact, another study using a lower H2O2

concentration reached the conclusion that H2O2 increases tau phosphorylation in

23

primary cortical neuron cultures, but the way the oxidative stress alters tau

phosphorylation is yet to be determined. The balance between kinases and

phosphatases does regulate the phosphorylated state of the tau protein and oxidative

changes in either of these enzymes are thought to create an imbalance between their

antagonist activities leading to tau hyper-phosphorylation [38].

The mechanisms by which the Aβ peptide causes cellular death are not fully

understood, however, as previously mentioned, oxidative stress is present in the

early onset of the Alzheimer’s disease (AD) pathology [53]. In in vitro studies, both

Aβ1-42 and Aβ25-35 peptides are known to play a role in oxidative stress leading to the

oxidation of several biomolecules [9,58]. The methionine in position 35 of the wild type

Aβ1-42 is necessary for the oxidative stress and neurotoxic proprieties of this peptide

and it is known to induce the reduction of copper ions (Cu2+) leading to the production

of H2O2 in the absence of cells [24]. The artificial peptide Aβ25-35 has similar oxidative

and neurotoxic proprieties but the mechanisms by which it acts are different. Aβ25-35

induces cell death much faster and its effects are more pronounced. Although this

peptide also displays the methionine 35 residue, its presence in the C-terminal alters

its proprieties and leads to no effect on the copper ion Cu2+, suggesting that the Aβ25-

35 induces oxidative damage by other mechanisms. Also, the presence of this residue

in the peptide is necessary for its oxidative proprieties as the truncated Aβ25-34

peptide, which lacks this terminal methionine, is known to be non-toxic and non-

oxidative [8,9].

A recent study by Arancibia et al has determined that BDNF induces a

neuroprotective effect against Aβ peptide toxicity, both in vivo and in vitro. The

24

collected data reported a dose-dependent toxic effect of β amyloid in cortical

neurons. Aβ25-35 was more toxic for cell survival than Aβ1-42 and although the

protection effect of BDNF had a dose-response result, it differed depending on the

peptide used for β amyloid toxicity. BDNF completely reversed the toxic action of

Aβ1-42 on neuronal survival whereas with Aβ25-35 this reversion was only partial [2]. As

previously mentioned, this neurotrophin can act as an antioxidant factor by increasing

the level of activity of several antioxidant enzymes [31]. As the neuronal loss after

amyloid β exposure, both in vivo and in vitro, can be explained by oxidative damage

caused by Aβ25-35, and that oxidative stress is present at the onset of AD, the

antioxidant defence provided by BDNF could explain its protective effect in Aβ toxicity

[55].

It has also been determined that BDNF and/or its TrkB receptor are impaired in

aging and in AD patients. Moreover, the production and signalling of BDNF in vivo

and in vitro is threatened by Aβ peptide which could ultimately lead to

neurodegeneration. On the other hand, neurons can be rescued from death by the

exogenous addition of this neurotrophin, as BDNF prevents Aβ-induced

neurodegeneration, both in vivo and in vitro [55].

25

2. Objectives

This study aims to determine if BDNF can protect neurons from structural and

oxidative DNA damage. Damage was induced by the Aβ25-35 peptide or by H2O2, a

known DNA damaging agent. As such, the following questions were posed:

o Do neurons accumulate DNA damage as they mature in culture?

o Does Aβ peptide induce structural and/or oxidative DNA damage at sub-

lethal concentrations?

o If Aβ25-35 has an effect on DNA integrity, can BDNF protect neurons

from this oxidative and/or structural damage?

o If Aβ25-35 has no effect, does BDNF protect neurons against the action

of another oxidative agent, such as H2O2?

26

3. Methodology

3.1 – Primary Cell Culture

Pregnant Sprague-Dawley rats, provided by a commercial company (Harlan

Interfauna Iberia, Barcelona, Spain), were anesthetized in a halothane chamber and

sacrificed by decapitation in accordance to the Portuguese animal handling laws.

Under sterile conditions, the 18 days of gestation embryos (E18) were removed from

the uterus and their brains harvested in cold dissection media (HBSS – Hanks’

Balanced Salt Solution – Ca2+ and Mg2+ free supplemented with 0.37% glucose).

After brain dissection and white matter removal, the cerebral cortex was placed in 2.7

mL of fresh dissection media. The cortexes were minced, Trypsin was added and the

minced tissue was incubated at 37ºC for 15 minutes. Cells were then precipitated by

a centrifuging step (188g, 5 minutes), the supernatant removed and cells

ressuspended in Neurobasal® (Gibco®) media supplemented with 10% FBS (Fetal

Bovine Serum), 25 U/mL Pen/Strep (Penicillin/Streptomycin), 0.5 mM glutamine, 2%

B27 supplement and 25 µM Glutamic Acid. The cells were further dissociated by

passing the solution through a pipette several times followed by filtration through a 70

µm cell strainer and ressuspension in Neurobasal® Media supplemented with 25

U/mL Pen/Strep, 0.5 mM glutamine, 2% of B27 supplement and 25 µM Glutamic

Acid. Cellular density was determined in a haemocytometer and neurons were plated

at 3x105 cells / mL (cellular density of 7x104 cells / cm2) in 6 or 24 well plates

previously coated with PDL (poly-D lysine). The cultures were maintained in a 37ºC

incubator with a 5% CO2 atmosphere.

27

3.1.1 – Culture Maintenance

The media used for cultured neurons was supplemented with B27, Pen/Strep and

Glutamine, but without Glutamic Acid. For the maintenance of cells in culture, the

media was changed twice a week, removing half of the culture media and

substituting it with fresh media supplemented with B27. Two days before the

experiment, all culture media is removed and fresh media is added in the absence of

B27 supplement. All cultures were maintained in a 37ºC incubator with a 5% CO2

atmosphere.

3.2 – Immunocytochemistry

For the immunocytochemistry experiments, 24 well plates with coverslips,

previously coated in PDL, were used. At the time of the experiment, the culture

media was removed and the coverslips transferred to another 24 well plate. The

coverslips were then washed twice with PBS (Phosphate Buffer Saline) for 5 minutes

to completely remove Neurobasal® media. Fixation of the DIV 5 and DIV 9 cultures

followed, being performed with a 4% paraformaldehyde solution in PBS for 15

minutes at room temperature. The fixation solution was completely removed by

aspiration and a solution containing 0.05% Triton X-100 in PBS added for 5 minutes

at room temperature. This permeating solution was then removed and substituted by

the blocking solution containing 0.25% gelatine in PBS for 40 minutes at room

temperature. After the removal, the coverslips were washed twice with PBS for 5

minutes. This was followed by 1 hour incubation with the monoclonal primary

antibody solution containing 0.05% Tween 20 and 0.1% gelatine in PBS. The primary

28

antibodies used were the anti-MAP2 (Microtubule Associated Protein 2) mouse

antibody (1:200) and the anti-GFAP (Glial Fibrillary Acidic Protein) rabbit antibody

(1:100). Coverslips were then washed twice with a PBS solution containing 0.05%

Tween 20 for 5 minutes, following which, incubation with the secondary antibody

solution for 1h, containing 0.05% Tween 20 and 0.1% gelatine in PBS was

performed. The anti-mouse secondary antibody (1:500) was conjugated with the

Alexa 488 fluorescent dye (green) and the anti-rabbit secondary antibody (1:500) to

the Alexa 568 fluorescent dye (red). Coverslips were again washed with a PBS

solution containing 0.05% Tween 20 and incubated with DAPI (4’, 6’ – diamidino-2-

phenylindole) (1:15000) for 5 minutes. This is followed by a washing step using the

0.05% Tween 20 in PBS solution and a final washing step using PBS. The coverslips

were then mounted in Mowiol® (Sigma®) before observation in the inverted

fluorescence microscope Axiovert® 200 (Zeiss®) and images captured using the

AxioCamMR3® (Zeiss®) digital camera.

3.3 – Cell Viability

To determine cell viability, a commercial kit named Alamar Blue® (Invitrogen®) was

utilized and the protocol was followed as instructed in the product’s manual. The DIV

1, DIV 5, DIV 9 and DIV 10 cultures were prepared in 24 well plates. The cells were

incubated for 1 hour with a 10% Alamar Blue® solution directly dissolved in the

culture media. All plates were automatically analysed in the Infinite M200® (Tecan®)

plate reader, using a gain of 85 and 25 flashes. Fluorescence values were obtained

using an excitation wavelength of 550 nm and an emission wavelength of 590 nm.

29

The absorbance values were obtained at 570 nm using the 600 nm wavelength as

reference.

3.4 – Comet Assay

3.4.1 – General Considerations

The comet assay, also called single cell gel electrophoresis (SCGE) was described

for the first time in 1984 by Ostling and Johanson. The basis of the assay, however,

relies on work published by Cook et al in 1976 as well as the work from Rydberg and

Johanson in 1978. In 1988 Singh et al described a version of the assay with

increased damage detection range by performing the electrophoresis in highly

alkaline conditions (pH>13) [16]. This version, the alkaline comet assay, quickly gained

popularity as a cheap, adaptable and highly sensitive technique to assess DNA

damage [42].

The comet assay is a versatile technique regarding the source of the biological

material. In fact, almost all types of cells can be used including cell lines, cultured

cells, frozen samples, tissue samples, plant cells and even sperm and prokaryotic

cells [42]. This technique is also very versatile regarding the type of DNA damage

measured as different versions of the assay can be used to observe different kinds of

lesions to the nuclear DNA. The original comet assay, with alterations from Olive et al

in 1990, labelled neutral comet assay, is able to measure both DSB and SSB, while

the alkaline version also measures AP sites in the genome. The comet assay can still

be further extended to measure even more types of DNA damage which is done by

incubating the DNA with lesion specific enzymes such as FPG, endonuclease III, T4

30

endonuclease V and Alk A. Other variants of the assay exist, such as the

Bromodeoxyuridine labelling version to detect replicating DNA, the version using

DNA synthesis inhibitors to detect intermediates of DNA repair and even Fluorescent

in situ hybridization (FISH) Comet to detect gene specific damage and repair [16,41].

A popular version is the oxidative comet assay, performed under alkaline

conditions (pH>13), in which the experimental conditions to be tested are performed

in duplicates with one of the two being incubated with FPG. Since this enzyme

recognizes and removes 8-oxoG lesions as well as ring-opened purines also called

Fapy (Formamidopyrimidines), this assay allows for the simultaneous determination

of structural and oxidative damage to the nuclear DNA [16].

The assay is also a very sensitive and cheap technique to perform, being able to

observe the DNA damage directly without having to rely on expensive antibodies. It

also obviates the need for special equipment such as for gas chromatography-mass

spectrometry (GC-MS) or high-pressure liquid chromatography (HPLC) while, in

addition, being free from these techniques oxidation artefacts which may result in

inaccurate measurements especially when measuring basal levels of oxidative DNA

damage [16]. As the assay is not standardized, its sensitivity and range differs

between different groups, however, Collins et al (2008) estimates this to be from 0.06

to 3 breaks per 109 Daltons of cellular DNA, corresponding to approximately 200 to

10000 breaks per diploid mammalian cell [18].

31

What experimental procedures comprise the alkaline comet assay however, and by

which mechanisms can the DNA damage be measured? The alkaline comet assay

entails the DNA electrophoresis of a population of cells embedded in a thin layer of

LMPA (Low Melting Point Agarose), which is liquid at 37ºC. This embedded

population of cells is subjected to lysis with a non-ionic detergent and high salt

concentrations, removing membranes, cytoplasm, nucleoplasm, disrupting the

A

B

Figure 7 – Diagrams explaining the comet assay technique and mechanisms. A –

Representation of main steps of the comet assay protocol [35]. B – Representation of the

comet assay comet formation process [48].

32

nucleosomes and removing almost all histones. What is left is the nucleoid, a scaffold

of proteins and supercoiled DNA as depicted in Figure 7 [16].

The supercoiled DNA may optionally be incubated with enzymes, such as FPG,

which will recognize and remove specific DNA damage, introducing a SSB. The

comet slides are then incubated in an alkaline solution causing DNA unwinding which

allows the relaxation of the supercoiled DNA. The electrophoresis then creates the

characteristic comet shapes as the more damaged the DNA is, greater DNA

migration into the comet tail will be observed. After staining with a fluorescent dye,

such as ethidium bromide, comet images are acquired and image analysis is

performed, subtracting the background fluorescence and analysing multiple

parameters such as the tail length and the percentage of DNA in tail as depicted in

Figure 8 [16,27].

Figure 8 – Representation of the analysis process used by comet analysis software. A

– Image of an acquired comet. B – Representation of the analysis performed by the image

analysis software on an acquired comet image [27].

A B

33

3.4.2 – Experimental Design

To answer the questions posed in the present work objectives, the experiments

were designed as follows:

o Do neurons accumulate DNA damage as they mature in culture?

To answer this question, the comet assay technique was performed on primary

cultured cortical neurons at DIV 5, DIV 9 and DIV 10. As described in Figure 9, at

DIV 3, the culture media was removed and replaced by fresh media without B27

supplement for the DIV 5 comet assay. Alternatively, half the culture media was

replaced by culture media with B27 supplement for the remaining experiments. At

DIV 7 and DIV 8, the culture media was completely removed and replaced by fresh

media without B27 supplement, and the comet assay was performed at DIV 9 and

DIV 10, respectively.

Figure 9 – Schematic representation of the comet assay timeline. Culture media

replacement and comet assay experiment dates.

34

o Does Aβ25-35 peptide induce structural and/or oxidative DNA damage at

sub-lethal concentrations?

For this second question, the same neuronal cultures were subjected to the comet

assay technique at DIV 5 and DIV 9 after a 24 hour exposure to sub-lethal

concentrations (0.3 µM, 1 µM and 3 µM) of Aβ25-35. As seen in the scheme of Figure

10, at DIV 3 the culture media was completely removed and replaced by fresh media

without B27 supplement. At DIV 4 the cultured neurons were exposed to the sub-

lethal concentrations of Aβ25-35 and at DIV 5 the comet assay was performed.

Alternatively, at DIV3, half the culture media was removed and replaced by media

with B27 supplement. At DIV 7 the media was completely removed, replaced by fresh

media without B27 supplement and at DIV 8 the cultured neurons were also exposed


exposure to Aβ25-35. Culture media replacement, Aβ25-35 exposure and comet assay

experiment dates.

35

to sub-lethal concentrations of Aβ25-35. The comet assay was then performed at

DIV 9.

As this second experiment could have had one of two outcomes, either DNA

damage was induced or not by the Aβ25-35 peptide, the following sub-experiments

were designed:

o If the Aβ25-35 has an effect on DNA integrity, can BDNF protect neurons

from this oxidative and/or structural damage?

As illustrated in Figure 11, the experimental conditions were the same as described

for the previous experiment with the exception of the incubation with BDNF. The

cultured neurons were incubated with this neurotrophin at 20 ng / mL for 48 hours


exposure to Aβ25-35 and a 48 hour incubation with BDNF. Culture media replacement,

BDNF incubation, Aβ25-35 exposure and comet assay experiment dates.

36

and the exposure to the Aβ25-35 peptide was performed 24 hours prior to the comet

assay.

o If the Aβ25-35 has no effect, does BDNF protect neurons against the

action of another oxidative agent, such as H2O2?

In the event that the Aβ25-35 induced no oxidative or structural damage, the cultured

rat cortical neurons were still incubated with BDNF at 20 ng / mL for 48 hours prior to

the comet assay. However, the cells were exposed to H2O2 instead of the Aβ25-35. As

seen in Figure 12, at DIV 3 half the culture media was removed and replaced by

culture media with B27 supplement. At DIV 8 the media was completely removed and

replaced by fresh media without B27 supplement in the presence or absence of


incubation with BDNF and exposed to H202. Culture media replacement, BDNF

incubation, H2O2 exposure and comet assay experiment dates.

37

BDNF. At DIV 10, the cultured neurons were exposed to 10 µM of H2O2 for 10

minutes prior to the comet assay.

3.4.3 – Technique Description

For the comet assay experiments, Superfrosted Rosa® slides were dipped in

absolute ethanol and passed over a blue flame. They were covered with a 1% NMA

(Normal Melting Agarose) in PBS solution, flattened with a 24x60 mm coverslip to a

thin gel layer and let o/n (overnight) to dry. After being subjected to the experimental

conditions in culture, neurons were washed twice with ice cold PBS, scrapped and

recovered to 15 mL Falcon tubes placed on ice. Following the centrifuging step

(700g, 10 minutes, 0ºC), the supernatant was removed and the cells were

ressupended in ice cold PBS. The neurons were then embedded in a 0.7% LMPA

(Low Melting Point Agarose) in PBS solution at 37ºC. The cell solution was carefully

placed over the NMA layer and flattened with a 24x60 mm coverslip. Coverslips were

removed and the slides placed in ice cold lysis solution (2.5 M NaCl, 100 mM EDTA,

10 mM Tris, 1% Triton X-100, pH 10) for 1 hour and 30 minutes. After retrieval from

the lysis solution, slides were washed three times with the FPG buffer (100 mM KCl,

40 mM HEPES, 500 µM EDTA, 200 µg/mL BSA, pH 8). Half were then incubated

with the FPG enzyme solution (1:3000 in FPG buffer) and the other half with the FPG

buffer alone for 30 minutes at 37ºC. The slides were transferred to the

electrophoresis chamber where they were immersed for 20 minutes in the

electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) to allow for DNA

unwinding. After this, an electric field with a voltage of 25 V (Volts) and a 300 mA

38

(mili Ampers) current was applied for 30 minutes (1.48 V / cm). Following removal of

the slides from the electrophoresis chamber, these were covered by the

neutralization solution (400 mM Tris, pH 7.5) for 5 minutes, repeating this step three

times. The agarose gels were then dehydrated in an alcohol series (70%, 95% and

100% ethanol solutions) for 5 minutes in each, and allowed to dry o/n at room

temperature. Staining was performed by covering the slides with a 0.4 µg / mL

Ethidium Bromide (EtBr) solution. Images were acquired in the Axiovert 200M®

(Zeiss®) microscope using the Coolsnap HQ CCD® (Roper Scientific®) 12-bit digital

camera. Image processing and analysis was achieved with Image J® (National

Institute of Health, USA) and with the Casp (Comet Assay Software Project) software.

Data and statistical analysis was performed using the GraphPad Prism® (GraphPad

Software®) program and expressed as mean ± SEM (Standard Error of the Mean).

The one way ANOVA (Analysis of Variance) statistical test followed by the Bonferroni

correction for multiple comparisons was chosen for analysis. Values were considered

statistically significant when the p-value was below 0.05. (For a more detailed

protocol of the performed technique please refer to Annex I)

3.4.3.1 – Electrophoretic Force (V / cm) Calculation

The electrophoretic chamber was measured and the determined dimensions were

introduced to an excel sheet available at: http://comics.vitamib.com/electrophoresis-

physics. The volume of buffer used was also introduced as well as the inner

electrophoresis chamber platform dimensions which allowed the calculation of the

electrophoretic force applied.

39

3.4.3.2 – Standard Cells

A culture of primary neurons was plated in a 143 mm2 Petri dish at a cell density of

9x105 cells / mL. At DIV 3, the cultured neurons were treated with 30 µM of H2O2 for

30 minutes, on ice. The media was removed and cells were washed twice with ice

cold PBS. This was followed by a PBS addition and cell scrapping. The cells were

then ressuspended and centrifuged at 800g for 10 minutes at 0ºC. The supernatant

was removed and ice cold Neurobasal media with 25 U/mL Pen/Strep, 0.5mM

glutamine and 10% FBS was added. The Falcon tube was placed on ice followed by

a cell counting procedure using an haemocytometer. Neurons were further

centrifuged at 800g for 10 minutes at 0ºC, the supernatant removed and

ressuspended at a final density of 3x106 cells / mL in Freezing media (DMEM with

20% FBS and 10% DMSO). The cell solution was then aliquoted and the aliquots

were frozen o/n in an isopropanol chamber until they reached -80ºC. They were then

transferred to a storing box and kept at -80ºC until necessary. (For a more detailed

standard cells protocol please refer to Annex II)

40

4. Results and Discussion

4.1 – The Oxidative Comet Assay: Implementation and Optimization

For the implementation and optimization of the comet assay in the laboratory

several issues had to be overcome. The major ones and their solutions are thus

addressed in the following topics.

The first attempts at implementing this technique used the THP-1 tumour cell line

as it was readily available and was more resistant in culture than the primary

neurons.

One of the initial problems was the discovery that the glass slides originally used

had a defect and auto-fluoresced. The solution was to use Superfrost Rosa® slides,

available in the laboratory, instead of plain ones. Early versions of the comet assay

used fully frosted slides instead of agarose-coated plain ones and are reported to

achieve a good gel anchorage. However, high background fluorescence due to a

light scattering effect on the frosted surface and the need of constant refocusing to

acquire comets on different planes was noted [17]. The usage of superfrosted slides in

conjunction with an NMA markedly reduced background fluorescence and did not

require constant refocus to acquire comet images. This is probably due to the

smoother surface of superfrosted slides, when compared with fully frosted ones, and

to the NMA coating that provides a separation and plane levelling from the

superfrosted slide to the comets. Albeit more expensive than the use of plain coated

slides, an increased gel anchorage was observed with this method.

41

The optimization of the agarose percentage on gels, both for the NMA and LMPA

was found to be of 1% and 0.7%, respectively. These percentages allowed a good

anchorage of the gels to the glass slides throughout the experiment.

For transporting the slides throughout the experiment, a dark moisture box was

built that protected the samples from light induced DNA damage and preserved both

moisture and fluorescence at different stages of the assay.

Although most comet assay protocols instruct the storage of the assay solutions at

4ºC, several fungal contaminations were observed and were prevented by storing the

solutions at -20ºC.

In order to test if the assay was working, the THP-1 cells were treated with various

concentrations of etoposide. This compound is a genotoxic agent that induces DNA

damage through the inhibition of the topoisomerase II. It is a highly toxic compound,

which quickly induces DNA damage in cells [26]. As such, it was used to assess if the

technical procedures allowed the observation and quantification of DNA damage, and

whether the assay could be used to discriminate between different levels of damage.

As seen in Figure 13, as the concentration of etoposide increases so does the tail

length and percentage of DNA in tail of the obtained comets. Of note that the comet

data does not fit a normal distribution but instead are better fitted in the Weibull

probability distribution. The consequence of this is that at least 30 comets must be

acquired per experimental condition as implicated in the central limit theorem. This

theorem implicates that all distributions are approached to the normal distribution as

the sample size increases [28].

42

In Figure 14 the mean values obtained for the tail length of the analysed comets

are shown. As can be seen in the panel B, the tail length parameter quickly rises and

soon reaches a plateau in which no further damage can be distinguished. Indeed

near half maximal damage is already atained with the first concentration of etoposide

used (1 µM). In the panel C the mean value of tail length at 50 µM is excluded which

removes the plateau effect on the graph. Although the tail length parameter is

extremely sensitive and discriminative between very low amount of damage, its

progression is not linear [16]. In the oxidative comet assay, the slide incubated with

FPG allows the detection of both structural and oxidative damage while the non

treated slide only allows the detection of the structural DNA damage. In principle, it

Figure 13 – Comet assay using the THP-1 cell line after incubation with etoposide for

one hour at several concentrations. A – 0 µM; B – 1 µM; C – 5 µM; D – 10 µM; E – 50 µM.

A B C

D E

43

would be possible to infer the oxidative DNA damage by simply subtracting these

values but if the data does not follow a linear progression such calculation will give

inacurate quantitive results, when subctracting different values of structural DNA

damage [16].

A quantitative analysis can still be performed in a particular condition: if the values

of tail length for structural DNA damage are constant between two different

Figure 14 – Graphic representations of the comet assay tail length data using the THP-

1 cell line after incubation with etoposide for one hour at several concentrations (n=1;

30 comets in average per slide). A – Histogram of the mean tail length values; B – Scatter

plot with a fitted tendency line to the mean tail length values; C – Scatter plot with a fitted

tendency line to the mean tail length values excluding the mean value at 50 µM.

CTRL 1 5 10 500

100

200

300

400

Tail Length

Etoposide (µM)

Tail

Len

gth

(µ

m)

A Tail Length

0 20 40 60

0

100

200

300

400

Etoposide (µM)

Tail

Len

gth

(µ

m)

B

Tail Length

0 5 10 15

0

100

200

300

400

Etoposide (µM)

Tail

Len

gth

(µ

m)

C

44

Figure 15 – Graphic representations of the comet assay percentage of DNA in tail data

using the THP-1 cell line after incubation with etoposide for one hour at several

concentrations (n=1; 30 comets in average per slide). A – Histogram of the mean % of

DNA in tail values; B – Scatter plot with a fitted tendency line to the mean % of DNA in tail

values; C – Scatter plot with a fitted tendency line to the mean % of DNA in tail values

excluding the mean value at 50 µM.

CTRL 1 5 10 500

20

40

60

80

100

% DNA in Tail

Etoposide (µM)

% D

NA

in

Tail

A % DNA in Tail

0 20 40 60

0

20

40

60

80

100

Etoposide (µM)

% D

NA

in

Tail

B

% DNA in Tail

0 5 10 15

0

20

40

60

80

100

Etoposide (µM)

% D

NA

in

Tail

C

experimental conditions, their oxidative values may be directly compared. This is due

to the fact that subtracting constant structural damage values to the total DNA

damage indicates that the deviation to the linearity of the total DNA damage values

will exclusively be derived from the oxidative damage.

Another situation in which a similar, although qualitative, analysis can be performed

is when the deviation to the linearity occurs in the same direction in both structural

45

and oxidative DNA damage for the two different conditions. If both structural and total

DNA damage values have a tendency to increase between the different experimental

conditions, then the oxidative DNA damage values must have the same tendency.

The same is valid if the tendency between the experimental conditions is a decrease

both in structural and total DNA damage values.

Regarding the percentage of DNA in tail, whose results can be seen in Figure 15, it

is indicated as the most versatile parameter as not only determines the greatest

amplitude of damage levels but does not saturate either into a plateau upon reaching

the maximum DNA damage discerned by the assay [16].

It is important to highlight, as becomes evident by comparing graphs B and C of

Figure 15, that the linear progression of this parameter is proven at least up to 70%

of DNA damage but according to Collins (2004), in theory, it can even reach 100%.

This allows for a quantitative determination of the oxidative DNA damage values,

unfortunately for low amounts of damage this parameter is not as accurate as the tail

length [16].

After determining that the assay was correctly functioning with the THP-1 cell line,

the procedure was adapted to the primary cultured rat cortical neurons. One of the

first problems in this adaptation was the insufficient number of cells available. This

was overcome with the use of a centrifuge with a swinging-bucket rotor which

allowed for a greater number of available neurons concentrated at the end of the

centrifugation tube.

46

A greater problem however, was the quality of the comets obtained. Instead of the

well-defined comets obtained earlier with the THP-1 cell line, these often appeared

“blurred” and “blasted” and unsuitable for analysis as shown in Figure 16. A paper by

the author that described the alkaline comet assay, proposed a parallel assay whose

purpose is to determine the percentage of apoptotic cells, allowing the differentiation

between necrotic and apoptotic ones [49]. The necrotic halos shown in that paper

have a great resemblance to the comets obtained in these initial experiments. The

causes for these “blurred” comets were then found to be related to the use of the

thermoblock and to the strong ressuspension during the cells agarose embedment

step. The thermoblock, which allowed for a less rushed cell embedment step using

the THP-1 cell line, proved to be contributing to neuronal necrosis probably due to a

quick temperature increase. The strong ressuspension in the LMPA agarose

increased this necrotic process as the neurons are highly sensitive to the shearing

forces of such a high density media.

Once the assay was successfully adapted to primary cultured neurons, it was

necessary to ensure that it was reliable and consistent in terms of results, as well as

to have an idea of the variability associated with the assay. To this end, Collins

A B

Figure 16 – Image of comets obtained from primary cultured cortical neurons exposed

to H2O2. A – Normal comet; B – “Blurred” or “Blasted” comet.

47

Tail Length

Structural Damage Oxidative Damage

0

100

200

300

400

Tail

Len

gth

(µ

m)

A % DNA in Tail

Structural Damage Oxidative Damage0

20

40

60

80

100

% D

NA

in

Tail

B

Figure 17 – Graphic representations of the standard cells tail length and percentage of

DNA in tail data mean values (70 comets in average per slide). A – Box and whiskers plot

of the mean tail length values (n=3); B – Box and whiskers plot of the mean % of DNA in tail

values (n=5).

suggests the use of standard cells [16]. These comprise a greater batch of the same

cells as the ones used in the assay, subjected to the same exposure compound,

aliquoted, frozen at -80ºC and used in all subsequent assays. The use of standard

cells would then give the same amount of damage in each assay and any deviation

from these values would indicate that some aspects of the assay had changed [16].

For the present work, the standard cells consisted of a large batch of cultured

neurons exposed to a 30 µM concentration of H2O2 for 30 minutes, then scraped,

counted, aliquoted into microcentrifuge tubes with Freezing Media and frozen at -

80ºC.(See Annex II)

The use of standard cells removes the variability associated with the use of

different cultures and, as such, detects the small intrinsic variations associated with

performing the assay at different times. The mean values for all measurements of

48

both structural and oxidative DNA damage in the standard cells can be observed in

Figure 17.

Regarding the structural DNA damage, in panel A and B of Figure 17, it can be

concluded that in different experiments performed in different days the results from

the standard cells are consistent, presenting a low variation either for the tail length,

with a mean value of about 150 µm, and the percentage of DNA in tail, with a mean

value of about 31%. For the oxidative DNA damage values in graph A of Figure 17,

the calculated mean tail length is 56 µm. However, as previously described, this

parameter is not accurate for calculating the oxidative damage as it does not follow a

linear progression. This mean value of 56 µm, although consistent, can only be

regarded as a qualitative measurement in arbitrary units, rather than a true

quantitative one. Of note that two experiments were removed from the tail length data

set as their respective slides had debris present and these were regarded by the

analysis software Casp as belonging to the comets. These debris than distorted the

analysis of the tail length parameter as their fluorescence intensity falsely increased

the tail length of the obtained comets, but as their DNA amount is very low, its effect

is negligible when considering the percentage of DNA in tail. Considering the panel B

in Figure 17, the oxidative DNA damage has a mean value of 36% DNA in tail. The

consistency of both this value and the mean tail length value, for oxidative DNA

damage, suggests a correct implementation of the incubation with FPG in the comet

assay. These results can thus provide a reliable proof of the assay effectiveness and.

correct implementation.

49

Several published papers and comet assay protocols specify an electrophoretic

voltage of 24 or 25 volts and a current of 300 mA, but fail to include the calculation of

the amount of voltage per cm across the gel. Thus, the electric field driving the DNA

migration can, and often is, different between different studies due to different

electrophoretic chamber dimensions [18]. Ideally the voltage per centimetre should be

0.8 V/cm as referred [48], however some papers use electrophoretic forces as low as

0.4 [34] and up to 1.5 V/cm [36,51]. The calculation for the present work indicates the

use of 1.48 volts per cm which is acceptable [48].

4.2 – Cell Culture Characterization: Are the cultured neurons viable at the

times of the experiment? What percentage of glial cells is present in culture?

In order to ensure the validity of the results obtained through usage of the comet

assay and determine the metabolic viability of the cultured neurons, two experiments

using Alamar Blue® were performed. At DIV 1, DIV 5, DIV 9 and DIV 10, the cultured

neurons were incubated with a 10% Alamar Blue® solution for one hour. At the end of

the incubation period, the fluorescence and absorbance values were determined

which correspond to the conversion of resazurin into resorufin by the reductive

environment produced by the living cells. These values are thus dependent of both

the viability and metabolic rates.

Since Alamar Blue® in effect measures the metabolic activity of cultured cells it is

not a true viability assay as many variables are at play. Its use has been employed to

50

assess cell viability after exposure to a toxic compound as well as to assess cell

proliferation and even the cells metabolic rate [11].

As observed in Figure 18, the obtained results evidence an increasing reductive

environment during the cells maturation in culture which would be compatible with

cell proliferation. However neurons are post-mitotic cells unable to proliferate. This

can have two different non-exclusive explanations, one being increased metabolic

activity in mature neurons, the other being the proliferation of non-neuronal cells such

as astrocytes and glial cells.

Previous experiments in the laboratory performed by A. Santos (2009) have

previously addressed the issue of astrocyte and glial contamination in the exact

same experimental conditions. This was assessed through an immunocytochemistry

assay using an anti-MAP-2 antibody which labels neurons and an anti-GFAP

antibody labelling both glial cells and immature neurons and also using DAPI to label

the cell nucleus [46].

Figure 18 – Graphics of the fluorescence and absorbance data obtained at various DIVs.

The fluorescence values were obtained at 590nm and the absorbance values at 570nm.

Fluorescence

DIV 1 DIV 5 DIV 9 DIV 100

5000

10000

15000

20000

Arb

itra

ry U

nit

s

Absorbance

DIV 1 DIV 5 DIV 9 DIV 100.030

0.035

0.040

0.045

0.050

0.055

0.060

Arb

itra

ry U

nit

s

51

His findings shows that at DIV 4, 80% of the total cells are neurons and at DIV 10

the neuronal percentage has been reduced to 50%, the rest corresponding to glial

cells either reactive labelled with GFAP, or non-reactive and thus only visible through

DAPI labelling [46]. This has been confirmed with immunocytochemistry experiments

performed in this study, results being depicted in Figures 19 and 20.

Figure 19 – Overview of a neuronal culture. Immunocytochemistry assay performed at DIV

5 (A) and at DIV 10 (B). Images obtained at a magnification of 100X. Neurons shown in green

labelled with the anti-MAP-2 antibody (λ=488nm) and astrocytes as well as immature neurons

shown in red labelled with the anti-GFAP antibody (λ=568nm). Non-reactive glial cells are

visible only through DAPI staining.

Figure 20 – Detail of an astrocyte at the

centre of the image. Immunocytochemistry

experiment performed at DIV 5. Image

obtained at a magnification of 400X.

Neurons shown in green labelled with the

anti-MAP-2 antibody (λ=488nm) and

astrocytes as well as immature neurons

shown in red labelled with the anti-GFAP

antibody (λ=568nm). Non-reactive glial cells

are visible only through DAPI staining.

A B

52

AraC is a nucleoside analog (cytosine arabinoside) that is used to induce apoptosis

in mitotic cells and as such obtain pure neuronal cultures, but is known that araC also

exerts an apoptotic effect in postmitotic neurons by two distinct mechanisms [1].

Initially at DIV 3 and at each subsequent media change, araC was added to the

culture medium at a final concentration of 10 µM. However, this greatly reduced the

culture viability and several morphologic changes were visible in the culture such as

neuronal aggregates. This alone greatly increased the difficulties of performing the

comet assay with mature neurons as the available number of cells after scrapping

became very low, and with a high prevalence of cellular debris.

Another reason against the usage of pure neuronal cultures for the comet assay

was indicated by the fact that the tail length of the comets appeared greater in the

preliminary experiments (data not shown) which could be derived from the action

mechanism of the araC compound. The purpose of the comet assay is to evaluate

DNA damage and thus it can be very sensitive to the presence of this compound in

culture.

Besides the increased technical difficulties of using pure neuronal cultures, the

araC compound is known to inhibit the DNA repair mechanisms [62] and thus may

alter the basal level of DNA damage present in cultured cells which explains the

increased levels of DNA damage measured by comet assay. This increased amount

of basal DNA damage in cultures would lead to difficulties in comparing results

between cultures of different DIVs as the period of exposure to araC would differ.

Even then, obtained results could not be attributed with certainty to the effects of the

53

Aβ peptide 25-35 or the H2O2, and the question of possible interaction or cumulative

effects with the araC would remain. In light of the above, the disadvantages of using

pure neuronal cultures far outweigh the advantages and as such araC was not added

to the cultures for the remaining experiments.

In the interest of culture characterization it would also have been useful to ascertain

the neuronal viability after exposure to the Aβ peptide 25-35 or the H2O2.

Experiments using Alamar Blue® to assess the neuronal viability after exposure to

H2O2 were indeed performed. However the results were not coherent as both the

fluorescence and absorbance values were wildly variable (data not shown). This can

probably be explained due to the oxidative nature of H2O2 and the possible effect of

BDNF on the regulation of the activity of antioxidant enzymes in neurons [32]. The

exposure to H2O2 possibly alters the reductive environment of cells and since Alamar

Blue® is a redox-sensitive dye thus making this assay unreliable to measure cell

viability [11,65].

An equivalent experiment for the Aβ peptide 25-35 was not conducted. However

there are published results for similar experimental conditions to those of this study

both for neuronal viability after exposure to H2O2 [22] and to the Aβ peptide 25-35

[2].

Neuronal cultured cells were exposed to either 10 µM of H2O2 for 10 minutes at DIV

10, in the comet assay experiments, or 30 µM for 30 minutes at DIV 3 for the

standard cells [see Annex II]. At the referred conditions of H2O2 exposure, Hoyt et al

report that the cell viability, for the comet assay experiments, would be around 50%,

while for the standard cells the cell viability would be approximately zero. Despite the

54

similarities between both studies, it is important to note that these calculations were

based on DIV 14 cultures and that cellular viability was only assessed after a 20 hour

period post exposure. These authors then calculated neuronal viability through a cell

counting method in which the non-exposed cultures were compared with cultures

exposed at various H2O2 concentrations and time periods [22].

Other authors such as Schwartz et al, used DIV 4 rat cortical neurons incubated

with 5 µM and 100 µM H2O2 for a up to 24 hour time period to study to induction of

repairable or non-repairable DNA damage. Their data reveals that the 5 µM proved

non-toxic for DIV 4 neurons while the 100 µM H2O2 induced a loss of viability of 60%

when incubated for a 24 hour period [47].

As such, these viability values cannot be directly compared to the present study.

Both during the comet assay and the standard cells preparation the cells are washed

twice in ice cold PBS after toxic exposure, the culture plate is placed over ice and all

subsequent procedures until cell lysis or cell freezing occur at near 0ºC. This slows

the cells metabolism and either the assay is performed with cells kept in this state, or

in the case of the standard cells, the neurons are frozen to -80ºC. Although some

necrosis is possible, it is thus unlikely that apoptotic procedures and nuclear

degradation can occur during the chosen toxic exposure time frame [4].

Regarding the effects of the Aβ peptide25-35 on neuronal viability, a paper by

Arancibia et al (2008) describes the cell death after exposure to various

concentrations of Aβ25-35 during a 48 hour period [2]. They determined that exposure

to the Aβ25-35 for a prolonged period of time has a significant effect upon neuronal

55


neuronal culture matures. Comet assay performed at DIV 5 (n=3), DIV 9 (n=6) and DIV 10

(n=2). The values refer to the medium tail length of the comets acquired. In average, 70

comets per slide were obtained at DIV 5 and DIV 9 and 120 comets at DIV 10.

viability however for low concentrations such as up to 5 µM the cell viability is still

greater than 80%. The authors then described these concentrations as sub-lethal.

Since the main purpose of the present study is to obtain data on the oxidative DNA

damage of neuronal cells, sub-lethal concentrations of Aβ25-35 of up to 3 µM were

selected and the exposure time limited to a 24 hour period.

4.3 – Do neurons accumulate DNA damage, either structural or oxidative, as

they mature in culture?

The evidence that cells accumulate oxidative damage in eukaryotic organisms as

they age has been in the foundation of the oxidative theory of ageing. Several studies

have found some evidence that supports this theory [59]. As neurons mature in

culture, does their nuclear DNA become more oxidized? In order to answer this

question, the DNA damage data from the control slides of DIV 5, DIV 9 and DIV 10

were compared.

As can be seen in Figure 21, the tail length has a tendency to increase as the

56

neurons mature in culture, suggesting that neurons do accumulate both oxidative and

structural DNA damage. However, it is important to remember that these conclusions

can only be drawn by the analysis of tail length values. Considering the percentage

of DNA in tail (see Figure 22), these conclusions are not evident as it appears that

both oxidative and structural DNA damage is maintained unaltered as the neurons

mature in culture. As referred above in 4.1 (The Oxidative Comet Assay:

Implementation and Optimization), the tail length parameter should be preferred as it

is known that for low amounts of damage, it presents a greater sensitivity despite that

after a certain amount of damage it also reaches a plateau.

Despite the consistent results, the question remains whether the accumulation of

oxidative damage does occur regardless of the use of the B27 supplement in the

culture media. As previous studies referred, this supplement might protect neurons

from apoptosis [41] when exposed to the Aβ25-35, and as such, the media was

DIV 5 DIV 9 DIV 10

0

20

40

60

80

100

Structural % of DNA in Tail

% D

NA

in

Tail

DIV 5 DIV 9 DIV 10

0

20

40

60

80

100

Oxidative % of DNA in Tail

% D

NA

in

Tail


neuronal culture matures. Comet assay performed at DIV 5 (n=3), DIV 9 (n=6) and DIV 10

(n=2). The values refer to the medium percentage of DNA in tail of the comets acquired. In

average, 70 comets per slide were obtained at DIV 5 and DIV 9 and 120 comets at DIV 10.

57

removed 48h prior to the comet assays and replaced with fresh media without B27.

This two day period of starving was a compromise between the total removal of the

B27 supplement from the culture media, which would lead to cell death and thus

impair the acquisition of results from mature neurons, and the maintenance of this

supplement which, as reported before, could protect the neurons and mask any DNA

damage induced by either the Aβ25-35 or the H2O2.

CTRL 1 µµµµM Aββββ 1 µµµµM Aββββ w/ B27

0

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Figure 23 – Graphic representation of structural and oxidative DNA damage caused in

the presence of the Aβ25-35 and in the presence or absence of the B27 supplement.

Comet assay performed at DIV 5 (70 comets in average per slide, n=3; except for the

experimental condition without B27 supplement – n=2). The values refer to the mean values

of the comet parameters, tail length and percentage of DNA in tail. A – Histogram of the

structural damage tail length mean values; B – Histogram of the structural damage

percentage of DNA in tail mean values; C – Histogram of the oxidative damage tail length

mean values; D – Histogram of the oxidative damage percentage of DNA in tail mean values.

58

Regardless of the chosen compromise, the presence of B27 could even alter the

basal levels of DNA damage which in turn could invalidate the obtained results.

However, experiments conducted with the Aβ25-35 in the presence and absence of

B27 at DIV 5 and DIV 9, revealed that this supplement appears to have no effect

whatsoever. As seen in the different panels shown in Figures 23 and 24, the amount


0

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A B

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Figure 24 – Graphic representation of structural and oxidative DNA damage caused in

the presence of the Aβ25-35 and in the presence or absence of the B27 supplement.

Comet assay performed at DIV 9 (70 comets in average per slide, n=3; except for the

experimental condition without B27 supplement – n=2). The values refer to the mean values

of the comet parameters, tail length and percentage of DNA in tail. A – Histogram of the

structural damage tail length mean values; B – Histogram of the structural damage



59

of DNA damage, either structural or oxidative, is the same either in the presence or

absence of the B27 supplement for all comet parameters.

An increase in the basal levels of both structural and oxidative DNA damage is

present when comparing between the DIV 5 and DIV 9, this increase is however the

same for all experimental conditions. This implies that the removal of B27

supplement 48 hours prior to assay did not exert an effect upon the basal levels of

DNA damage.

Although further studies would be necessary to obtain statistical significance, it is

probable that neurons do accumulate both structural and oxidative DNA damage as

they mature in culture or have increasingly higher levels of basal DNA damage due

to less effective DNA repair mechanisms.

4.4 – Does Aβ25-35 peptide induce structural and/or oxidative DNA damage at

sub-lethal concentrations?

Several studies in recent years [2,37] have found a significant neuronal death when

primary cultured neurons are exposed to both Aβ1-42 and Aβ25-35 peptides. However,

the concentrations used ranged around 20 µM for periods of 48 hours [2,37].

In the present work, the Aβ25-35 concentration was far below, reaching only 3 µM

and the exposure time was reduced to 24 hours. These conditions were chosen as

the objective of this work was to determine whether the Aβ25-35 caused oxidative DNA

damage in sub-lethal concentrations.

60

The mechanisms by which the Aβ peptide exerts its effects it is not yet fully

understood. In vitro studies have, however, demonstrated the ability of Aβ1-42 to

induce the production of H2O2 using Cu2+ and other ions even in the absence of cells.

On the other hand, in the same conditions, the Aβ25-35 proved unable to do so [24]. In

spite of this, Aβ25-35 can cause protein oxidation [58] and thus we asked whether it

could cause nuclear DNA damage.

The interest of this peptide was even reinforced by the report that in AD brains

there is a twofold increase in DNA strand breaks and that the levels of oxidized DNA

bases are increased, particularly 8-OHdG [58].

As seen in Figure 25 and 26, the data suggests that incubation with Aβ25-35 (3 µM)

for 24 hours has no effect on either structural or oxidative DNA damage. Regarding

the DIV 5 data in Figure 25, it shows no difference on the levels of DNA damage

between the control and the experimental conditions. The same situation is observed

at DIV 9 in Figure 26. Although there is a difference between the basal levels of

damage between DIV 5 and DIV 9 neurons, as the DNA at DIV 9 has greater

structural and oxidative damage, this corresponds to the normal culture maturation

process and it is not related to the Aβ25-35 exposure. Of note the necessity of

evaluating the oxidative damage through the percentage of DNA in tail parameter

when comparing between DIVs, as the oxidative tail length parameter cannot be

compared due to the different amounts of structural damage and the non-linearity of

this parameter previously described.

61

These results can be explained by two non-mutually exclusive phenomena. Either:

1) the Aβ25-35 exposure concentrations were too low to cause DNA damage, the cells

being able to completely repair DNA damage at the studied concentrations; 2) or the

Aβ25-35 although able to cause protein oxidations is not able to induce DNA damage.

Figure 25 – Graphic representation of structural and oxidative DNA damage caused by

sub-lethal concentrations of Aβ25-35. Cultured cells exposed to Aβ25-35 at DIV4; Comet assay

performed at DIV 5 (70 comets in average per slide, n=3). The values refer to the mean

values of the comet parameters, tail length and percentage of DNA in tail. A – Histogram of

the structural damage tail length mean values; B – Histogram of the structural damage



B

CTRL 0.3 µµµµM Aββββ 1 µµµµM Aββββ 3 µµµµM Aββββ

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62

Some published data indicates the ability of the Aβ25-35 to induce structural DNA

damage to primary cultured cortical neurons. However, this is not easily accessed as

the paper is written in chinese with only an english abstract available. The available

data reports that the use of 25 µM of Aβ25-35 only induced a very small level of

structural DNA damage and the oxidative DNA damage was not determined. Further


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Figure 26 – Graphic representation of structural and oxidative DNA damage caused by

sub-lethal concentrations of Aβ25-35. Cultured cells exposed to Aβ25-35 at DIV8; Comet assay

performed at DIV 9 (70 comets in average per slide, n=3). The values refer to the mean

values of the comet parameters, tail length and percentage of DNA in tail. A – Histogram of

the structural damage tail length mean values; B – Histogram of the structural damage



63

studies, at a higher concentration of Aβ25-35, are thus necessary to determine its

effect upon nuclear DNA damage.

4.5 - Does BDNF protect against H2O2 induced DNA damage?

As the Aβ25-35 peptide did not induce DNA damage in cultured rat cortical neurons

at the sub-lethal concentrations tested, the neuroprotective effect of BDNF upon

induced DNA damage could not be studied. I therefore assessed the putative

protective effect of BDNF by exposing neurons to a stronger oxidizing agent, H2O2, a

known product of Aβ1-42 in vivo.

The results illustrated in Figure 27 clearly show increased levels of DNA damage

on neurons exposed to 10 µM H2O2 for 10 minutes. As with the results from the

exposure to the Aβ25-35 peptide care must be taken to interpreter the tail length

parameter information. Regarding the structural damage evaluated through tail length

it is important to note that neurons exposed to H2O2 as well as neurons pre-incubated

with BDNF for 48 hours show similar levels of structural damage. However, H2O2 is a

strong oxidizing agent and therefore, the tail length parameter has already reached

its plateau phase, precluding any conclusion based upon this parameter. This is

consubstantiated when analysing the oxidative tail length parameter, as reading the

data at face value would imply that H2O2 as well as a BDNF pre-incubation followed

by H2O2 exposure would decrease the basal levels of oxidative damage. This is an

artefact, in reality the increased tail length in the control condition, visible in the panel

C, corresponds to the tail length parameter reaching its plateau in the FPG incubated

64

slide (structural + oxidative damage) and having the non-plateau value of the non-

FPG incubated slide subtracted from it, the other conditions having reached the

plateau in the non-incubated slide.


exposure to H2O2 with or without a pre-incubation with BDNF. Comet assay performed at

DIV 10 (120 comets in average per slide, n=2). The values refer to the mean values of the

comet parameters, tail length and percentage of DNA in tail. A – Histogram of the structural

damage tail length mean values; B – Histogram of the structural damage percentage of DNA

in tail mean values; C – Histogram of the oxidative damage tail length mean values; D –

Histogram of the oxidative damage percentage of DNA in tail mean values. * – p<0.05; ** –

p<0.01; Φ – p<0.05.

CTRL H2O2 H2O2 + BDNF0

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65

Due to the strong oxidizing nature of H2O2, the percentage of DNA in tail parameter

is the most suitable for analysis since it allows the discrimination of the widest range

of DNA lesions for the assay. Figure 28 shows a detail of the data set seen in Figure

27 regarding this parameter and as can be seen, H2O2 exposure increased both

structural and oxidative DNA damage. These values are statistically significant with a

p-value of less than 0.01. Most importantly, however, is the fact that the H2O2

exposed and BDNF incubated neurons had reduction in the DNA damage levels

when compared to those exposed to H2O2 but not incubated with BDNF. Although

this reduction is small and not statistically significant regarding the structural damage,

for the oxidative DNA damage a statistically significant decrease (p<0.05) was

observed. It is apparent that the BDNF protection is more effective in oxidative DNA

damage rather than in structural damage. This probably occurs due to the increased

activity levels of antioxidant enzymes, especially the glutathione reductase and

glutathione peroxidase, that is known to occur after pre-incubation with BDNF [32].


0

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30


** *

% D

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in

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φ

**

*

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in

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exposure to H2O2 in the presence or absence of BDNF. Comet assay performed at

DIV 10 (120 comets in average per slide, n=2). The data refers to the mean values of the

percentage of DNA in tail parameter. * – p<0.05; ** – p<0.01; Φ – p<0.05.

66

A control experiment without exposure to H2O2 but with incubation with BDNF was

performed (data not shown, n=1). However, debris were present in the FPG

incubated slide and a single experiment could not be directly compared to the other

data sets. As this lead to the compromise of the oxidative tail length parameter these

values were not represented in Figure 27. While the percentage of DNA in tail

parameter indicates that BDNF has no adverse effect upon neuronal DNA and may

in fact slightly protect against basal structural DNA damage (data not shown), further

experiments are necessary to confirm this.

Recent studies regarding DNA repair and apoptosis of cortical neurons in the

presence of H2O2 [47,57] have been reported as well as BDNF protection of apoptosis

in the presence of the same compound [5]. Terminally differentiated neurons have

been shown to activate the cell cycle machinery and to undergo the G0 → G1

transition to efficiently repair induced DSB [47,57]. Blockage of this transition, in

neurons exposed to lethal concentrations of H2O2 impaired both DNA repair as well

as apoptosis [47,57], however, blockage of the G1 → S transition prevented apoptosis

without impairing DNA repair [47]. Neurons exposed to sub-lethal concentrations of

H2O2 were also shown to undergo the G0 → G1 transition, but not the G1 → S [47].

There is also evidence that G0 → G1 transition may be influenced by the cytoplasmic

concentration of H2O2 via ERK signalling [7].

Studies that look upon the signalling pathways of BDNF neuroprotection often use

co-administration with high concentrations of H2O2 to study its effect on apoptosis.

There is in fact evidence that points to the anti-apoptotic effect of BDNF signalling via

MAPK/ERK in oxidative stress conditions and via PI3-K/Akt, as well as MAPK/ERK,

67

in excitotoxic stress [5]. BDNF is thought to have an inhibitory effect upon the

MAPK/ERK pathway activated by oxidative stress [5]. This is possibly derived from

the effect of KSR1 on ERK1/2 preventing its nuclear localization and leading to an

inhibition of phosphorylation of the pRb and release of the E2F1 which mediates the

G1 → S transition and leads to apoptosis [5,12,54].

The data obtained in this study however, suggests that BDNF protection of nuclear

DNA occurs independently of BDNF anti-apoptotic effects. Although BDNF is most

likely also exerting its anti-apoptotic effect upon neurons, the experimental design

and time frame does not allow for apoptosis to occur.

68

5. Conclusions

The comet assay was correctly implemented in primary rat cortical neurons, which

allows future studies on DNA damage in neurons, and even glial cells, to be

performed in the laboratory.

Although not statistically significant, a tendency depicting that mature neurons have

higher, although still low, levels of DNA damage, both structural and oxidative, when

compared with DNA damage levels of more immature ones was found. This was

expected as a body of evidence refers that aged cells in vivo have their nuclear DNA

in a more damaged state, especially regarding oxidative DNA damage.

The Aβ25-35 peptide was not found to induce DNA damage of any kind at the tested

sub-lethal concentrations. This may be due to the low concentrations used,

comparing with most in vitro studies, and higher concentrations should be tested to

account for this possibility. Or, the Aβ25-35 peptide may be unable to induce nuclear

DNA damage of any kind, especially since it does not appear to induce the formation

of the highly diffusible and damaging H2O2.

H2O2 was found to induce both structural and oxidative DNA damage as was

expected. The BDNF was found to produce a significant protective effect upon the

nuclear DNA of neurons. To this date and to my knowledge, no other studies have

attempted to determine the BDNF effect on oxidative DNA damage. The collected

data thus presented the first direct observation that BDNF can protect against

nuclear DNA damage, particularly of the oxidative kind.

69

6. Future Work

Further experiments to characterize the culture regarding its maturation should be

performed to confirm the data and attain statistical significance. Neurons at latter

DIVs such as 15 should also be tested.

The Aβ25-35 peptide did not cause oxidative DNA damage at the tested sub-lethal

concentrations. The experiments should be repeated in higher concentrations of the

peptide to see if oxidative damage appears, or if only structural damage, which may

be derived from the earlier stages of apoptosis.

The Aβ1-42 peptide should also be tested due to its ability to form H2O2 in the

presence of copper. Its mechanism of action upon the cell is different and although

oxidative DNA damage was not present at sub-lethal concentrations with the Aβ25-35

peptide, it can be present with the Aβ1-42 peptide.

The BDNF DNA damage protection is probably due to the increase in activity of the

antioxidant enzymes GPx and GR, but this evidence could be consolidated by

repeating the experiments in the presence of selective enzyme inhibitors, or

glutathione depleting agents such as, ethacrynic acid and buthionine sulfoximine

[21,45]. Identification of the signalling pathway, PI3K/Akt, PLC-γ or MAPK/ERK,

responsible for DNA damage protection through the use of inhibitors for the above

pathways such as LY-294002, U-73122 and U-0126 respectively would also be of

interest.

70

In addition, although BDNF plays a role in preventing apoptosis of DNA damaged

neurons, which effect does it have on DNA repair? If BDNF prevents neurons from

exiting G0 into G1, which is thought to be dependent upon pRb and the cyclin

C/CDK3 complex [57], it could have a detrimental effect upon DNA repair as neuronal

cells re-enter the cell cycle to repair their DNA. However, if BDNF only blocks the

G1→S transition, dependent upon pRb and the cyclin E/CDK2 complex [57], its effect

could either be beneficial, with an elevated activity of repair mechanisms when

compared with neurons not incubated with BDNF, or neutral if the DNA repair

efficiency is the same. This could be measured by comet assay, for example with

neurons pre-incubated with a glutathione depleting agent in the presence or absence

of BDNF and exposed to H2O2 at low, non-lethal, concentrations. Performing the

oxidative comet assay at different time frames, would allow for the determination of

BDNF effect upon DNA repair mechanisms [64].

71

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46 Santos, A. J. (2009). Influência dos receptores A2A da adenosina no efeito neuroprotector do BDNF na morte neuronal induzida. Master Thesis. Faculdade de Ciências - Universidade de Lisboa.

47 Schwartz, E. I., Smilenov, L., Price, M. A., Osredkar, T., Baker, R. A., Ghosh, S., Shi, F., Vollmer, T. L., Lencinas, A., Stearns, D. M., Gorospe, M. & Kruman, I. I. (2007). Cell Cycle Activation in Postmitotic Neurons is Essential for DNA Repair. Cell Cycle, 6(3), 318-329.

48 Shaposhnikov, S., Thomsen, P. D., & Collins, A. R. (2011). Chapter 10: Combining Fluorescent In Situ Hybridization with the Comet. DNA Damage Detection In Situ, Ex Vivo, and In Vivo: Methods and Protocols, Methods in Molecular Biology, 682, USA, Springer Science+Business Media, LLC.

49 Singh, N. P. (2005). Chapter 4: Apoptosis assessment by the DNA diffusion assay. Methods in Molecular Medicine, 111(2). Totowa, NJ, Humana Press Inc.

50 Skaper, S. D. (2012). Chapter 1: The Neurotrophin Family of Neurotrophic Factors: An Overview. Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, 846, USA, Springer Science+Business Media, LLC.

51 Srujana, K., Begum, S. S., Rao, K. N., Devi, G. S., Jyothy, A., & Prasad, M. H. (2010). Application of the comet assay for assessment of oxidative DNA damage in circulating lymphocytes of Tetralogy of Fallot patients. Mutation Research, 688, 62-65.

52 Stanciu, M. & Defranco, D. B. (2002). Prolonged Nuclear Retention of Activated Extracellular Signal-regulated Protein Kinase Promotes Cell Death Generated by Oxidative Toxicity or Proteasome Inhibition in a Neuronal Cell Line. The Journal of Biological Chemistry,277(6), 4010-4017.

53 Su, B., Wang, X., Nunomura, A., Moreira, P. I ., Lee, H., Perry, G. Smith, M. A & Zhu, X. (2008) Oxidative Stress Signaling in Alzheimer's Disease. Current Alzheimer Research, 5(6), 525-532.

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76

8. Annexes

Annex I – Comet Assay Working Protocol for Structural and Oxidative DNA

Damage

• Buffers:

o PBS :

(Ca2+ Mg2+ free, pH 7.4)

Stock 10X

Stock 1X

Working Solution

- 80.0 g NaCl

- 2.0 g KCl

- 11.5 g Na2HPO4

- 2.0 g KH2PO4

- Add 900 mL of H2O Elix

- Adjust pH to 7.4

- Adjust volume to 1000 mL

- Sterilize by autoclaving

- Store at room temperature

- 100 ml of Stock Solution 10X

- Add 850 mL H2O Elix

- Adjust pH to 7.4



- Transfer 100 mL to a

Labelled Sterile 100 mL

Schott Flask in the laminar

flow unit


- Refrigerate in an Ice/Water

Bath 30 min before use

o Lysis Solution :

(2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, pH 10)

Stock 1X

Working Solution (Fresh before use)

- 146.1 g NaCl

- 37.224 g EDTA

- 1.2110 g Trizma base

- 8.0 g NaOH (Use plastic spatula;

Hygroscopic)


- Dissolve for 20 minutes with

agitation

- Adjust pH to 10


- Prepare and label 5x 200 mL Schott

flasks

- Freeze and Store at - 20°C

- Thaw an aliquot of 200 mL of the Stock

Solution in a Water Bath (37ºC)

- Prepare two P1000 Blue Tips with the

point cut

- Add 2 ml of Triton X-100 to the bottle with

the tips

- Stir the solution gently and horizontally

- Place on Ice/Water Bath for more than 30

min prior to use Note: Due to the high density of Triton X-100, firstly

cover the inner surface of the prepared tip by pipetting

up and down once. Then pipette 1 mL Triton X-100

very slowly to ensure the correct volume is added to

the solution.

77

o FPG Solutions :

(100 mM KCl, 40 mM HEPES, 500 µM EDTA, 200 µg/mL BSA, pH 8)

FPG Stock Solution10X

FPG Reaction Solution 1X - 66.724 g HEPES

- 52.185 g KCl

- 1.303 g EDTA

- 1.4 g BSA

- Adjust pH to 8.0 with KOH (5 M)


- Aliquot the solution into (10 + 10) 50

and 15 mL Falcon tubes

- Store the remaining volume in 1500 µL

eppendorfs in equal number to available

FPG enzyme stock aliquots

- Freeze and Store at -20ºC

- Thaw two Falcon Tubes (50 + 15 mL) of

FPG Stock Solution

- Transfer the solution to a 1 L Schott flask


- Correct pH to 8.0 with KOH (5 M)

- Add water to a volume of 650 mL

- Store overnight at 4ºC in the cold room

FPG Stabilization Solution 1X

Note: The FPG Stabilization Solution is necessary at

very small volumes at each time. Its use is to, after

dispensing the FPG Enzyme Stock Solution, kindly

provided by Dr. Andrew Collins, into 5 µL aliquots,

dilute one of the aliquots at 1:100 and further dispense

into 25 aliquots of 20 µL (100x diluted). All aliquots

must be immediately stored at -80ºC as FPG is

unstable. The 100x diluted aliquots are further diluted

at 1:30 with reaction solution and used one per assay.

- Thaw an eppendorf of FPG Stock

Solution and transfer 1 mL into a 50 mL

Falcon tube.

- Add 1 mL pure glycerol

- Adjust pH to 8 with KOH (1 M)


o Electrophoresis Solution :

(300 mM NaOH, 1 mM EDTA, pH > 13)

NaOH Stock 1X (Fresh every two weeks)

EDTA Stock 1X (Fresh every two weeks) - 27.79 g NaOH


- Dissolve for 10 minutes with agitation in

the hood


- Remove and discard 10 mL solution

- Transfer the 190 mL to a labelled 200

mL Schott flask

- Store at 4ºC for up to two weeks

- 1.23 g EDTA


- Adjust pH to 10 while dissolving with

agitation


- Transfer 10 mL to a labelled 15 mL Falcon

tube

- Store at 4ºC for up to two weeks

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H2O Elix

Working Solution (Fresh before use) - Measure 2 L of H2O Elix


- Refrigerate all Stocks and the H2O Elix to

4ºC overnight in the cold room

- Mix all Stocks and water into a 2 L

Erlenmeyer

o Neutralization Buffer :

(400 mM Tris, pH 7.5)

Stock 1X

Working Solution (Fresh before use) - 24.25 g Tris Base

- Add 400 mL H2O Elix

- Dissolve with agitation

- Adjust pH to 7.5 with HCl (6-10 M)


- Transfer 50 mL to 10x Labelled 50 mL

Falcon tubes

- Freeze and Store at - 20°C

- Remove an aliquot of Lysis Solution

- Thaw the Neutralization Solution overnight

at room temperature prior to use

o Staining Solution :

(0.4 µg/mL EtBr)

Working Solution (Fresh before use) - Fill one Falcon tube with 25 mL H2O Elix

- Involve the Falcon with aluminium paper

- Pipette 1 µL of EtBr stock solution (10 mg/mL)

to the Falcon

- Close Falcon and vortex

79

• Agarose:

o NMA – Normal Melting Agarose :

(1%)

Aliquots

Working Solution

- Weight 250 mg of NMA accurately in

an eppendorf

- Repeat this process for the amount

of aliquots necessary for future

experiments

- Transfer the 250 mg aliquot to a 25 mL

Schott flask

- Add 25 mL of PBS (Working Solution)

- Use a portion of the solution to thoroughly

wash the eppendorf, removing the

remaining NMA to the Schott flask

- Heat the solution in the microwave with the

lid half open for periods of 30s until all lumps

are completely dissolved

- Store at room temperature until necessary

o LMPA – Low Melting Point Agarose :

(0,7%)

Aliquots

Working Solution

- Weight 175 mg of LMPA accurately

in an eppendorf

- Repeat this process for the amount

of aliquots necessary for future

experiments

- Transfer the 175 mg aliquot to a 25 mL

Schott Flask

- Add 25 mL of PBS (Working Solution)

- Use a portion of the solution to thoroughly

wash the eppendorf, removing the

remaining LMPA to the Schott flask

- Heat the solution in the microwave with the

lid half open for periods of 30s until all lumps

are completely dissolved

- Store at room temperature until necessary

80

• Protocol:

o Preparation of microscope slides:

Preparation

- Place 200 mL of Absolute Ethanol in a 250 mL goblet

- Modulate the Bünsen air entry to produce a blue flame

- Place 12 Superfrost Rosa® slides and around 15 coverslips on tissue

papers for easy access

- Prepare an ice tray, smoothing the ice surface with a metal plate and

leaving the latter to ensure an even temperature

Slide Cleaning and Coating

- Dip both the slides and coverslips in the ethanol

- Wipe them gently with a tissue paper

Do the following procedure to no more than 4 slides at the time:

- Heat the NMA solution in the microwave with an open lid and use shortly

after it stops boiling

- With a tweezer, dip the slides one at a time in ethanol and burn them over

the blue flame to remove dust and machine oil

- Add 210 µL NMA and the 24x60 mm coverslip to the slide accordingly to

the outline of Figure A.

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- Place the slide in the ice tray for 5 minutes

- Remove the coverslip with a dissection tweezer accordingly to the outline

in Figure B.

- Place the slide on a flat surface

- Repeat the procedure for the other 3 slides

Repeat the above procedure for the remaining slides

- Allow the slides to dry overnight on a flat surface or, preferably, on a

previously prepared (dry at this time) aluminium-foil covered moisture box

and place the lid to avoid dust deposition and ease of transport

- Label the slides

Figure A – Outline of the slide cleaning and coating first steps. 1 – Add the NMA to

one side of the slide; 2 – Introduce the cover slip and drag the NMA to the left; 3 – Allow

the cover slip to spread the NMA across the slide without any air bubbles.

Figure B – Outline of the slide cleaning and coating last steps. 1 – Place the slide in

the ice tray for 5 minutes; 2 – Remove the coverslip with a dissection tweezer.

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o Cell preparation and lysis:

Cell Culture

- Remove the culture media

- Wash the cells two times with ice cold PBS to stop cell metabolism and

especially DNA repair

- Place the culture plate on an ice tray

- For each experimental condition, scrape the cells with a cell scraper and 1

mL ice cold PBS (take care to scrape cells only once with the scraper at

90º angle and firm hand to avoid cell membrane rupture)

- Transfer the PBS with the cells to a labelled 15 mL falcon tube

- Wash the cell plate well with another mL of PBS and add to the falcon

- Place the falcon on an easily movable ice box (with a bit of water on the

box bottom)

- Rinse the cell scraper and repeat for the other experimental conditions

Cell Suspension

- Refrigerate a 15 mL falcon tube centrifuge with a swinging-bucket rotor to

0º C for at least 30 minutes before initiating the following step

- Centrifuge at 700 g for 10 minutes (acceleration 7; brake 3)

- Return the falcon tubes to the ice box

- For each tube, remove the supernatant by tilting

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- Add 100-80 µL PBS and very carefully ressuspend the pellet (for the

experimental conditions at DIV 5 and DIV 9-10, it corresponds to a cellular

density of approximately 7.5x105 cells per mL)

- Return the tube to the ice box and repeat for the other conditions

Standard Cells Suspension (Optional)

- Refrigerate a eppendorf centrifuge to 4º C for at least 30 minutes before

initiating the following step

- Take a Standard Cells aliquot (prepared as in Annex II) from the -80ºC

freezer

- Add 400 µL of ice cold PBS to the eppendorf but do not ressuspend

- Centrifuge at 800g for 10 minutes (4ºC)

- Remove 300 µL from the upper liquid carefully

- Ressuspend ( 120 µL final volume, cellular density 5 x 105 cells per mL)

- Immediately place the eppendorf on ice or ice/water bath

Agarose Embedment and Cell Lysis

- Regulate a water bath to 37º C and allow it to stabilize the temperature

- Prepare the working lysis solution and refrigerate it on an ice/water bath for

at least 30 minutes prior to use

- Heat the LMPA solution in the microwave with an open lid and place on the

water bath at 37º for at least 15 minutes prior to use

- Place 12 0,5 mL eppendorfs on an eppendorf support on the water bath at

37º C to normalize their temperature

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Figure C – Outline of the agarose embedment and cell lysis steps. 1 – Place the LMPA

in the centre of the slide, setting it into an oval shape; 2 – Press the coverslip directly on top

of the agarose; 3 – The LMPA should cover the NMA completely.

- Prepare an ice tray, smoothing the ice surface with a metal plate and

leaving the latter to ensure an even temperature

Do the following procedure for each slide, one each time:

- Very carefully ressuspend the cells in the falcon tube

- With one micropipette add 10 µL of cell suspension to a heated eppendorf

- With another micropipette (with care to pipette the LMPA down and up

again to remove air bubbles) add 75 uL LMPA to the heated eppendorf

- Do NOT ressuspend the cell and agarose mixture (this is CRITICAL

especially when working with sensitive cells such as neurons as the

shearing forces of the cells subjected to movement in an higher density

medium will burst, and nuclear membrane rupture occurs resulting in DNA

degradation from cytoplasmic nucleases)

- Place the corresponding slide on a flat surface

- Transfer the 85 µL from the eppendorf to the slide and press a clean 24x60

mm coverslip over it according to the outline of Figure C.

85

- Place the slide on the metal plate over the ice tray for at least 5 minutes

Repeat the above procedure for the remaining slides

- Transfer the working lysis solution to an opaque coplin jar

- Remove coverslips from each slide on the tray and place them on the

coplin jar, on ice, for 1h 30 min

- Transport the coplin jar to the cold room

o Oxidative damage determination:

Preparation

- Prepare the FPG Reaction Solution the day before and leave it overnight

on the cold room

- Place all the necessary material in the cold room: 200 µL micropipette and

tips; an ice tray with a metal plate on top; 1 L goblet for waste; Falcon tube

support; several paper tissues and 24x60 mm clean coverslips on a paper

tissue for easy access

- Soak the transport and incubation moist box with some water and place it

in the incubator at 37ºC

- Remove an aliquot of 100x diluted FPG from the -80ºC freezer and

transport it to the cold room on ice.

- Dilute it with 580 µL (1:30) with FPG Reaction Solution, ressuspending a

bit and place it on ice once again.

- Fill one other eppendorf with FPG reaction solution only and place it on ice

86

FPG Incubation

- After 1h 30 min, remove the slides from the coplin jar and remove the lysis

solution to the waste

- Fill the coplin jar with approximately 200 mL of FPG Reaction Solution and

wash the slides for 5 minutes

- Repeat the previous step twice

- Remove the slides one at a time (starting with the structural damage only)

- Over the bench on a paper tissue add 115 µL of FPG Reaction Solution or

FPG Reaction Solution + FPG from the eppendorfs as appropriate , cover

with a coverslip and immediately place over the metal plate on the ice tray

- Repeat the procedure for all the slides

- Cover the ice tray with aluminium foil to minimize the exposure to light

- Transport the slides out of the cold room to near the incubator

- Place the slides in the moist box in the incubator for 30 minutes

o Electrophoresis:

Preparation

- Place the stock solutions and Elix water for the electrophoresis solution in

the cold room overnight to normalize their temperature

- Prepare the working lysis solution in an 2000 mL Erlenmeyer by gentle

mixing with a plastic pipette

87

- Ensure the electrophoresis chamber is perfectly level with a carpenters

bubble

- Cover the top of the electrophoresis chamber with aluminium foil or a black

cloth to prevent light-induced DNA damage

- Add the working electrophoresis solution to the chamber wells only

DNA Unwinding and Electrophoretic Run

- Remove the slides from the moist box in the incubator to the metal plate on

the ice tray to stop the reaction and cover with aluminium foil to preserve

from light induced DNA damage

- Transport the slides to the cold room and remove the coverslips

- Place the slides side-by-side on the centre of the electrophoresis chamber

(fill with extra empty slides if necessary

- Add more working electrophoresis solution to cover the slides (Total

Volume approximately 2100 mL) and use a tweezer to gently press on the

slides to dislodge any bubbles

- Replace the chamber lid and wait 20 minutes as the DNA unwinds

- Input the electrophoresis parameters (24 V for 30 minutes)

- Adjust the electrophoresis solution volume with a plastic pipette (by quickly

starting and stopping the electrophoresis) such as at the end of the

unwinding step the current amounts to 300 mA

- Start the electrophoresis (30 minutes)

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- When finished, remove the slides to a flat tray with 7-10 paper tissues for

transport and proceed to neutralization (no care to shield the slides from

light induced damage is necessary from this point onwards)

o Neutralization and Dehydration:

Neutralization

- Using a pipette, drop wise coat Neutralization Buffer on top of the slides

and allow to sit for 5 minutes

- Drain the slides onto the paper tissues on the tray and repeat the

procedure twice more

Dehydration

- Previously chill an ethanol series (70%, 95% and Absolute Ethanol) on ice

( 200 mL each in Schott Flasks in order to completely cover slides on the

coplin jar)

- Add the 70% ethanol solution to the coplin jar and transfer the slides to it

for the duration of 5 minutes

- Remove the slides and ethanol solution and repeat the procedure twice

more, with the 95% and Absolute Ethanol solutions

- Air dry the slides overnight or on an oven at 50ºC for 30 minutes

- Store the slides in dry area protected from dust or proceed to staining

89

o Staining and Image Acquisition:

Staining

- Prepare the Staining Solution

- Clean at least 20 coverslips ( dip coverslips on ethanol and wipe with a

paper tissue once to remove grease and dust from stock coverslips) and

place them over a paper tissue for easy access

- Prepare the aluminium foil covered moist box for transport of stained slides

by adding water

- Add sufficient ice to a big ice box capable of transport of the moist box

Do the following procedure to no more than 4 slides at the time:

- Add 40 µL of Staining Solution to each slide and cover with a clean

coverslip making sure all the gel is covered with Staining Solution

- Wait 5 minutes and remove the coverslip

- Remove the excess stain with an Elix water wash bottle

- Add 50 µL of Elix water to the slide and cover with a clean coverslip

according to the outline of Figure D.

- Transfer the slide to the moist box to protect it from drying and light

- Repeat the above procedure for the remaining slides

- Place the moist box in the ice box until the image acquisition step

90

Figure D – Outline of the staining steps. 1 – Add the staining solution to one side of the

slide; 2 – Use a coverslip to spread the staining solution over the slide; 3 – Wait 5 minutes

with the coverslip on top of the slide; 4 – Remove the coverslip; 5 – Wash the staining

solution using a wash bottle and do not let the staining solution touch the back of the slide;

5 – Add the water to one side of the slide and place a clean coverslip on top.

Image Acquisition

- Turn the fluorescence microscope and camera (Axiovert 200M, Roper

Scientific Coolsnap HQ CCD (12-bit))

- Open the control program and use the recommended parameters:

o 400 ms of exposure time as acquired images have been tested and

are not saturated for the experimental conditions;

o 20x objective;

o Pseudo-colour software filter for ease of focus

- Acquire between 50 and 120 comet images per slide (the later number is

recommended as more precise results can be obtained)

91

- Acquire each slide without undue delay as some fluorescence is lost when

exposed to the light from the microscope, the same applies to water

evaporation

Note: Although special care has been taken to ensure the microscope is

not contaminated with ethidium bromide such as using a 5x more diluted

staining solution than normally recommended by Kumaravel (2009) and

removing the excess stain and substituting with water, thoroughly clean the

microscope platform with absolute ethanol to ensure no residual

contamination remains

Storage

- Remove the coverslips with a pair of dissection tweezers

- De-stain the gels with Absolute Ethanol from a wash bottle

- Allow the slides to dry and store in a dry place at room temperature

- For re-examination, re-staining, as above, is necessary

o Data Processing and Analysis:

Image Conversion

- Prepare a copy of the directory of images to be converted (Example:

Results) and name it (Example: Converted Results)

- Open ImageJ

92

Automatic Procedure:

- Drag and open provided Macro “CometConvert.ijm” with ImageJ (or

“CometInvert&Convert.ijm” if a horizontal flip is necessary for comet left-to-

right orientation)

- Select each experimental condition directory at a time within the Converted

Results, and for convenience, select the same directory as destination

- Do NOT alter the minimum and maximum intensities from 0 and 4096

unless greater resolution in the results is necessary (in such situation the

absolute minimum and maximum bit intensity of all images needs to be

analysed for each experiment set and can be defined here)

- Repeat the previous steps for all the experimental conditions

Manual Procedure:

- Open all images to be analysed , one experimental condition / image series

at each time

- Select Image → Adjust → Brightness / Contrast → Set → Imput

parameters Min 0 and Max 4096 → Propagate to All Images

- (If necessary) Select Image → Transform → Flip Horizontally

- Select File → Save as → TIF

- Repeat procedure for all the experimental conditions

- Note: Image conversion is necessary as the TIF files produced upon

acquisition are 16-bit files and the analysis software only accepts 8-bit files.

Since a 12-bit camera was used, only the first 12 bits of the 16-bit files

have information. The algorithm consists of removing the empty bits,

93

flipping the image horizontally if necessary, and converting the information

from 12-bits to the equivalent in 8-bits. Some resolution is lost in this

process but it is negligible as the main advantage of using a 12-bit camera

is to prevent image saturation.

Data Analysis

The analysis software used was CASP (Comet Assay Software Project),

freely available at www.casplab.com)

- Open CASP software

- Select File → Select Files → Select converted images to be analysed

(each experimental condition/image series at each time)

- Select Options → Adjust → Insert parameters Comet Threshold 0.03 and

Profile 2 (No tail cluster) → Set as Default → OK

- Select View → Results Window → Maximize program window → Organize

windows in a comfortable display

- Select a comet from one image, defining the frame size (It must be large

enough to include all comets in the set to be analysed)

- Select Start Measurements

- Use the following commands in order to proceed quickly:

[If necessary Ctrl-W (Flip Comet Frame)] → Ctrl-A (Assay Comet) → Ctrl-Z

(Store Comet) → Next Image

- Select File → Export Results → Name appropriately and add .txt to the end

of the filename → Save

- File → New Series

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- Repeat procedure for each experimental condition

Data Processing

- Open provided Exel file “Modelo Analise Automatica”

- Name each tab after each experimental condition

- Select Data → Obtain External Data → From Text → Select each data file

at a time → Select parameters Delimited and File Origin 437: United States

OEM → Next → Select Space →Next → Conclude

- Copy data from entry tab to corresponding data set

- Repeat procedure for each experimental condition

Note: If the images were not obtained with Axiovert 200M 20x objective the

bit size value must be altered for a correct determination of the Tail Length

parameter (20x objective bit size = 0.48 µm)

Final Note: To better understand the image acquisition and analysis steps as well as

possible troubleshooting please refer to Vilhar B. (2004) Available at:

http://botanika.biologija.org/exp/comet/comet_guide01.pdf

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Annex II – Standard Cells Protocol

o Treatment with H2O2

- Culture a large batch of neuronal cells in a Petri dish.

- At DIV 3, treat the cells with 30 µM H2O2.

- Incubate 30 min on ice.

- Remove the medium and wash twice with ice cold PBS.

- Add new PBS and scrape cells on ice to a Falcon tube (50 mL).

o Centrifugation

- Centrifuge at 800g at 0ºC for 10 min.

- Remove the supernatant with a pipette.

- Add 5 mL ice cold Neurobasal (with 25 U/mL Pen/Strep, 0.5mM

glutamine and 10% FBS).

- Ressuspend with the pipette (use blue tips and blue-yellow tips).

- Place the Falcon on ice.

o Cell Count:

- Remove 15 µl of the cell suspension to a 500 µL eppendorf with 30 µl

of Trypan Blue and ressuspend gently.

- Replace Falcon on ice.

- Counting cells in the hemocytometer:

- Cells per mL = average count of 4 fields x 3 x 104

- Calculate the ressuspension:

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- Total Cells = Cells per mL x Volume in the Falcon

- Volume for Ressuspension = Total Cells / Desired Cell Density

(3x106)

o Ressuspension:

- Centrifuge at 800g at 0ºC for 10 min.

- Remove the supernatant with pipettor.

- Add the calculated Volume for Ressuspension of ice cold Freezing

Medium (DMEM with 20% FBS and 10% DMSO).

- Ressuspend with the pipettor (use blue tips and blue-yellow tips).

- Place the Falcon on ice.

o Freezing:

- Aliquot 20 µL of the ressuspension to 1500 µL microfuge tubes.

- Slowly freeze overnight in a plastic box with isopropanol with support

for microcentrifuge tubes in the -80ºC freezer.

- Transfer the tubes to box in the freezer at -80°C or to Liquid Nitrogen.

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