Dysregulated redox balance associated with glutamate excitotoxicity in autism spectrum

disorders

Prof. Afaf El-AnsaryDepartment of BiochemistryScience College, King Saud Universityelansary@ksu.edu.sa

Background

� Autism spectrum disorders(ASD) are characterized by threecore behavioral domains: socialdeficits, impairedcommunication, and repetitivebehaviors.

� Glutamate excitotoxicity hasbeen found in various preclinicalmodels of this disorder.

� Inefficient detoxification systemleads to oxidative stress, gutdysbiosis, and immunedysfunction has been alsoaccepted as etiologicalmechanismof autism.

This work is an attempt to understand the relationship between glutamate

excitotoxicity and impaired detoxification as two mechanisms recently related to as two mechanisms recently related to

autism.

� Central Nervous System (CNS)• Brain & Spinal Cord

� Peripheral Nervous System (PNS)� Peripheral Nervous System (PNS)• Afferent (sensory) Nerves –

Carry sensory information to the CNS

• Efferent (motor) Nerves –Transmit information to muscles or glands

Cells of the Nervous System

� Neurons• Signal integration/generation; direct

control of skeletal muscle (motor axons)

� Supporting Cells (Glia cells)� Supporting Cells (Glia cells)• Astrocytes (CNS – blood brain barrier)• Oligodendrocytes (CNS – myelination)• Schwann cells (PNS – myelination)• Microglia (activated astrocytes)

Cellular Events in Neurodevelopment

Events:• Division• Division• Migration• Differentiation• Neurogenesis• Formation of synapses• Myelination• Apoptosis

Active throughout childhood & adolescence

� Neurons are post-mitotic cells

� High dependence on oxygen

• Little anaerobic capacity

• Brief hypoxia/anoxia-neuron cell death

Why is the Brain Particularly

Vulnerable to Injury?

• Brief hypoxia/anoxia-neuron cell death

� Dependence on glucose

• Sole energy source (no glycolysis)

• High metabolic rate

� Many substances go directly to the brain via inhalation

Blood Supply to the Brain

Blood-brain Barrier

� The BBB consists of around 500 miles of bloodvessels throughout the brain, all packed withendothelial cells that are highly selective aboutwhat gets through to the brain. With fewexceptions, only fat-soluble small moleculesexceptions, only fat-soluble small moleculespenetrate - alcohol, caffeine, and nicotine areamong them.

BBB can be broken down by

� Hypertension: high blood pressure opens the

BBB

� Hyperosmolarity: high concentration of

solutes can open the BBB. solutes can open the BBB.

� Infection: exposure to infectious agents can

open the BBB.

� Trauma, Ischemia, Inflammation, Pressure:

injury to the brain can open the BBB.

� Development: the BBB is not fully formed at

birth.

Glutamate Synapses

�Excitatory synapse ofbrain

�Required to generateaction potentials

�Both AMPA and�Both AMPA andNMDA receptors arecritical for normal brainfunction

�NMDA-high Ca++permeability

Glutamate/glutamine cycle� It is well known that glial cells,

mainly astrocytes surroundglutamatergic synapses, andexpress glutamate transportersand the glutamate-metabolizingenzyme, glutamine synthetase(GS).

� Glutamate is transported intoglial cells and amidated by GS tothe non-toxic amino acidglutamine. Glutamine is thenreleased by glial cells and takenup by neurons, where it ishydrolyzed by glutaminase toform glutamate again, completingthe glutamate/glutamine cycle.

�� AppropriateAppropriate clearanceclearance ofof

synapticsynaptic glutamateglutamate isis

requiredrequired forfor thethe normalnormal

functionfunction ofof brainbrainfunctionfunction ofof brainbrain

excitatoryexcitatory synapsessynapses andand

hencehence forfor preventionprevention ofof

neurotoxicityneurotoxicity recordedrecorded inin

patientspatients withwith autismautism..

Brain oxidative stress

� The central nervous systempresents high vulnerability to freeradical damage due to its elevatedoxidative metabolic rate andenriched content of unsaturatedlipids, as well as to its elevatedrate of free radical generationrate of free radical generationderived from neurotransmittersmetabolism, and poor radicalscavenging mechanisms.

� It is also hypothesized thatautistic patients as poordetoxifiers have reduced abilityto eliminate mercury. Higherlevels of mercury were recentlyfound to be associated withsocial impairment and severity ofautism.

oCoal power plants

oCement production

ANTROPOGENIC

EMISSIONS

oVolcanoes

oRock erosion

oFires

NATURAL EMISSIONSoFolk medicines

oCosmetics

oAmalgams

oVaccines

OTHER POTENTIAL SOURCES

Methylation

Plankton

Excitotoxicity-Glutamate Mediated Cell Death

��GlutamateGlutamate inducesinduces aa delayeddelayed cellcell deathdeath inin neuronsneurons

��ThisThis cellcell deathdeath requiresrequires extracellularextracellular calciumcalcium andand isisblockedblocked byby antagonistsantagonists ofof NMDANMDA receptorsreceptors

�Action potentials are initiated in the nerve axon after�Action potentials are initiated in the nerve axon afterglutamate excitatory activation of receptors in theneuron's dendrites and cell body.

��HypothesisHypothesis:: ProlongedProlonged oror inappropriateinappropriate activationactivation ofofNMDANMDA receptorsreceptors underliesunderlies glutamateglutamate excitotoxicityexcitotoxicity ofofneuronsneurons ..

20 male patients with autism age of 8 ±±±± 4 were

enrolled through the Autism Research and

Methodology

enrolled through the Autism Research and

Treatment (ART) Center clinic. The diagnosis of

ASD was confirmed in all subjects using the

Autism Diagnostic Interview-Revised (ADI-R)

criteria, the Autism Diagnostic Observation

Schedule-Generic (ADOS-G) criteria and the

Developmental, Dimensional and Diagnostic

Interview (3DI). Another age and sex matching 30

as a control group.

Samples collection

AfterAfterAfterAfter overnightovernightovernightovernight fast,fast,fast,fast, 10101010 mlmlmlml bloodbloodbloodblood samplessamplessamplessamples werewerewerewerecollectedcollectedcollectedcollected fromfromfromfrom autisticautisticautisticautistic andandandand controlcontrolcontrolcontrol groupsgroupsgroupsgroups inininin testtesttesttesttubestubestubestubes containingcontainingcontainingcontaining anticoagulantanticoagulantanticoagulantanticoagulant.... TubesTubesTubesTubes werewerewerewerecentrifugedcentrifugedcentrifugedcentrifuged atatatat 1000100010001000 rpmrpmrpmrpm atatatat roomroomroomroom temperaturetemperaturetemperaturetemperature forforforfor15151515 minutes,minutes,minutes,minutes, plasmaplasmaplasmaplasma waswaswaswas obtainedobtainedobtainedobtained andandandand deepdeepdeepdeepfreezedfreezedfreezedfreezed (at(at(at(at ----80808080°°°°C)C)C)C) andandandand RBCsRBCsRBCsRBCs werewerewerewere keptkeptkeptkept atatatat ----20202020°°°°CCCCuntiluntiluntiluntil analysisanalysisanalysisanalysis timetimetimetime....

Biochemical assays:

Glutamate,Glutamate, glutamine,glutamine, thioredoxinsthioredoxins I&III,I&III,ThioredoxinThioredoxin reductasereductase,, peroxidoxinsperoxidoxins,,glutathioneglutathione statusstatus andand glutathioneglutathione--ss--transferasetransferase werewere measuredmeasured inin plasmaplasmasamplessamples ofof autisticautistic childrenchildren comparedcompared totocontrolscontrols usingusing ELISAELISA kitskits.. MercuryMercury waswasmeasuredmeasured usingusing atomicatomic absorptionabsorption..

1. Independent t-test

• Independent t-test to compare between the 2 studied groups.• Results were expressed as means ± S.D. Statistical comparisons were performed with independent t-tests using (SPSS).• Significance were assigned at the level of P < 0.05.

2. ROC analysis2. ROC analysis

Area under the curve, cutoff values, and degree of specificity and sensitivity were calculated.

Parameter Group N Mean ± S.D. Percent Change P value

Glutamic (µmol/l)Control 20 111.91 ± 4.51 100.00

0.001Autistic 20 152.80 ± 6.47 136.54

Glutamine (µmol/l)Control 20 241.82 ± 12.93 100.00

0.001Autistic 20 111.34 ± 5.69 46.04

Glutamic / Glutamine RatioControl 20 0.46 ± 0.03 100.00

0.001Autistic 20 1.37 ± 0.06 296.18

Glutamate Dehydrogenase(GLDH) (U/l)

Control 20 1.71 ± 0.47 100.000.001

Autistic 20 0.93 ± 0.36 54.22

Table 1: Table 1 Mean ± SD of the measured chemicals in plasma or red blood cells of patients with autism compared with age-matched controls

�Higher Hg can be easilyrelated to of GSH/GSSG status.It is well known that sulfhydryl-containing enzymes are inhibitedby MeHg.

�With particular emphasis on thetoxicity induced by MeHg, it isknown that its mercury atom

Thioredoxin 1 (ng/ml)Control 20 44.71 ± 7.43 100.00

0.001Autistic 20 74.70 ± 9.04 167.09

Thioredoxin reductase(mU/ml)

Control 20 1.83 ± 0.52 100.000.001

Autistic 20 3.31 ± 1.11 180.87

Peroxiredoxin 1 (ng/ml)Control 20 19.58 ± 4.76 100.00

0.001Autistic 20 34.56 ± 8.32 176.55

Peroxiredoxin 3 (ng/ml)Control 20 24.30 ± 2.69 100.00

0.001Autistic 20 43.05 ± 5.86 177.16

GSH /GSSGControl 20 26.07 ± 5.03 100.00

0.001Autistic 20 8.03 ± 2.46 30.79

Glutathione S Transferaseactivity (µmol/min/ml)

Control 20 0.69 ± 0.20 100.000.001

Autistic 20 0.41 ± 0.12 59.26

Mercury (µg/L)Control 20 4.64 ± 0.68 100.00

0.001Autistic 20 6.93 ± 0.74 149.40

known that its mercury atomdirectly interacts with the thiolgroup of GSH, leading to theformation of an execretable GS-HgCH3 complex

A mechanism links dysregulated redox statusto glutamate excitotoxicity could be easilyproposed.�Simply, in the brain, ion gradients acrossneural membranes is important for restingmembrane and this usually maintained byATP-dependent ion pumps, such as a Na+/K+

pump.�ATP depletion induces impairment in therepolarization of neural membranes after adepolarizing stimulus.�Defective repolarization can relieve a�Defective repolarization can relieve avoltage dependent Mg+ block of NMDAchannels, leading to persistent receptoractivation by endogenous glutamate.�This hypothesis is derived from an in vitroexperiment showing that the inhibition ofenergy metabolism makes glutamateneurotoxic at concentrations that ordinarilyshow no toxicity.�Calcium entry via NMDA receptors cantrigger neuronal cell death

AUCBest

Cutoff value

Sensitivity %

Specificity %

Glutamic(µmol/l)

1.000 131.110 100.0 % 100.0 %

Glutamine (µmol/l)

1.000 169.800 100.0 % 100.0 %

Glutamic / Glutamine Ratio

1.000 0.906 100.0 % 100.0 %

Glutamate Dehydrogenase (GLDH) (U/l)

0.915 1.575 100.0 % 75.0 %

Table 2: ROC-Curve of all parameters in autistic groups

Thioredoxin 1 level (ng/ml)

0.993 57.400 100.0 % 95.0 %

Thioredoxinreductase activity (mU/ml)

0.894 2.000 80.0 % 75.0 %

Peroxiredoxin 1 level (ng/ml)

0.977 27.250 95.0 % 100.0 %

Peroxiredoxin 3 level (ng/ml)

1.000 33.000 100.0 % 100.0 %

GSH / GSSG 1.000 16.340 100.0 % 100.0 %

Glutathione S Transferase activity (µmol/min/ml)

0.900 0.505 85.0 % 95.0 %

Mercury (µg/L) 0.988 5.997 95.0 % 100.0 %

Predictor Variable Beta P valueAdjusted

R2Model

F value P value

Glutamine (µmol/l) 0.302 0.001

Predictor Variable Beta P valueAdjusted

R2Model

F value P value

Glutamic (µmol/l) 0.006 0.001

0.995 1938.823 0.001Glutamine (µmol/l) -0.005 0.001

Thioredoxin reductaseactivity (mU/ml)

0.013 0.038

GSH / GSSG -0.003 0.018

�Based on this, theexcitotoxicity suggested in thepresent study as etiologicalfactor in autism can be relatedto the impaired Prx I and III,Trx, Trxreductase ,GSH/GSSG, and GST asmarkers of impaireddetoxification mechanisms.

Table 3: Multiple Regression using Stepwise method for Glutamic (µmol/l)as a dependent variable

Table 4: Multiple Regression using Stepwise method for Glutamine (µmol/l)as a dependent variable

Glutamine (µmol/l) 0.302 0.001

0.964 352.514 0.001Glutamic / Glutamine Ratio

92.604 0.001

Peroxiredoxin 1 level (ng/ml)

-0.273 0.011

Predictor Variable Beta P valueAdjusted

R2 square

Model

F value P value

Glutamic (µmol/l) 0.969 0.001

0.990 1243.754 0.001

Glutamic / Glutamine

Ratio-177.627 0.001

Thioredoxin 1 level

(ng/ml)-0.274 0.044

�This could find support in thestudy of Leveille et al(2009)who reported thatexcitotoxic insults can renderthe neurons more vulnerable toperoxidoxin hyperoxidationthrough the oxidation of cystineto cysteine.

Table 5: Multiple Regression using Stepwise method for Glutamic / Glutamine Ratioas a dependent variable

AUTISM

CONCLUSION CONCLUSION

�Mercury as neurotoxic metal could inhibit glutamatetransporters resulting in raising the levels of extracellularglutamate by inhibiting antioxidant enzymes andglutathione.

�Mercury aggravates free radical generation and lipid �Mercury aggravates free radical generation and lipid peroxidation.

�Majority of children with autismexhibited high Hg,mitochondrial dysfunction, and oxidative stress,conditions that magnify glutamate excitotoxicity.

� This should call attention to glutamate signaling being a major mechanism in damage to the autistic brain.

�Stimulation of glutamate transporters could be a good strategy to treat this disorder. strategy to treat this disorder.