Dysregulated redox balance associated with glutamate excitotoxicity in autism spectrum
disorders
Prof. Afaf El-AnsaryDepartment of BiochemistryScience College, King Saud [email protected]
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.
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