Autism: A Neuroepigenetic Disorder. Deth, R et al

8/3/2019 Autism: A Neuroepigenetic Disorder. Deth, R et al.

1/12AUTISM SCIENCE DIGEST: THE JOURNAL OF AUTISMONEISSUE 03 REPRINTED WITH PERMISSION www.autsmone.org

Dr. richarD Deth is Professor of Pharmacology at Northeastern University,where he has maintained a research laboratory since 1976. Currently hislab is ocused on understanding the relationship between antioxidant statusand methylation reactions, including its role in autism and other neurologicaldisorders, such as ADHD, schizophrenia, and Alzheimers disease. Dr. MostafaWaly received his PhD from Northeastern University in 2003 and is currently anAssistant Professor of Nutrition at Sultan Qaboos University in Oman. Dr. ChristinaMuratore received her PhD from Northeastern University in 2010 and is currentlya postdoctoral fellow at Harvard University. Nathaniel Hodgson and MalavTrivedi are currently doctoral students at Northeastern.

Malav Trivedi

Nathaniel Hodgson

Christina Muratore, PhD

AUTISM SCIENCE DIGEST: THE JOURNAL OF AUTISMONEISSUE 03 REPRINTED WITH PERMISSION www.autsmone.org

Mostafa Waly, PhD


2/12www.autsmone.org REPRINTED WITH PERMISSION AUTISM SCIENCE DIGEST: THE JOURNAL OF AUTISMONEISS

IntroductIonWhere to begin in trying to understandautism? Autism is so complex and so variable

rom child to child. The brain, too, is complex.Is it possible to comprehend the eventsthat cause certain children to stray rom thepath o normal neurodevelopment? What

is neurodevelopment anyway? There areso many autism theories: its the gut, its theimmune system, its the mitochondria, its theenvironment, its the genes, its vaccination.But wait! Weve learned so much in the pastdecade. What is all this new science tellingus about the cause(s) o autism? Lets take astep back and try to assemble the pieces othe puzzle that is autism.

First o all, lets be clear that this is not acontest among di erent theories, with winnersand losers. Every legitimate scientifc andclinical (and parental) observation is correctand is a potentially important piece o thepuzzle. In other words, its A and B and C,

not A or B or C. That said, the most use ulperspective is the one that encompasses thelargest number o observations and leavesthe ewest unaccounted or. It is also obviousthat we will learn more rom observationsmade directly on individual autistic childrenthan rom some population-based statistical

construct ar removed rom the actual clinicaldisorder. This is especially true becausethe high level o genetic variability amonghumans tends to wash out actors not presentin the majority of the population. In reality, thehuman population is a collection o minoritypopulations when it comes to genetics;although autism rates are increasing, theautistic population is still just one of thoseminorities. Finally, among observations,interventions that improve autism have specialdistinction and value, both or the beneftthey provide and or the insights they conveyabout the disorder.

Remembering that autism is a develop-

Autism:A neuroepigenetic

disorder

It is so ob io s t t we wi e rn more rom obseron in i i tistic c i ren t n rom some pop t

constr ct r remo e rom t e ct c inic isor er. bec se t e i e e o enetic ri bi it mon m

ctors not present in t e m jorit o t e pop ti

mental disorder, it is imperative to frstunderstand the actors that guide normadevelopment. It then becomes possibleto identi y dys unctional actors that cbe linked to autism via studies o autistiindividuals. Moreover, it is generallynot universally) accepted that autism re

molecular events gone awry, so the levelknowledge and the vocabulary we use mbe molecular. This represents a challengein communication since the generalpopulation is not widely conversant in thlanguage o molecules and biochemistryHowever, when the need is great, one canlearn a new language. Such is the case

or parents o autistic children as is cleaevident at meetings o organizations sucas AutismOne, the Autism Research Insand the National Autism Association

ollows, then, is our perspective on the oo autism, hope ully providing a summcurrent knowledge.

By RIChaRd C. dE h, Phd,1 Na haNIEl W. hOdgSON, 1 Malav S. RIvEdI,1 ChRIS INa R. MuRa ORE, Phd,2 aNd MOS a a I. Waly, Phd 3

A f ia i :

1 Department of Pharmaceutical Sciences, Northeastern University, Boston, MA02115; 2 Center for Neurological Diseases, Harvard Medical School, Boston,MA 02115; and3 Sultan Qaboos University, Muscat, Oman



EpIgEnEtIc Actors controldEvElopmEntHuman development begins with the unitingo sperm and egg, triggering an ongoingseries o rapid cell divisions. The new cellsgradually diverge in their gene expressionand their properties, giving rise to variousstem cell lineages and ultimately to di erenttissues and organs. Throughout this trulyamazing process of development, the DNAsequence o each cell is essentially identical,whether assessed in the ertilized egg orin the liver or brain o the ully developedadult. Di erences between cell types re ectalternative patterns o gene expression, aprocess known as transcription, which giverise to different patterns of messenger RNA(mRNA), coding for a different pattern ofproteins in each cell type, a process knownas translation. For example, the casein proteinis an important component o milk, andits gene is only available or transcription

(mRNA formation) in milk-producing cells ofthe emale breast, even though every cellhas the casein gene. In contrast, genes orproteins that support the most undamentalmetabolic activities in all cells (such as theenzymes involved in glucose metabolism) areactively transcribed in all cells.

Several mechanisms combine to determinewhether genes are actively transcribedor not. The frst involves the binding otranscription actors (TFs), which eitherinitiate or repress mRNA synthesis (Figure1). The level o TF activity is controlled byexternal actors (such as growth actors,hormones, and neurotransmitters) actingvia their individual signaling pathways,including transcription o their own genes.For example, when estrogens activate theirreceptors, two o the activated receptors orma complex that binds to specifc locations onestrogen-responsive genes, leading to theirtranscription.1 O course, the gene or theestrogen receptor must have previously beentranscribed or estrogens to exert their e ect,making certain cells estrogen-responsive.

A second mechanism or regulating genetranscription involves the attachment o methylgroups (single carbon atoms) to specifclocations in the genes, a process known asDNA methylation and epigenetic regulation.2 The methylation places are not random butare at cytosine bases preceding guanosinebases, called CpG sites. Collectionso CpG sites (called CpG islands) arecommonly located where they can in uencetranscription o nearby genes. The additiono a methyl group provides a new bindingsite or proteins called methyl CpG binding

domain proteins (MBDPs), of which themost well-known example is MeCP2, whoseactivity is critical or epigenetic regulationin nerve cells. Mutations in MeCP2, whichinter ere with its CpG binding, cause Rettsyndrome, a genetic orm o autism.3,4

The binding of MBDPs to methylatedDNA typically interferes with transcriptionand also provides a binding plat orm orother proteins that acilitate the wrappingof DNA around histone proteins, forminga tight complex called heterochromatin,which does not allow gene transcription.2 Histones can also be methylated, which

urther tightens the chromatin complex,although other modifcations, such as theaddition o an acetyl group, exert theopposite e ect. Together this is re erred toas epigenetic regulationbecause it involveschemical modi cation of the DNA (that is,CpG methylation) and histones, resulting instable changes in gene transcription without

mutation of the DNA sequence.Although they can be removed, methylationmarks on DNA can last a surprisingly longtime, perhaps or an entire li etime, such asinactivation o one o the X chromosomesin emales.5 Even more surprisingly, these

epigenetic patterns can be transmitted romgeneration to generation via germline cells(egg and sperm). Transmission or three ormore generations has been documented.6,7 When cells divide, the methylation pattern frst stripped o and then aith ully recrein the new cells via a process that is poorlyunderstood at present. However, it appearsthat the in ormation needed to recreate theDNA methylation pattern is inherent wthe cytoplasm o the cell since trans er onew nucleus takes on the character o thecytoplasm.

Epigenetic methylation patterns are highlsensitive to the cellular environment. Indeeit appears that the ongoing revision omethylation marks provides a mechanism bwhich cells can alter their gene expression tadapt to changes in the environment, broadldefned. Changes in the cellular environmencan include changes in the levels o signalimolecules (growth actors, hormones, and

neurotransmitters), nutritional constituentsand xenobiotics or other toxic substances.Given the nature o epigenetic regulation, iclear that the e ects o toxic exposure caoutlast the actual period o exposure.8

In part because o their long-lasting natu

Transcription factor regulation

Epigenetic regulation

DNA + Histone = HeterochromatinGenes are silenced and transcription is blocked

mRNA

Protein(e.g., enzyme)

DNA

DNA

Transcription Translation

Start site formRNA synthesis

Growth factors

Neurotransmitters Hormones

TF binding region Gene sequence

RNApolymerase

Histoneproteins

HMT

SAM

SAMDNMT

MBDP(e.g., MeCP2)

Me Me

CpG CpG

Me Me

MeMe

TF

Upper panel: Transcription factors (TFs) bind to speci c DNA locations preceding theregulate, leading to binding of RNA polymerase which synthesizes the corresponding mRNA (mRNA).

Lower panel: Epigenetic regulation involves methylation of DNA at CpG sites, leadingthe binding of methyl binding domain proteins (MBDPs), which in turn bind enzymeshistone methyltransferase (HMT). DNA wraps tightly around methylated histones, blocktranscription. S-adenosylmethionine (SAM) is the donor of methyl groups for both DNhistone methylation, making it a critical actor or epigenetic regulation.

fi e 1. r gul on of g n ns p on by ns p of o s nd p g n m n sms



epigenetic mechanisms are central to normaldevelopment. Inherent within our DNA is aprogrammed sequence o changes in the

actors that regulate gene expression duringdevelopment. Disruption o methylation byxenobiotics and other toxic substances canthere ore cause developmental disorders viatheir epigenetic in uence.

EpIgEnEtIc rEgulAtIon IssEnsItIvE to rEdox stAtusDNA methylation is only one of more than200 methylation reactions.9 All o thesereactions are under the in uence o the

olate and vitamin B12-dependent enzyme,methionine synthase (MS). As illustrated inFigure 2, MS is a part of the methionine cycleo methylation, converting homocysteine(HCY) to methionine (MET) using a methylgroup derived rom 5-methyltetrahydro olate(5-MeTHF), with vitamin B12 being directlyinvolved in transferring the methyl group. MET

formation by MS increases the level of themethyl donor S-adenosylmethionine (SAM)and lowers the level o the methylationinhibitor S -adenosylhomocysteine (SAH),whose conversion to HCY is reversible. Thus,increased MS activity powerfully promotesmethylation by a ecting both componentsof the SAM to SAH ratio and increasingthe value of SAM/SAH. Conversely, adecrease in MS activity inhibits methylationreactions by lowering the SAM/SAH ratio.MS activity thus affects each of the morethan 200 methylation reactions, exerting anexceptionally broad in uence over manycellular activities, as illustrated in Figure 3.Indeed, we showed that a 2- old increasein MS activity, caused by insulin-like growth

actor 1 (IGF-1), resulted in a similar 2- oldincrease of global DNA methylation,indicative o a broad epigenetic in uence.10 Accordingly, any actor that signifcantly altersMS activity will exert signi cant epigenetice ects, especially during development.

The vitamin B12 cofactor within MS servesas a sensor o cellular redox (reduction-

oxidation) status, helping to maintain redoxhomeostasis or the balance betweenreduced and oxidized states. When levelso the antioxidant glutathione (GSH) arelow, oxidative stress prevails and MSactivity is inhibited, resulting in a decreaseof SAM/SAH and inhibition of methylationreactions. However, during oxidative stressthe lower MS activity diverts more HCYto the transsul uration pathway, increasing

ormation o cysteine and GSH, andrestoring antioxidant levels to normal. Thisrelationship between GSH levels and MS

fi e 2. r dox nd m yl on p w ys n n u ons

Transsulfuration

NEURONALCELL

GLIAL CELLS(Astrocytes)

Glutathionesynthesis

GSSG GSH

-Glutamylcysteine

Cysteine

Cystathionine

()

>200 methylation reactions(e.g., DNA methylation)

DIETARY PROTEIN

Adenosine

METHIONINESYNTHASE

SAH

SAM

THF

MET

Methyl-THF

HCY

ATP PP + Pi

Cysteine Cysteinylglycine GSH

The amino acid cysteine is rate-limiting or glutathione (GSH) synthesis, and it is providedeither by uptake from astrocyte-derived cysteine or by transsulfuration of homocysteThe methionine cycle o methylation (lower right) depends upon both dietary methionine(MET) and remethylation of HCY by methionine synthase. Since formation of HCYS-adenosylhomocysteine (SAH) is reversible and SAH inhibits methylation, decreasedmethionine synthase activity (e.g., caused by oxidative stress) both augments GSH synthesiinhibits methylation reactions. Thus, redox status and methylation activity are closely linked

More than 200 methylation reactions are sensitive to redox status via its in uenceto SAH ratio, and selected examples relevant to autism are illustrated. Oxidative stress will ieach o these reactions, exerting an exceptionally broad in uence over cell unction, includepigenetic regulation o gene expression.

COGNITIVESTATUS NITRIC OXIDESYNTHESIS

ARGININEMETHYLATION

CATECHOLAMINEMETHYLATION

CREATININESYNTHESIS

ENERGYSTATUS

MEMBRANEPROPERTIES

PHOSPHOLIPIDMETHYLATION

DNA / HISTONEMETHYLATION

SEROTONINMETHYLATION

MELATONIN

SLEEP

EPIGENETICREGULATION

OFGENE

EXPRESSION

REDOXSTATUS:

GSHGSSG

METHYLATIONSTATUS:

SAMSAH

> 200METHYLATION

REACTIONS

fi e 3. r dox-s ns v m yl on ons



activity is there ore a critical mechanism ormaintaining cellular redox status. Any actorthat lowers GSH levels will cause a decreasein global DNA methylation, with epigeneticconsequences.11

The in uence of oxidative stress on DNAmethylation provides an opportunity orphysiological regulation o gene expressionvia epigenetic regulation, which may be adriving mechanism or development. In thisregard, it is interesting that the mature ovumat ertilization is relatively rich in glutathione,12 while sperm are enriched in the antioxidantselenium and selenium-containing proteinswhich supply electrons to maintain GSH inits reduced state.13 Thus, when the combinedDNA in the fertilized egg is exposed to anovel redox state distinct rom either theun ertilized egg or the sperm, it will initiateglobal changes in DNA methylation and geneexpression. These changes may be critical orinitiation o the program o development.

Pluripotent stem cells arising rom theertilized egg ultimately develop into thevarious specialized tissues in the maturebody. Growth actors play an importantrole in guiding the stable changes in geneexpression that distinguish di erent celltypes. In part, growth actors cause thesedevelopmental changes by altering theepigenetic marks on DNA.14 Most growth

actors exert their e ects via activation othe PI3 kinase signaling pathway, whichis the same pathway that insulin activatesto stimulate glucose uptake by cells. Werecently ound that IGF-1 and several otherneurotrophic growth actors cause signifcantchanges in redox status, including an increasein GSH levels, as a result o their ability toincrease uptake o the amino acid cysteine,which is rate-limiting or GSH synthesis. Theincrease in GSH levels was accompaniedby an increase in methylation capacity.Coupled with our earlier observation thatIGF-1 stimulates MS activity and increasesDNA methylation,15 these fndings outlinea signaling pathway by which growth

actors can use changes in redox statusand epigenetic mechanisms to alter geneexpression (Figure 4). Further studies haveshown that the proin ammatory cytokinetumor necrosis factor-alpha (TNF-) has ane ect opposite to growth actors, loweringcysteine uptake and inhibiting methylation,while augmenting transsul uration, illustratingthe potential or reciprocal epigeneticregulation. The ability of TNF- to increasetranssul uration has previously been describedby others.16 Notably, brain levels of TNF- are increased in autism.17,18

rEdox sIgnAlIng AndEpIgEnEtIc rEgulAtIon In thEhumAn brAIn

The ability of growth factors and TNF- toalter cysteine uptake, GSH, and methylationactivity are illustrative of redox signaling,in which changes in oxidative state mediatethe cellular e ects o a wide variety ophysiologic substances. Recognizing theability o redox signaling to alter geneexpression, we can now examine the special

eatures o this mode o epigenetic regulationin the human brain. As we do so, it is use ul toappreciate that the human brain representsthe pinnacle o natural evolution and is

characterized by mechanisms that havebeen exploited across biological time. Thekey to a highly responsive redox/epigeneticsignaling system is the presence o a strongthreat o oxidation and dynamic regulation oantioxidant production, with the human brainproviding both eatures.

As previously noted, availability o cysteineis limiting or GSH synthesis. However, thelevel o cysteine in cerebrospinal uid (CSF)is only one tenth the level in blood,19 meaningthat the raw material or making antioxidantsis much scarcer in the brain. Indeed, the

GSH level in neurons is the lowest reportedor any cell type,20 despite the act that the

brain utilizes oxygen at a ten times higherrate than other tissues. Neurons obtaincysteine rom neighboring astrocytes, whicrelease GSH that is subsequently brokendown to cysteine.21 This cysteine is thenavailable or uptake by neurons, dependingupon the activity o the cysteine transportecontrolled by growth actors. Higher levelso growth actor allow more cysteine intocells, increasing GSH synthesis and leadingto higher MS activity and increased Dmethylation. The increase in antioxidantlevel also allows cells to sa ely increase

their oxidative metabolism (that is, theirmitochondrial activity), and the resultantincrease in ATP (adenosine triphosphate)supports a higher level o neuronal activitythis way, redox signaling not only regulatesgene expression but also controls the level oneuronal unction.

A second brain-specifc eature thataugments redox signaling is the brainsdecreased level o transsul uration activity,limiting the conversion o homocysteine tocysteine.22 As a result, neurons are more at rio oxidative stress and more dependent upon

METHYLATION

> 200 different reactionsNeural networksynchronization

Epigeneticregulation

Redox status

GSH GSSG

HCY

SAH

SAM

MET

Methioninesynthase

( )

( + )( + )( )

( + )

CYSTEINE

TNF-alpha (inflammatory cytokine)GROWTH FACTORS

TranssulfurationCysteine influx

By altering the activity o cysteine uptake or transsul uration pathways, physiological actorsas neuronal growth factors (e.g., IGF-1) or proin ammatory cytokines (e.g., TNF-) can shi t therelative contribution o each pathway to GSH synthesis. However, activation o transsul uratiby TNF- is accompanied by a decrease in methionine synthase activity, while growth actoractivation o cysteine uptake causes an increase, producing opposite e ects on methylation aepigenetic regulation.

fi e 4. r dox s gn l ng by g ow f o s nd tNF-



it acilitates attention by synchronizing theactivity o neural networks.28 Subsequently itwas shown that the D4 dopamine receptoris indeed critically involved in neuronalsynchronization during attention.29 Since D4receptor-mediated phospholipid methylationis absolutely dependent upon MS activity,this raises the possibility that ADHD mightre ect a decrease in MS activity due tooxidative stress.

systEmIc oxIdAtIvE strEssAnd ImpAIrEd mEthylAtIon InAutIsm

During the past seven years, 12 separatestudies have been published rom laboratoriesaround the world, each reporting a highlysignifcant decrease in plasma level oGSH in autistic children, with the averagedecrease amounting to 35%.30-42 Since theblood compartment is in ready equilibriumwith extracellular uid (except in the brain),this indicates a body-wide defcit. Thestudies ound increased levels o oxidizedglutathione (GSSG) in conjunction witha decrease in the GSH to GSSG ratio,indicative o body-wide oxidative stress.Levels o cysteine, the primary extracellularantioxidant, were also signifcantly decreasedin each o the studies in which it wasmeasured. Other work shows that intracellularlevels o GSH are decreased in lymphoblastsfrom autistic subjects,43 indicating that bothextracellular and intracellular compartmentsare under oxidative stress.

To our knowledge, no studies have ailedto fnd a decrease in GSH and cysteine inautistic children. Moreover, the number ofsubjects needed to demonstrate a statistically

signifcant decrease in plasma GSH issurprisingly small (no more than 20-30/group). This indicates that oxidative stressis very common in autism, a fnding thatcontrasts starkly with the rare occurrenceo de novo genetic mutations. Given theunanimity o evidence or oxidative stress asa common eature o autism, it is remarkableand even shocking that this fnding hasreceived so little attention in the generalmedical community and in the publicawareness domain. This active ignorance isespecially troubling since there are metabolic

treatment strategies to correct oxidative The sensitivity of MS to oxidative

leads to a decrease in SAM formatian increase in SAH ormation. Both o have been observed in the plasma o autchildren in conjunction with a decrethe SAM to SAH ratio, which is ino impaired methylation capacity.44,45 Thuoxidative stress and impaired methylatiobe viewed as confrmed hallmarks o autsupporting a redox/methylation hypoof autism.11 In this scenario, global Dmethylation in blood cells decreasesaccordingly, indicating that epigenetic

regulation o gene expression is a ectewell.42 Brain levels o olate and methyl ola

lower than in other tissues.46,47 Moreover,decreased availability o methyl olateincreases the probability that the B12cofactor in MS will be oxidized. Bethe vulnerability of the B12 cofactorto oxidation is greater when methyl olatlevels are low, MS inhibition (and tincreased vulnerability o methylation) greater in individuals carrying disablingsingle nucleotide polymorphisms (SNin the methylenetetrahydro olate reduct(MTHFR) gene. In conjunction withbrain-specifc redox eatures noted abovthe metabolic and therapeutic benefts o

olinic acid, methyl olate, and methyl-Bautism re ect their role in reactivatio

ollowing B12 oxidation.30,44,48

SNPs that adversely affect vitaminavailability or the ability to reactivata ter oxidation also increase vulnerabilio epigenetic regulation to oxidativestress, and these SNPs as well as a

in catecholamine-O-methyltrans erase(COMT) have been reported to be mprevalent in autistic subjects.45,4951 It shoube noted, however, that the presence oSNPs represents apotentialvulnerability oxidative stress that will occur only undethreatening environmental conditions. Unormal conditions, these very samemay endow individuals with benefcialepigenetic responsiveness to redox signaCystathionine beta-synthase (CBS), whiinitiates transsulfuration of HCY toexhibits a large number of SNPs bu

cysteine uptake to sustain GSH levels. Thisincreases the power and importance o growth

actors in regulating redox and methylationstatus and, together with the limited availabilityo extracellular cysteine, makes epigeneticregulation exceptionally dynamic.

The restricted availability o GSH inthe brain is partly compensated or by anincreased dependence upon selenoproteinsto sustain antioxidant activity. Selenoproteins(a class o proteins that contain selenium inthe orm o the amino acid selenocysteine)per orm a variety o important redox-relatedbiological unctions. To meet the increased

demand or selenoproteins, the brain (aswell as the testes) has developed a highercapacity to retain precious selenium.Moreover, when selenium levels are low,other tissues become depleted whilebrain levels remain high. Un ortunately,selenoproteins are exquisitely sensitive toinhibition by mercury, and any mercurythat penetrates the brain will inter ere withredox signaling and potentially disruptnormal epigenetic regulation.23,24 The verytight binding o mercury by selenoproteins(as measured by an a fnity constanto 1045), especially to selenoprotein P,contributes to the long-term retention omercury in the brain and its accumulationacross the li espan. Indeed, it has beenproposed that selenoprotein P, which has 10selenocysteines and is not directly involvedin redox regulation, serves a specifc roleto bind mercury, thereby protecting otherselenoproteins rom its toxicity.25

In addition to its conversion of HCY toMET (described previously), MS carriesout a second reaction, providing methyl

groups to the D4 dopamine receptor, whichsubsequently trans ers them to membranephospholipids when stimulated by dopamine.Our lab was frst to discover this novelactivity, which appears to only be carried outby the D4 dopamine receptor.15,26,27 Geneticvariants o the D4 receptor (such as the7-repeat variant) have been linked to novelty-seeking behavior and are also risk actors

or at tention-defcit/hyperactivity disorder(ADHD). Although the role o dopamine-stimulated phospholipid methylation remainsincompletely understood, we proposed that

un ort n te , se enoproteins re exq isite sensiti e to inn n merc r t t penetr tes t e br in wi inter ere wi

potenti isr pt norm epi enetic re tio



been studied as extensively as MTHFR. Onestudy ound that the risk o having a child withautism was higher if the mother carried SNPsin either MTHFR or CBS and did not takeprenatal vitamins.52

ExplorIng thE rEdox/mEthylAtIon hypothEsIsIn collaboration with colleagues at the SultanQaboos University School of Medicine inOman, we recently evaluated the requency oautism in Oman.53 Our study, which involved30 autistic and 30 control children (with 15males and 15 emales in each group), alsoassessed the childrens nutritional status, serumlevels o redox and methylation-related sul urmetabolites, and vitamin B12 and olic acidlevels. In contrast to autistic children in the US,malnutrition was common in Omani childrenand associated with highly signifcant defcitsin B12 and olic acid levels (Figure 5). Similarto the US, we observed a signifcantly lower

level o GSH in autistic children (48% ocontrols or combined groups), with the extento the decrease being greater in male versusfemale subjects, compared with same-sexcontrols. We speculate that autism in Omanichildren may result mainly rom an antioxidantdefcit associated with a nutritional defciencyin the cofactors for MS, whereas autism inthe US appears to result rom environmental

toxin exposure in a genetically vulnerablepopulation. The occurrence o lower GSHlevels arising rom di erent etiological actorsis very strong evidence or the undamentalimportance o impaired antioxidant capacityin autism.

The act that mitochondrial dys unction iscommon in autism can be directly relatedto the presence o oxidative stress and toan increase o GSSG. When the level oGSSG increases, it can react with cysteineresidues in proteins, leaving hal o the GSSGattached to the sul hydryl (SH) group on theprotein, a process called glutathionylation.54 The other hal o GSSG is released asGSH, providing more antioxidant. A classicexample o how glutathionylation worksinvolves complex I in the mitochondrialelectron transport chain. When GSSGbuilds up during oxidative stress, complexI is increasingly glutathionylated, causinga decrease in the ow o electrons toward

oxygen reduction and ATP generation,leading to mitochondrial dys unction.55 However, this is actually a use ul survivalmechanism for cells under oxidative stress;shutting down mitochondrial unction lowersthe amount o reactive oxygen species(ROS) production, thereby decreasing thestrain on already low levels o antioxidant.Thus, mitochondrial dys unction can occur

secondary to oxidative stress and theaccumulation o oxidized glutathione, whicis a metabolic eature o autism.

Recently, thanks to research grants romthe Autism Research Institute, the NatiAutism Association, and SafeMinds, wehad the opportunity to evaluate the level oMS mRNA in postmortem human corteautistic subjects and age- and sex-matccontrols. As illustrated in Figure 6, we

ound a remarkable pattern o progressivedecrease in mRNA levels across the liin the control subjects, amounting to mthan a 400- old decrease. The decreaseoccurred in two phases. High initial mlevels rapidly decreased up until the end othe teens and decreased more graduallytherea ter. This biphasic pattern appearsto correspond to the period o rapid lineargrowth lasting through puberty, ollowedby the relatively static state o adulthood.Despite the dramatic decrease in mRNA

levels, levels of MS protein remainedrelatively static, suggesting the possibilitythat the rate o new protein synthesis rommRNA decreases with age and that theproteins are lasting longer as we get older.This is in keeping with the general concepto gradually decreasing metabolic activitywith advancing age, but at this time it isunclear whether MS has a special rolecoordinating the decrease.

In comparison, we ound an abnormalage-dependent pattern in autism. The highinitial phase of mRNA was essentiallywith mRNA levels in 4- to 10-year-old

Autism in Oman appears to be largely a consequence o malnutritionand nutritional actors, including signifcantly lower levels o vitaminB12 and olic acid, unlike autism in the US. Thus, di erent causesconverge on methionine synthase, indicating its central role in autism.

fi e 5. S um l v ls of fol d nd v m n B12

n u s subj s f om Om n

An age-dependent decrease in MS mRNA of more than 400-foloccurs across the li espan. An initial stage o rapid decrease lasts untithe end o adolescence, ollowed by a slower decline therea ter.

fi e 6. L v ls of MS mrNa n pos mo mum n o x d s w g

S um Fol L v ls

S um B12 L v ls

ag (y s)

Male Con olMale Au is icfemale Con olfemale Au is ic

Male Con olMale Au is icfemale Con olfemale Au is ic

S e u m

F o l t e

n g / m

l

S e u m

B 1 2

p g

/ m

l

** p < 0.001

** p < 0.0001

** **

****

6

5

4

3

2

1

0

600

500

400

300

200

100

0

300

200

100

00 10 20 30 40 50 60 70 80 90 100

M S c o

b m

r N a ( b

i t y u n

i t s ) t as = 3.4 yea s

t slow = 29.4 yea s

r 2 = .91



subjects being similar to levels in 30-year-old control subjects (Figure 7). On average,the mRNA levels in the autistic sampleswere reduced by about 50% at eachage versus paired control subjects. Thesedata provide clear evidence that in autismMS mRNA status is altered in the brain.Although additional study is required to ullyunderstand the unctional signifcance o thedecrease, at this point it seems reasonableto conclude that the normal age-dependentdecrease in metabolic activity o thedeveloping cortex is accelerated in autism.Progressive changes in redox status arelikely to underlie the normal age-dependentdecrease, although this remains to be proven,and the oxidative stress that is characteristico autism may accelerate this progression,with untoward epigenetic consequences.Essentially, this perspective postulates aredox-driven epigenetic clock mechanismo development in which both intrinsic genetic

actors (in the cases o Rett and Angelmansyndromes) and extrinsic actors (such asredox-active xenobiotic substances) candisrupt the mechanism and contribute toneurodevelopmental disorders such asautism.

rEdox E Ects o glutEn- AndcAsEIn-dErIvEd opIAtE pEptIdEsThe overwhelming number o parental andpublished reports indicating benefcial e ectso a gluten- ree/casein- ree (GF/CF) diet,5659 coupled with evidence o gastrointestinal(GI) in ammation,5961 suggests that redox/methylation dysregulation in autism has itsroots in the digestive tract. Moreover, thisevidence suggests that the deleterious e ectso gluten and casein in sensitive individualsmight be associated with their previouslydescribed ability to st imulate opiatereceptors. To investigate this possibility, weexamined the redox e ects o three peptidesthat are relatively stable end products ogluten and beta-casein o either human orbovine origin. As illustrated in Figure 8, thesepeptides share a similar but non-identicalamino acid sequence, and their prolineresidues make them comparatively resistantto urther hydrolysis, although they arehydrolyzed by dipeptidyl peptidase-4(DPP-IV or CD26).62 Recent studies suggest arole or casein-derived opiate peptides andlow DPP-IV activity in sudden infant deathsyndrome (SIDS).63,64

In cultured cell studies using either

human intestinal epithelial cells or humaneuroblastoma cells, we ound that allthree peptides, as well as morphine,inhibited the uptake o cysteine in a dosdependent manner a ter a 30-minute peo incubation (Figure 9). While morphinwas clearly the most power ul inhibitor,bovine (cows milk) peptide was strongethan the human peptide in both epitheliaand neuronal cells. The inhibitory e ecthe bovine peptide was also evident as adecrease in cellular levels o cysteine anGSH (Figure 10). These peptide e ectswere completely blocked by naltrexone analoxone, confrming involvement o opreceptors. Although cultured cell studiesmay not ully re ectin vivoresponses, thesresults indicate that a GF/CF diet may exits benefcial e ect in autism by acilitaintestinal cysteine uptake, thereby increaavailability o this essential raw materia

or GSH synthesis. In addition, the greainhibitory activity o the bovine peptidethe human peptide supports the generallyheld belie that breast eeding o ers ubenefts to early development65 that mayre ect the epigenetic consequences o aenhanced antioxidant capacity.

Messenger RNA (mRNA) levels are lower inautistic subjects versus age-matched controls,

with the di erence being most pronounced atyounger ages.

fi e 7. L v ls of MS mrNan pos mo m um n o x of

u s subj s

Human breast milk Wheat, barley, rye

Beta-Casein

-L - V - Y - P - F - P - G - P - I -X

Gliadins

-gliadin-7Tyr-Pro-Gin-Pro-Gin-Pro-PhTyr-Pro-Phe-Val-Glu-Pro-Ile

Tyr-Pro-Phe-Pro-Gly-Pro-Ile

-Casein (40% of milk protein)

Human -Casomorphin-7

Bovine-Casomorphin-7(0.4 g/L milk)

A1(His at 67)

A2(Pro at 67)

59 66-67

The proteins beta-casein rom milk and gliadin rom wheat, barley, and rye are broken downthe intestine to peptides that have opiate activity. The seven amino acid- containing peptides human milk, cows milk (bovine), and wheat have similar but not identical sequences, includior three proline residues (Pro) that increase their stability.

fi e 8. Food-d v d op p p d s

Te o erw e min n mber o p rent n p b is e rebene ci e ects o ten- ree/c sein- ree (g /C ) i

e i ence o strointestin (gI) inf mm tion, s ests t tsre tion in tism s its roots in t e i esti

M S c o

b m

r N a ( b

i t y u n

i t s )

400

300

200

100

0

ag (y s)0 10 20 30 40

Con olAu ism



Gluten intolerance is the classic eature oceliac disease. The prevalence o glutenintolerance is increasing in the US,66 paralleling, to some extent, the increasein autism rates. It is there ore tempting tospeculate that the population as a wholeis experiencing an environmental exposurethat causes the uptake and availabilityo cysteine or antioxidant synthesis tobe increasingly critical or a signifcantnumber o persons. In this context, autismcan be viewed as a neurodevelopmentalmani estation o this exposure, with parallels

to celiac disease, which has GI tract,immune, and neurological components. Asrecently reviewed,67 there are numerousreports o signifcant improvement in otherneurological and neuropsychiatric disorders

ollowing institution o a gluten- ree diet,accompanied by reports o re-emergenceo symptoms upon re-exposure to gluten.Where casein is concerned, cerebral olatedefciency (CFD) is a pertinent example.CFD is associated with autoantibodies to the

olate transporter, whose levels decrease ona milk- ree diet.68,69 Finally, it is worthwhile to

note that intestinal uptake o selenocysteineis also critical or normal redox regulation,and levels o selenium are low in autisticchildren.70,71 It is possible that gluten- andcasein-derived opiate peptides may inhibitselenocysteine uptake, but this has yet to beinvestigated.

WhAt Is cAusIng thE rIsE InAutIsm rAtEs?Overwhelming evidence indicates thatoxidative stress is a core eature ocontemporary autism, and the increasing

autism rates imply that one or moreenvironmental actors are responsible. Itseems clear that the a ected metabolicpathways causing autism are those a ectingGSH synthesis and maintenance o itsreduced state. Candidate environmental

actors, there ore, are agents capable odisrupting the ow o antioxidant electronsto GSH or inter ering with availability ocysteine or GSH synthesis. Reports ogender-related di erences in GSH synthesisare consistent with the predominance oautistic males.72,73 These observations

suggest the ollowing criteria or theselection o candidate environmental acto

1. Candidate causative actors should becapable o inter ering with the metabolicsystems that maintain a normal redox status

2. Treatments that improve autism shouldhelp to identi y these metabolic pathways.

3. The a ected metabolic pathwaysshould provide a rationale or the 4:1 maleto emale gender bias in autism.

4. Genetic variants a ecting the targetmetabolic pathway should in uence the risko autism.

5. The toxicological profle o candidateactors should be consistent with observed

signs and symptoms o autism.

6. Candidate actors must be widespreadin their distribution and capable o increasiautism rates in essentially all regions o theUS and in most developed countries.

Cysteine uptake was measured in cultured human intestinalepithelial cells (Caco2 cells) a ter a 30-minute incubation witheither morphine, human or bovine -casomorphin-7 (BCM7),or the gliadin-derived peptide gliadinomorphin at the indicatedconcentrations. Morphine caused the greatest decrease in uptake,followed by bovine BCM7, human BCM7, and gliadinomorphin.

fi e 9. Mo p n nd op p p d s n bys n up k by n s n l p l l lls

Cellular levels o sul ur metabolites were measured in human neuronalcells (SH-SY5Y cells) following a 4-hour incubation with 1 MBCM7. Signi cant decreases in cysteine, GSH, and methionine wobserved, while homocysteine and cystathionine increased. This patternis consistent with decreased cysteine uptake and inhibition o methionisynthase activity.

fi e 10. con n on-d p nd n ng s n sm bol s n n u on l lls us d by bov n Bc

O erw e min e i ence in ic tes t t oxi ti e stress iso contempor r tism, n t e incre sin tism r tes im

more en ironment ctors re responsib e.

Mo p ineBovine BCM7human BCM7gliadinomo p in

c y s

t e i n e

U p

t k e

( n m o

l / m g o

f p o

t e i n )

6

4

2

0

con n ons (M)0 -10 -8 -6 -4

Cys eineMe ionine

homocys einegSh

Cys a ionine

n m o

l / m g

o f p o

t e i n

con n on (M)0 -10 -9 -8 -7 -6

100

75

50

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0



7. There should be a temporal associationbetween increased autism rates andincreased exposure to the causative

actor(s), and in cases o regressive autism,the onset o symptoms should temporallycorrelate with exposure.

The list o candidate environmental actorsthat meet the listed criteria is not long.Metals such as lead, mercury, cadmium,and arsenic are well recognized or theirtoxicological actions on redox pathways,including binding to selenoproteins suchas glutathione peroxidases or thioredoxinreductases or to critical thiol groups inredox-active proteins such as thioredoxin orglutaredoxin.7477 Aluminum also promotesoxidative stress, apparently via inhibitiono isocitrate dehydrogenase, the primarysource of NADPH for GSH reduction inmitochondria.78 None of these metalsparticipate in normal human metabolism,

and their presence above some arbitrarythreshold level is incompatible with li e. Assuch, these metals are not only at the topo the autism candidate list, but also at thetop o ederal agency lists o importantenvironmental toxins.79

Mercury merits special considerationbecause o its extremely potent inhibitiono selenoproteins74 and its broad exposure

rom airborne, ood, amalgam, andvaccination sources.80 Mercury wasrecently demonstrated to cause epigeneticabnormalities in embryonic stem cells,demonstrating its potential or contributingto developmental disorders.81 Interestingly,selenium also caused abnormalities,indicating the importance o redox inepigenetic regulation. While the ethylmercurypreservative thimerosal has been substantiallyremoved rom most childhood vaccines in theUS, it un ortunately remains in many vaccinesdistributed outside the US. The potential oorganomercurials such as thimerosal to enterthe brain and to be trapped or long periodso time ollowing their dealkylation represents

another troubling property o this metal.A number o other xenobiotic compoundsadversely impact GSH metabolism and

promote oxidative stress, making themadditional candidates or causing autism.These include plasticizing agents such asbisphenol-A and phthalates,82,83 pesticidessuch as diazinon or chlorpyri os,84 andherbicides such as atrazine.85 Widespreadcommercial use o these compounds impliesextensive population-level exposure, buttheir toxicological profles generally do notmatch the metabolic abnormalities oundin autism as closely as do heavy metals.Acetaminophen (Tylenol), however, is anexception in that it lowers GSH levels,86 inter eres with selenium metabolism,87,88 and exhibits gender-dependent toxicity.72,87 It has been suggested that increased useo acetaminophen or treatment o post-vaccination ever and in ammation, ollowingthe 1980 recommendation to not use aspirindue to the risk o Reyes syndrome, may havecontributed to a rise in autism rates.8991

The highly controversial possibility that

vaccination itsel may contribute to autismmust also be given consideration sincethe undamental nature o vaccination isto provoke an immune response that, bydefnition, is associated with in ammationand some level of oxidative stress. Multiplevaccinations there ore represent multipleprovocations o the ability o antioxidantsystems to maintain or restore redoxequilibrium, which may exceed the capacityo some individuals. Recent studies haverevealed the undamental role o GSH-basedredox signaling in the immune response.92,93

For example, antigen-presenting cells not onlyphysically interact with naive T-cells throughthe T-cell receptor complex, but they alsorelease GSH into the local environment, romwhich cysteine is available or T-cell uptake,similar to the release o GSH by astrocytes,which provides cysteine or neuronal uptake.When T-cells take up cysteine, they becomemetabolically active and divide. However,regulatory T-cells suppress the immuneresponse by competing with the naive T-cells

or cysteine, indicating the importance o

redox regulation.92

Currently utilized vaccineadjuvants, such as aluminum hydroxide,augment immune response by promoting

oxidative stress and in ammation.94,95 The persistent epigenetic consequenceso this action may contribute to the redoabnormalities ound in autism.96,97 Moreovthe ever-increasing number o mandatedvaccinations demands a very high standao sa ety testing, higher than is currentlemployed, including long-term studies wunvaccinated populations.

Because so many di erent environmactors impinge upon the GSH system, t

e ects are additive and possibly synergespecially i they impair di erent pathNonetheless, agents that have the higlikelihood o population-wide exposure are capable o being retained in the body

or long periods o time deserve speciaattention as possible causes o autism.

summAry And pErspEctIvEThe rationale or considering autism as neuroepigenetic disorder is clear. Withou

minimizing the classifcation o autism aa neurodevelopmental disorder, the termneuroepigenetics increases our clarity abwhich molecular events are abnormal.Neuroepigenetics encompasses formsautism that are primarily genetic in origias well as those caused primarily byenvironmental actors, with contributionboth in most cases. Since neuroepigenetidisorders are potentially amenable totreatment, it is important to recognize theepigenetic roots o autism and to pursueearly intervention treatments directed townormalizing redox and methylation statuand eliminating or minimizing exposure causative actors. It is equally importantdevelop and widely employ laboratorytests or oxidative stress and impairedmethylation, making them routinely avai

or early identifcation and treatment othese metabolic disorders. O course,the most important goal is to identi y thenvironmental cause(s) o autism andeliminate them. No matter which facidentifed, the autism epidemic is a wake-

up call about the dangers associated withenvironmental toxic exposures arising rour own activities.

Since ne roepi enetic isor ers re potenti men bis import nt to reco nize t e epi enetic roots o tism n

inter ention tre tments irecte tow r norm izin re ox nn e imin tin or minimizin expos re to c s ti e



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Autism: A Neuroepigenetic Disorder. Deth, R et al