Searching for ways out of the autism maze: genetic, epigenetic and environmental clues

INMED/TINS special issue

Searching for ways out of the autismmaze: genetic, epigenetic andenvironmental cluesAntonio M. Persico1,2 and Thomas Bourgeron3,4

1Laboratory of Molecular Psychiatry and Neurogenetics, University ‘Campus Bio-Medico’, Via Longoni 83, I-00155, Rome, Italy2IRCCS ‘Fondazione Santa Lucia’, Department of Experimental Neurosciences, Via del Fosso di Fiorano 64/65, I-00143, Rome, Italy3Laboratory of Human Genetics and Cognitive Functions, Institut Pasteur, 25 Rue du Docteur Roux 75015, Paris, France4University Paris VII, 2 Place Jussieu 75013, Paris, France

Our understanding of human disorders that affect higher

cognitive functions has greatly advanced in recent

decades, and over 20 genes associated with non-

syndromic mental retardation have been identified

during the past 15 years. However, proteins encoded

by ‘cognition genes’ have such diverse neurodevelop-

mental functions that delineating specific pathogenetic

pathways still poses a tremendous challenge. In this

review, we summarize genetic, epigenetic and environ-

mental contributions to neurodevelopmental alterations

that either cause or confer vulnerability to autism, a

disease primarily affecting social cognition. Taken

together, these results begin to provide a unifying

view of complex pathogenetic pathways that are likely

to lead to autism spectrum disorders through altered

neurite morphology, synaptogenesis and cell migration.

This review is part of the INMED/TINS special issue

Nature and nurture in brain development and neuro-

logical disorders, based on presentations at the annual

INMED/TINS symposium (http://inmednet.com/).

Introduction

Autistic disorder was first described by the psychiatristLeo Kanner in 1943 [1] and is diagnosed on the basis ofthree behaviorally altered domains: social deficits,impaired language and communication, and stereotypedand repetitive behaviors [2]. Beyond this unifyingdefinition lies extreme clinical heterogeneity, rangingfrom debilitating impairments to mild personality traits.Hence autism is not a single disease entity, but rather acomplex phenotype encompassing either multiple ‘autisticdisorders’ or a continuum of autistic-like traits andbehaviors defined as ‘autism spectrum disorder’ (ASD),which includes autistic disorder (Kanner’s ‘autism’),childhood disintegrative disorder, pervasive developmentdisorder not otherwise specified (PDD-NOS, or ‘atypicalautism’) and Asperger syndrome. The dramatic rise inASD incidence from 2–5 to 15–60 per 10 000 children

Corresponding author: Persico, A.M. ([email protected]).

www.sciencedirect.com 0166-2236/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved

during the past two decades can be explained largely bythe use of broader diagnostic criteria and increasedattention by the medical community [3,4]. The limits ofan exclusively genetic etiology, and the possible contri-butions of environmental and epigenetic factors toincreased ASD incidence (Box 1), are highlighted by,among other evidence, the dramatic behavioral andneuroanatomical differences displayed by geneticallyidentical monozygotic twins discordant for an ‘autism’diagnosis [5]. Furthermore, in only w10% of the affectedindividuals is autism ‘syndromic’ – that is, secondary to aknown genetic disorder [6] such as chromosomalrearrangement (e.g. duplication of 15q), fragile X syn-drome, tuberous sclerosis and neurofibromatosis, orsecondary to exposure to identified teratological agents(Box 1). This highlights the current limitations of geneticdiagnostic protocols routinely employed in clinical set-tings. For the vast majority of patients, the origin of ‘non-syndromic’, ‘primary’ or ‘idiopathic’ autismremains unknown.

Altered neurodevelopment is widely recognized as theunderlying neuropathological cause of ASD. The CNS ofindividuals with autism might process information byactivating neural networks distinct from those employedby non-autistic individuals, particularly for sociallyrelevant stimuli [7,8]. The neuroanatomical substrates ofthis altered information processing appear as hetero-geneous as clinical manifestations and etiological under-pinnings. The few post-mortem studies of autistic brainsperformed to date suffer from methodological limitationsincluding diagnostic heterogeneity and small samplesizes; they typically describe brains of older individuals,who are likely to display chronic adaptive changes at leastas much as primary developmental pathology, and in someinstances the studies might not have employed the mostup-to-date techniques. Nonetheless, they have uncoveredvarious neurodevelopmental alterations, encompassingmany aspects of CNS formation, such as reducedprogrammed cell death and/or increased cell proliferation,altered cell migration with disrupted cortical and sub-cortical cytoarchitectonics, abnormal cell differentiation

Review TRENDS in Neurosciences Vol.29 No.7 July 2006

. doi:10.1016/j.tins.2006.05.010

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Box 1. Environmental and epigenetic factors in autism spectrum disorders

An etiological role for environmental factors in ASD has been

conclusively demonstrated only for: (i) prenatal or perinatal

exposure to viral agents, such as rubella and cytomegalovirus

[90,91], and (ii) prenatal exposure to thalidomide and valproic acid

[92,93]. Among other factors still under scrutiny, aside from

organophosphates (see main text), vaccinations have recently

drawn attention owing to their high content of thimerosal, a

preservative containing mercury, and to the proposed causal link

between ASD and the measles–mump–rubella vaccine. However,

epidemiological studies performed to date have excluded a wide-

spread causal role for vaccines in ASD [4,94].

During the past decade, several lines of evidence have pointed

towards epigenetic factors as contributing to ASD, in conjunction with

genetic variants. First, females who carry heterozygous mutations of

MeCP2 develop Rett syndrome, a pervasive developmental disorder

characterized by autism, loss of speech, hand wringing and seizures

[14]. MeCP2 brings about transcriptional silencing by binding to

methylated promoters, and recruiting co-repressor and histone

deacetylase complexes. A decrease in MeCP2 activity produces

transcriptional de-repression localized at specific promoters crucial

to brain development and plasticity, including those regulating levels

of brain-derived neurotrophic factor (BDNF), the transcription factor

distal-less homeobox 5 (DLX5), ubiquitin-protein ligase E3A (UBE3A)

and the GABA receptor subunit GABRB3 [95–97]. MeCP2 mutations

have also been implicated as rare genetic causes of idiopathic autism

[98]. Second, several chromosomal regions subject to imprinting (i.e.

the epigenetic silencing of either the paternal or the maternal allele)

are linked to ASD. For example, two separate loci on chromosome 7q

might contribute to ASD: the locus closer to the centromere might

express the paternal allele, whereas the locus closer to the telomere

might express only the maternal allele [99]. The imprinted region of

the Prader–Willi and Angelman syndromes on chromosome 15q is

also associated with ASD and includes the UBE3A gene, whose

abnormal methylation pattern has been described in at least one

autistic brain [100]. Finally, females with Turner syndrome, who have

monosomy of the X chromosome (X0), display an increased

susceptibility to ASD compared with normal XX females [101],

compatible with the existence of imprinted genes on the

X chromosome.

In summary, there is compelling evidence that environmental and

epigenetic factors can have a role in ASD susceptibility. Novel

technological tools are warranted to address the difficult question of

epigenetics in humans. Additionally, more results from combined

epidemiological and genetic studies are needed to understand better

the contributions of genetic, epigenetic and environmental factors in

these highly heterogeneous disorders.


with reduced neuronal size, and altered synaptogenesis[9,10]. These anomalies might explain the unbalancedlocal versus long-distance and inhibitory versus excitatoryconnectivity that is likely to underlie altered social-information processing in autism [11,12]. However, thisanatomical heterogeneity has undoubtedly hinderedthe discovery of etiological factors in ASD and hasprompted researchers to seek new insights throughgenetic approaches.

Family and twin studies have conclusively describedautism as the ‘most genetic’ neuropsychiatric disorder,with concordance rates of 82–92% in monozygotic twinscompared with 1–10% in dizygotic twins, sibling recur-rence risk at 2–3%, and heritability estimates of O90%[6,13]. However, three different levels of complexity haveemerged in recent years, namely a high degree of geneticheterogeneity (i.e. different contributing genes in differentpatients), a polygenic or oligogenic mode of inheritance inmost cases (i.e. many susceptibility-conferring genevariants at different loci are required for an individual todevelop the disease), and the presence of significant gene–gene and gene–environment interactions. To date, genomescans, linkage and association studies, chromosomalrearrangement analyses and mutation screenings haveidentified: (i) genomic regions likely to contain autismsusceptibility loci on human chromosomes 1q, 2q, 5q, 6q,7q, 13q, 15q, 17q, 22q, Xp and Xq; (ii) genes whosemutations represent a rare cause of ‘non-syndromic’autism (NLGN3 and NLGN4) or yield ‘syndromic’ autism(FMR1, TSC1, TSC2, NF1 and MECP2); and (iii)candidate vulnerability genes, with potential commonvariants enhancing risk but not causing autism per se(Table 1).

Within the framework of this clinical, neuroanatomicaland genetic heterogeneity, the study of simple monogenicforms of the disease (i.e. one gene, one disease), althoughrelatively uncommon, can powerfully improve ourunderstanding of the underlying causal processes. At the

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same time, genetic and functional studies of vulnerabilitygenes can provide the genetic, neuroanatomical andneurobiological information necessary to delineateplausible scenarios and to design novel hypothesis-drivenstudies of gene–gene and gene–environment interactions.

Neurodevelopmental genes and autism

Figure 1 depicts proteins that have been implicated inASD to date. All of these proteins are involved inneurodevelopment and many have roles in synapticfunction. These proteins can be schematically dividedinto at least eight distinct ensembles (Table 1), dependingon their involvement in (i) chromatin remodelling andregulation of transcription, (ii) actin cytoskeletondynamics, (iii) synaptic scaffolding, (iv) neurotransmission,(v) second-messenger systems, (vi) apoptosis, (vii) celladhesion, or (viii) paracrine cell–cell communication.

(i) Chromatin remodeling and regulation of transcription

Two X-linked genes, MeCP2 and FMR1, are involved inautism ‘secondary’ to Rett and fragile X syndromes,respectively. Rett syndrome, the first pervasive develop-mental disorder with a known genetic etiology, is mostfrequently caused by mutations in the gene encodingMeCP2, a methylated DNA-binding protein that regulateschromatin structure and gene expression [14] (Box 2). Thecognitive-regression characteristics of Rett patientssuggest that MeCP2 should be essential in synapsemaintenance and remodeling, more so than in synapseformation. Fragile-X mental retardation protein (FMRP)is encoded by the FMR1 gene, the 5 0-untranslated region(5 0-UTR) of which contains a polymorphic CGG repeatthat can undergo triplet-repeat expansion, resulting inpromoter hypermethylation and FMR1 gene silencing[15]. The clinical outcome is fragile X syndrome (FXS), themost common known cause of inherited mental retar-dation. FXS is often accompanied by autistic features,with w2–3% of autistic males having FXS and 20–40% of


Table 1. Genes involved in ASDa,b

Genes Chr Function Evidence Disorder Observation Refs

Chromatin remodeling and gene regulation

MECP2 Xq28 Methyl-binding protein M MR, Rett,

ASD

Girls with autistic features, one male

with ASD

[14]

FMRP Xq28 RNA-binding protein M MR, FXS,

ASD

20–40% of boys with FXS have ASD [15,16,18]

EN2 7q36 Transcription factor L, A ASD [21–23]

HOXA1 7p15 Transcription factor A ASD [25–27]

WNT2 7q31 Transcription factor L, A ASD [24]

Actin cytoskeleton dynamics

TSC1/TSC2 9q34/16p13 Inactivation of GTPase M TCS ASD in 43–86% of TS patients [6]

NF1 17q11 Inactivation of GTPase M NF1 Learning disabilities in 30–45% of NF1

patients

[30]

cAMP-GEF 2q31 Activation of GTPase L, A ASD Rare variants observed in ASD [31]

Synaptic scaffolding proteins

SHANK3 22q13 Dendrite induction CR MR, ASD Binding partner of NLGN [32]

Receptors and transporters

GRIN2A 16p13 NMDA receptor subunit L, A ASD Highly significant association [46]

GRIK2 6q16–21 Kainate receptor subunit L, A ASD Two independent studies [47]

GABAR 15q12 GABA receptor subunit CR ASD Duplication of 15q is the major CR in ASD [45]

SLC6A4 17p11 Serotonin transporter L, A, M ASD Evidence for allelic heterogeneity in ASD [41]

SLC25A13 2q31 Aspartate–glutamate

carrier

L, A ASD Two positive and one negative

association

[48]

OXTR 3p25–26 Oxytocin receptor L, A ASD [49]

AVPR1 12q14 Vasopressin receptor L, A ASD [50]

Second-messenger systems

PRKCB1 16p11.2 Protein kinase L, A ASD [52]

CACNA1C 12p13.3 Ca2C channel M TS, ASD Multiorgan dysfunction [55]

NBEA 13q13 PKA anchor protein L, CR ASD [51]

Cell adhesion molecules

NLGN4 Xp22.3 Synapse formation L, CR, M MR, ASD Typical autism, Asp [61–65]

NLGN3 Xq13.1 Synapse formation L, M MR, ASD Typical autism, Asp [61–65]

NrCAM 7q31 Neuronal migration L, A ASD [70]

Secreted proteins

RELN 7q22 Neuronal migration L, A ASD [77]

LAMB1 7q31 Cell migration L, A ASD [70]aGenes carrying causal mutations are indicated in bold; the remaining genes are strong candidates found within a linkage region and/or associated with ASD, but no mutation

or other disease-causing alteration has yet been reported.bAbbreviations: A, association; ASD, autism spectrum disorder; Asp, Asperger syndrome; Chr, chromosome; CR, chromosomal rearrangement; FXS, fragile X syndrome; L,

linkage; M, mutation; MR, mental retardation; NF1, neurofibromatosis type 1; TCS, tuberous sclerosis; TS, Timothy syndrome.

Review TRENDS in Neurosciences Vol.29 No.7 July 2006 351

FXS patients meeting criteria for an ASD diagnosis [16].Interestingly, recent data point towards variation of theFMR1 gene in its highly conserved 3 0-UTR as contributingto autism vulnerability [17].

The FMRP protein is involved in mRNA transport andtranslation at the synapse [18]. Several FMRP targetshave been identified, such as PSD95, MAP1B and BC200[18,19], but many more remain to be discovered. Interest-ingly, FMRP is modulated by neuronal activity via Rac1, arho GTPase that is crucial for the control of cytoskeletaldynamics [20].

Finally, interesting results have come from other genesencoding transcription factors and body-patterningproteins, including engrailed-2 [21–23], WNT2 [24] andHoxA1 [25–27], and their potential roles in ASD warrantfurther investigation.

(ii) Actin cytoskeleton dynamics

Several genes that encode factors involved in cytoskeletaldynamics, such as GTPase-activating proteins (GAPs) andguanosine exchange factors (GEFs), are mutated inindividuals with mental retardation or ASD [28]. First,tuberous sclerosis, a dominant disorder characterized bybenign tumor-like growths often presenting with mentalretardation, epilepsy and autism, is caused by mutationsin the tumor-suppressor genes TSC1 or TSC2 (Box 2).


In general, ASD is associated with a localization of tubersto the temporal cortex. However, loss of a single TSC1gene copy in mice is sufficient to perturb cytoskeletaldynamics and dendritic spine structure, highlightinggeneralized neurotrophic roles for these genes, in additionto cell growth regulation [29]. Second, neurofibromatosistype 1 is a genetic disorder due to mutations in the NF1gene, affecting the growth properties of neural-crest-derived cells and producing learning and memory deficitsin Drosophila and mice (Box 2). Individuals with ASDhave a greater risk of neurofibromatosis type 1 than thegeneral population [30]. Finally, rare non-synonymousvariants (i.e. encoding proteins differing by a single aminoacid) of cAMP-GEFII, which is located in the chromosome2 region linked to autism, were identified in ASD, but theirfunctional consequences still need to be elucidated [31].

(iii) Scaffolding proteins

At the synapse, appropriate connectivity between cytos-keleton and membrane proteins is mediated by scaffoldingproteins, which are crucial for dendritic morphology. TheSHANK3 gene, located in a 22q13 terminal region that isdeleted in a patient who has ASD and delayed onset ofexpressive speech, encodes a synaptic scaffolding proteinthat is involved in the induction and maintenance ofdendritic spines [32]. SHANK3 is also a binding partner


GTP

GDP

AAAAAA

AAAAAA Me Me Me

Me Me Me

NLGN

NLGNGRIK2NMDA

TSC1NF1 cAMP-GEFII

REELIN

LAMB1

SHANK3SHANK3

FMRP

FMRP

Actin

ELNWNT2HOXA1

MECP2

NLGN2GABAR

NRXNNRXN

5-HTT

AGC1

GTPasecycle

NrCAM

NBEA

CACNA1

PKCβ

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Figure 1. Proteins that are known or suspected to be altered in function or amounts in autism spectrum disorders (ASD). MECP2 binds to methylated DNA (Me). ELN, WNT2

and HOXA1 are transcription factors. The fragile-X mental retardation protein FMRP is an RNA-binding protein involved in transport and translation of specific mRNA at the

synapse. AGC1 is the mitochondrial aspartate–glutamate carrier encoded by the gene SLC25A12. The GTP-activating proteins tuberous sclerosis 1 (TSC1) and

neurofibromatosis type 1 (NF1) and the guanosine exchange factor cAMP-GEFII influence the actin cytoskeleton by controlling the rho GTPase cycle. Neurobeachin (NBEA),

the protein kinase PKCb in its two isoforms I and II, both encoded by the same PRKB1 gene, and the Ca2C channel CACNA1 regulate signal transduction. The scaffolding

protein SHANK3 organizes the architecture of the postsynaptic density by binding cytoskeletal, membrane and other scaffolding proteins. The neuroligin (NLGN), neurexin

(NRXN) and NrCAM cell-adhesion molecules and the extracellular proteins reelin and laminin b1 (LAMB1) are involved in synapse formation and maintenance, cell–cell

recognition and/or cell migration. Finally, the serotonin transporter (5-HTT) and GABA and glutamate receptor subunits (NMDA and GRIK2) participate in neuronal

transmission. GABA receptor subunits and NLGN2 are expressed at GABAergic synapses, whereas the other illustrated synaptic proteins implicated in ASD are found

preferentially at glutamatergic synapses.


for neuroligins (NLGN) [33]; as we will go on to discuss,these cell adhesion molecules are mutated in some cases ofASD. SHANK3 could thus belong to the ‘NLGN autismpathway’, connecting the actin cytoskeleton to the scaffoldof the postsynaptic density at glutamatergic synapses(Figure 1).

(iv) Neurotransmitter receptors and transporters

Converging lines of evidence currently indicate thatvariants of genes encoding neurotransmitter receptorsand transporters might be susceptibility factors ormodulators of the behavioral phenotype, but notsufficient or direct causes of ASD. The most studiedgene in this category is SLC6A4, which encodes theserotonin (5-HT) transporter (5-HTT). The neurotrophicroles of 5-HT during prenatal development [34], and thefamilial trait of elevated 5-HT blood levels that isconsistently found in at least 25% of autistic patients[35], spurred interest in potential dysregulation ofSLC6A4 gene expression in ASD. Enhanced 5-HTblood levels derive from changes in the density of


functional 5-HTT molecules on platelet membranes,with no change either in 5-HTT affinity for 5-HT or infree 5-HT plasma levels [36]. Taken together, thecontradictory results of association studies of SLC6A4in ASD seem most compatible with a very small effectof SLC6A4 gene variants on 5-HT blood levels and onASD affection status [37], perhaps with greater effectson the cortical gray matter overgrowth that charac-terizes many autistic children after 2 years of age[38–40] and on the dimension of stereotyped andrepetitive behaviors [41]. Extensive mutation screeningof this gene recently detected excessive transmission toaffected offspring of rare SLC6A4 functional alleles,some resulting in gain of function [41,42]. Similarfindings in other neuropsychiatric disorders [43] pointtowards dimensional, rather than categorical, roles forSLC6A4 gene variants in stereotypic behaviors.

The second most studied neurotransmitter-relatedgenes with regard to ASD are those of the GABA receptorcluster on chromosome 15q11–13. Chromosomalrearrangements in this region might be the most frequent


Box 2. Animal models of autism

Mice that have genetic diseases associated with autism

spectrum disorders† Fragile X syndrome. Mice lacking FMRP display the main

morphological, behavioral and neuroanatomical characteristics

of fragile X syndrome and several features of autism in humans:

(i) macroorchidism; (ii) hyperactivity; (iii) mild deficits in spatial

learning (which are especially evident in tasks involving sudden

changes), blunted startle reflex responses and enhanced

prepulse inhibition, all of which are reminiscent of the reduced

responsiveness to external stimuli also observed in autism; (iv)

significantly decreased seizure threshold [102]; and (v) long,

thin and immature dendritic spines with abnormal synaptic

connections [103].

† Rett syndrome. Several mouse models exist for Rett syndrome,

either lacking MeCP2 [104,105] or carrying one of the truncating

mutations identified in Rett syndrome patients [106]. After the first

six weeks of postnatal life, heterozygous mutant female mice

develop a progressive neurological phenotype including the main

features of Rett syndrome: tremors, motor impairment, hypoactiv-

ity, increased anxiety-related behaviors, seizures, and stereotypical

forelimb motions. They also show microcephaly and a general

reduction in neuronal cell size.

† Tuberous sclerosis complex. Mice carrying an astrocyte-specific

inactivation of the TSC1 gene display progressive, age-dependent

astrocyte proliferation leading to altered hippocampal cytoarchi-

tectonics, seizures and death [107].

† Neurofibromatosis type 1. Nf1 mutations have been found to affect

visual–spatial learning, attention and motor coordination, leaving

unaltered other forms of learning and memory, such as classical

conditioning [108,109]. Increased numbers of astrocytes in the

cortex, hippocampus and brainstem, enhanced GABA-mediated

inhibition, and specific deficits in long-term potentiation [110] might

all contribute to these behavioral deficits. Interestingly, only 40–60%

of the mutant mice are affected, and learning deficits can be rescued

by pharmacological manipulations that decrease Ras function [110].

Animal models of autism based on animal behaviorSeveral animal models have been proposed exclusively on the basis of

animal behaviors resembling specific features of human ASD [111].

Profound alterations in socio-emotional behavior are present in lesion

models involving the amygdala in monkeys or rats, the medial

cerebellum in neonatal rats, and Borna virus infections in neonatal

rats [112,113]. Furthermore, modulation of the oxytocin–vasopressin

system, which is involved in communication, ritual and social

behaviors, has also produced behaviors resembling autism in rodents

[114]. For example, oxytocin-deficient or oxytocin-receptor-deficient

mice fail to develop social discrimination and memory, whereas other

forms of learning and memory appear unaffected [115]. Interestingly,

levels of oxytocin might be significantly lower than normal in autistic

children [116], and a positive association between oxytocin receptor

gene variants and autism has been reported [49].


cytogenetic abnormality in ASD [44]. Extensive genotyp-ing of 14 GABA receptor subunit genes found significantevidence of gene–gene interactions involving GABRA4and GABRB1 [45], a particularly interesting finding inlight of the high incidence of seizures and electroenceph-alogram (EEG) abnormalities in autistic patients (seelater in this review).

Finally, several studies have pointed at other genes inthis category, such as those encoding the NMDA subunitGRIN2A [46], the kainate receptor GluR6/GRIK2 [47], themitochondrial aspartate–glutamate carrier AGC1 [48] andthe oxytocin and vasopressin receptors OXTR [49] andAVPR1a [50]. The involvement of glutamatergic receptorsin synapse maintenance and plasticity, and the pivotalrole of neurohypophyseal hormone receptors in animalmodels of social interaction (Box 2), together with thestatistical strength of these association findings, raiseconfidence about their potential relevance toASD pathogenesis.

(v) Second-messenger systems

A de novo translocation identified in one autistic patientwas shown to disrupt the gene encoding neurobeachin(NBEA), an anchoring protein able to recruit proteinkinase A to the tubulovesicular endomembranes locatedin neuronal cell bodies and dendrites [51]. Another studyfound that the PRKCB1 gene, which encodes the twoprotein kinase C (PKC) isoforms bI and bII, is associatedwith autism and speech delay [52]. PKC-bII joinsacetylcholinesterase monomers and receptor for acti-vated C-kinase 1 (RACK1) in a protein complexsurprisingly shown to modulate fear-induced conflictbehavior in rodents [53]. Moreover, PKC-bII controlsthe differentiation of antigen-presenting dendritic cells,whose dysfunction could contribute to the altered


immune responsiveness described in autism [54]. Finally,Ca2C signaling might be involved in autism patho-genesis, as suggested by recent findings for Timothysyndrome, a multisystem disorder accompanied byautistic behaviors [55]. Activity-dependent Ca2C influxhas been shown to control the number of excitatorysynapses in hippocampal neurons and cerebellar granulecells, through transcriptional regulation mediated byMADS-box transcription enhancer factor 2 (MEF2)[56,57]. Perturbed Ca2C signaling could thus translateinto altered synaptogenesis; as we will discuss later, thisis one of the neurodevelopmental paths likely to resultin autism.

(vi) Apoptosis

Reduced programmed cell death has been evoked as aputative explanation for the increased cell numbers andmaintenance of misplaced cells described in neuropatho-logical studies of ASD. Surprisingly, to date no geneticstudy of programmed cell death genes has been reported.It will be interesting to see whether functional variants ofthe BCL2 gene explain the reduced amounts of BCL2protein found in cerebellar and cerebral cortices of someautistic brains [58].

(vii) Cell adhesion molecules

NLGNs are cell adhesion molecules localized post-synaptically at glutamatergic synapses (NLGN1,NLGN3 and NLGN4X/Y) or GABAergic synapses(NLGN2) [59,60] (Figure 1). These proteins are encodedby five NLGN genes in humans: NLGN1–NLGN4X,plus NLGN4Y, which interestingly diverged from itsX-linked homolog NLGN4X during primate evolutionand is present only in males, owing to its localizationon the Y chromosome. Mutations in the coding


Table 2. Distribution and characteristics of microscopic neuroanatomical alterations in brains of autistic patients and reeler micea

Brain regions Autistic patients Refs Reeler mice Refs

Cerebral cortex Increased cell density [117] Inversion of cortical lamination [121,122]

Smaller cortical minicolumns [118] Neuronal disorganization [121–125]

Ectopic neurons [117] Altered intracortical course of afferent fibers, [124,125]

Neuronal disorganization [117] with quantitatively normal thalamocortical

Areas of increased cortical thickness [117] and callosal connections

Poor lamination in the anterior

cingulate cortex

[119]

Cerebellar cortex Decreased Purkinje cell number [117,119] Decreased Purkinje cell number [126]

Modest decrease in granule cell

counts

[119] Purkinje cells are disorganized

Ectopic subcortical Purkinje cells

Climbing fibers innervate more than one

Purkinje cell

Deep cerebellar nuclei Increased cell size before age 12 and

decreased cell counts after age 22

[119] Decreased cell counts and dysplasia in lateral

nucleus (dentate nucleus in humans)

[126]

Dysplasia in the dentate nucleus [117] Subcortical ectopic gray matter

Subcortical ectopic gray matter [117]

Inferior olivary

nucleus

Increased cell size before age 12 and

decreased cell size after age 22

[119] Olivary dysplasia [126,127]

Olivary dysplasia [117,119]

Entorhinal cortex Increased cell density and reduced

neuronal size

[119] Cytoarchitectonic disturbances [128]

Facial nucleus Cell density decreased by 95% [120] Heterotopic neurons [127,129,130]

Less distinct boundaries

Hippocampus (CA4

and subiculum)

Increased cell density and reduced

neuronal size

[119] Altered fiber input from entorhinal cortex

Cytoarchitectonic disturbances

[131]

Decreased dendritic branching [132,133]

Amygdala (central,

medial, cortical nuclei)

Increased cell density and reduced

neuronal size

[119] Cytoarchitectonic disturbances [128]

aFor reviews, see Refs [9,10,121].


sequences of X-linked NLGN3 and NLGN4 wereidentified in individuals with autistic disorder, Aspergersyndrome and mental retardation [61–63]. Althoughmutated NLGN genes explain !1% of ASD diagnoses[61–65], they have provided crucial information on thesynaptic abnormalities possibly present in ASD. In vitrostudies suggest a major role for NLGNs in synapseformation, because their postsynaptic expressioninduces the formation of fully functional presynapticterminals in contacting axons. Functional studies of theNLGN3 R451C and NLGN4 D936X mutations clearlyindicate defective trafficking and synapse inductionproperties of the mutated proteins [66–68]. Further-more, the association of NLGNs with scaffoldingproteins seemingly regulates the glutamate–GABAbalance [69], which could be altered in neuronalnetworks of the w30% of ASD patients who displayseizures or altered EEG patterns. Finally, the geneencoding NrCAM, which is located on the chromosome7q31 region linked to autism and encodes a celladhesion molecule involved in neuronal migration, isan excellent candidate for susceptibility to ASD, but itsrole in the disorder remains to be proven [70].

(viii) Secreted molecules

Linkage between chromosome 7q and ASD is one of themost replicated genetic findings in ASD research. TheRELN gene, found within the 7q22 region, encodes thereelin protein, which has a pivotal role in neuronalmigration and in the prenatal development of neuralconnections [71]. Reeler mice, which are devoid of reelinowing to spontaneous deletions of the gene, display altered


neural migration yielding cytoarchitectonic alterations innumerous brain regions (Table 2), and a behavioralphenotype characterized by action tremor, dystonicposture and ataxic gait. Absence of reelin in humansproduces lissencephaly with severe mental retardation,resembling neither the reelermouse phenotype nor autism[72]. RELN gene variants that decrease reelin geneexpression might instead confer vulnerability to ASD, assuggested by reduced levels of reelin mRNA and protein inboth brain and serum of autistic patients [73,74], and bythe large overlap of subcortical brain regions displayingcytoarchitectonic alterations in ASD and reeler mice(Table 2). Interestingly, the ‘long’ variants of a poly-morphic GGC repeat found in the 5 0-UTR of the RELNgene, encompassing R12 repeats immediately adjacent tothe AUG initiator codon, were shown both in vitro [75] andin vivo [76] to blunt RELN gene expression by 25–50%.These same ‘long’ variants were also found associated withASD in an initial study [77] and in three independentsamples [78–80], but not in four others [81–84]. Geneticheterogeneity is usually viewed as the most plausiblesource of non-replications in the psychiatric geneticliterature; however, these results could also be interpretedwithin the framework of a region-specific gene–environ-ment interaction model [85]. Reelin acts through variousreceptors, including the very low-density lipoprotein(VLDL) receptor, apolipoprotein E receptor 2 (APOE-R2)and a3b1 integrins, and also exerts a proteolytic activityon extracellular matrix proteins, which is crucial forneuronal migration [86]. This proteolytic activity wasspecifically and potently inhibited by difluorophosphate(DFP) [86], an organophosphate compound toxicologically


Low PON1 activityHigh PON1 activity

= >12 GGC

= 7–10 GGC

= Threshold for correct neuronal migration

= Exposure to organophosphates

Ree

lin p

rote

ase

activ

ity

TRENDS in Neurosciences

Figure 2. A model of gene–environment interactions involving the reelin and

paraoxonase genes (RELN and PON1, respectively), and prenatal exposure to

organophosphates. RELN variants carrying either ‘normal’ (7–10 repeats) or ‘long’

(R12 repeats) GGC alleles genetically determine whether levels of reelin are normal

or reduced, respectively. In principle, both conditions are compatible with normal

neurodevelopment. However, prenatal exposure to organophosphates can

transiently inhibit the proteolytic activity of reelin, which might then fall below

the threshold required for correct neuronal migration, also depending on baseline

levels of RELN gene expression determined genetically and epigenetically. In

addition, exposure to identical doses of organophosphates can affect reelin to a

different extent depending on the amount and affinity spectrum of the organopho-

sphate-inactivating enzyme paraoxonase produced by the PON1 alleles of each

individual.


identical to organophosphates that are routinely used asagricultural pesticides and household insecticides [87].Consequently, a subgroup of vulnerable individualscarrying genetic or epigenetic variants expressing reducedamounts of reelin, if exposed prenatally to organopho-sphates during critical periods in neurodevelopment,would be more likely to suffer from altered neuronalmigration resulting in ASD (Figure 2). Because householduse of organophosphates is significantly more widespreadand intensive in North America than in Europe [87,88],this pathogenetic model predicts the lack of geneticassociation found in Italy [77], France [81], England [82]and Germany [82], and fits with four out of six positive-association results from North American patient samples[77–80]. The association in Caucasian-American, but notin Italian, families between autism and functionalvariants of PON1, which encodes paraoxonase, theenzyme responsible for organophosphate inactivation,provides further indirect evidence in favor of this model[85] (Figure 2). Nonetheless, more research will berequired to substantiate fully the existence of gene–environment interactions involving RELN andrelated genes.

Three paths to ASD: reduced cell migration, excitatory–

inhibitory imbalance and abnormal synaptogenesis

Through the evidence already summarized in this review,we can begin depicting three major pathways involved inASD pathogenesis. The first affects cell migration, thesecond impinges on the glutamate–GABA equilibrium,and the third encompasses synapse formation andmaintenance, as well as dendritic morphology. On onehand, the evidence surrounding the reelin pathway, in


conjunction with neuropathological studies, underscoresthe role of altered neuronal migration in generating theaberrant neural networks that underlie altered infor-mation processing in autism. Second, converging evidencefrom functional studies of MeCP2 and NLGN, and fromchromosomal rearrangements involving the GABAreceptor gene cluster, underscores the crucial role ofunbalanced excitatory–inhibitory networks in abnormalCNS excitability and function in autism [12,89]. Finally,several independent lines of evidence suggest thatabnormal synapse formation and dendritic spines couldcontribute to ASD. Increased numbers of dendritic spines,which also appear longer and thinner than normal, wereinitially described in patients with FXS and weresubsequently confirmed in mouse models of FXS (Box 2).Similarly increased numbers of dendritic spines werefound in brains of autistic patients with severe mentalretardation [10]. In addition, the effect on dendriticmorphology of the mutations ofMECP2 in Rett syndrome,of TSC1 or TSC2 in tuberous sclerosis and of NLGN inautism all indicate that the appropriate development ofsynaptic contacts at axodendritic regions is crucial for thecorrect processing of socially-relevant information.

Several major questions are still unanswered. Doesgenetic susceptibility mainly affect synapse formation,maintenance or pruning? Does it affect the glutamate–GABA equilibrium mainly by boosting glutamate-mediated neurotransmission or by reducing numbers ofGABAergic synapses? The involvement of these neurobio-logical pathways is not dramatically different in mentalretardation, epilepsy and ASD: where does the specificitylie? Some degree of overlap is expected, because w70% ofASD patients also present with mental retardation, and30% have seizures. However, only a subset of patientswith FXS, tuberous sclerosis, neurofibromatosis type 1or epilepsy also presents with ASD. Frontal and temporalcortical networks encompassing mirror neuronsseemingly represent the crucial neurobiological under-pinning of social cognition [89], but is a stochasticdistribution of neuropathological lesions solely respon-sible for their impact on social behaviors, or are modifiergenetic, epigenetic and environmental factors also atwork?

Studies of genetic, epigenetic and environmentalfactors are finally beginning to provide some clues tosolving the complexities of ASD pathogenesis. Furtherprogress is likely to come frommerging of results obtainedusing different methodologies, including functionalstudies in animal models of genetic variants responsiblefor ASD in humans.

AcknowledgementsA.M.P. is supported by Telethon-Italy (grant GGP02019), the FondationJerome Lejeune (Paris, France), the Cure Autism Now Foundation (LosAngeles, CA USA) and the National Alliance for Autism Research(Princeton, NJ, USA). T.B. is supported by the Cure Autism NowFoundation (Los Angeles, CA, USA), Fondation France Telecom,Fondation biomedicale de la mairie de Paris, AUMOLGEN FP6 andEUSynapse FP6. We gratefully acknowledge Pat Levitt and AndreGoffinet for helpful comments, and all the patients and their familiesparticipating in these studies who have generously contributed to theadvancement of the autism field.



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Searching for ways out of the autism maze: genetic, epigenetic and environmental clues