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

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    Our understanding of human disorders that affect higher

    Review TRENDS in Neurosciences Vol.29 No.7 July 2006ASD incidence from 25 to 1560 per 10 000 childrenIntroductionAutistic 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 (Kanners autism),childhood disintegrative disorder, pervasive developmentdisorder not otherwise specified (PDD-NOS, or atypicalautism) and Asperger syndrome. The dramatic rise in

    individuals 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 neurodevelopment is widely recognized as theunderlying neuropathological cause of ASD. The CNS ofcognitive functions has greatly advanced in recentdecades, and over 20 genes associated with non-syndromic mental retardation have been identifiedduring the past 15 years. However, proteins encodedby cognition genes have such diverse neurodevelop-mental functions that delineating specific pathogeneticpathways still poses a tremendous challenge. In thisreview, we summarize genetic, epigenetic and environ-mental contributions to neurodevelopmental alterationsthat either cause or confer vulnerability to autism, adisease primarily affecting social cognition. Takentogether, these results begin to provide a unifyingview of complex pathogenetic pathways that are likelyto lead to autism spectrum disorders through alteredneurite morphology, synaptogenesis and cell migration.This review is part of the INMED/TINS special issueNature and nurture in brain development and neuro-logical disorders, based on presentations at the annualINMED/TINS symposium ( special issue

    Searching for waysmaze: genetic, epigenvironmental clueAntonio M. Persico1,2 and Thomas Bourge1Laboratory of Molecular Psychiatry and Neurogenetics, Univer2IRCCS Fondazione Santa Lucia, Department of Experimental N3Laboratory of Human Genetics and Cognitive Functions, Institu4University Paris VII, 2 Place Jussieu 75013, Paris, Franceut of the autismnetic and


    Campus Bio-Medico, Via Longoni 83, I-00155, Rome, Italyrosciences, Via del Fosso di Fiorano 64/65, I-00143, Rome, Italyasteur, 25 Rue du Docteur Roux 75015, Paris, France

    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 autismdiagnosis [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 cell migration with disrupted cortical and sub-cortical cytoarchitectonics, abnormal cell differentiation

    Corresponding author: Persico, A.M. ( 0166-2236/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tins.2006.05.010

  • ASD to date. All of these proteins are involved in


    epidemiological and genetic studies are needed to understand better

    Review TRENDS in Neurosciences Vol.29 No.7 July 2006350with 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 8292% in monozygotic twinscompared with 110% in dizygotic twins, sibling recur-rence risk at 23%, and heritability estimates of O90%

    Box 1. Environmental and epigenetic factors in autism spectru

    An etiological role for environmental factors in ASD has beenconclusively demonstrated only for: (i) prenatal or perinatalexposure 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 fromorganophosphates (see main text), vaccinations have recentlydrawn attention owing to their high content of thimerosal, apreservative containing mercury, and to the proposed causal linkbetween ASD and the measlesmumprubella 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 pointedtowards epigenetic factors as contributing to ASD, in conjunction withgenetic variants. First, females who carry heterozygous mutations ofMeCP2 develop Rett syndrome, a pervasive developmental disordercharacterized by autism, loss of speech, hand wringing and seizures[14]. MeCP2 brings about transcriptional silencing by binding tomethylated promoters, and recruiting co-repressor and histonedeacetylase complexes. A decrease in MeCP2 activity producestranscriptional de-repression localized at specific promoters crucialto brain development and plasticity, including those regulating levelsof brain-derived neurotrophic factor (BDNF), the transcription factordistal-less homeobox 5 (DLX5), ubiquitin-protein ligase E3A (UBE3A)[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 genegene and geneenvironment 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-syndromicautism (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

    www.sciencedirect.comneurodevelopment 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 cytoskeletonsame 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 genegene and geneenvironment interactions.

    Neurodevelopmental genes and autismFigure 1 depicts proteins that have been implicated in

    the contributions of genetic, epigenetic and environmental factors inthese highly heterogeneous disorders.disorders

    and the GABA receptor subunit GABRB3 [9597]. MeCP2 mutationshave 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 7qmight contribute to ASD: the locus closer to the centromere mightexpress the paternal allele, whereas the locus closer to the telomeremight express only the maternal allele [99]. The imprinted region ofthe PraderWilli and Angelman syndromes on chromosome 15q isalso associated with ASD and includes the UBE3A gene, whoseabnormal methylation pattern has been described in at least oneautistic brain [100]. Finally, females with Turner syndrome, who havemonosomy of the X chromosome (X0), display an increasedsusceptibility to ASD compared with normal XX females [101],compatible with the existence of imprinted genes on theX chromosome.

    In summary, there is compelling evidence that environmental andepigenetic factors can have a role in ASD susceptibility. Noveltechnological tools are warranted to address the difficult question ofepigenetics in humans. Additionally, more results from combineddynamics, (iii) synaptic scaffolding, (iv) neurotransmission,(v) second-messenger systems, (vi) apoptosis, (vii) celladhesion, or (viii) paracrine cellcell communication.

    (i) Chromatin remodeling and regulation of transcriptionTwo 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,withw23% of autistic males having FXS and 2040% of

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

    Review TRENDS in Neurosciences Vol.29 No.7 July 2006 351Actin cytoskeleton dynamicsTSC1/TSC2 9q34/16p13 Inactivation of GTPase MNF1 17q11 Inactivation of GTPase M

    cAMP-GEF 2q31 Activation of GTPase L, ASynaptic scaffolding proteinsSHANK3 22q13 Dendrite induction CRReceptors and transportersGRIN2A 16p13 NMDA receptor subunit L, AGRIK2 6q1621 Kainate receptor subunit L, AGABAR 15q12 GABA receptor subunit CRSLC6A4 17p11 Serotonin transporter L, A, MSLC25A13 2q31 Aspartateglutamate

    carrierL, A

    OXTR 3p2526 Oxytocin receptor L, AAVPR1 12q14 Vasopressin receptor L, ASecond-messenger systemsPRKCB1 16p11.2 Protein kinase L, ACACNA1C 12p13.3 Ca2C channel MNBEA 13q13 PKA anchor protein L, CRCell adhesion moleculesTable 1. Genes involved in ASDa,b

    Genes Chr Function Evidence

    Chromatin remodeling and gene regulationMECP2 Xq28 Methyl-binding protein M

    FMRP Xq28 RNA-binding protein M

    EN2 7q36 Transcription factor L, AHOXA1 7p15 Transcription factor AFXS 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 [2123], WNT2 [24] andHoxA1 [2527], and their potential roles in ASD warrantfurther investigation.

    (ii) Actin cytoskeleton dynamicsSeveral 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).

    NLGN4 Xp22.3 Synapse formation L, CR, MNLGN3 Xq13.1 Synapse formation L, MNrCAM 7q31 Neuronal migration L, ASecreted proteinsRELN 7q22 Neuronal migration L, ALAMB1 7q31 Cell migration L, A

    aGenes carrying causal mutations are indicated in bold; the remaining genes are strong ca

    or other disease-causing alteration has yet been reported.bAbbreviations: A, association; ASD, autism spectrum disorder; Asp, Asperger syndrom

    linkage; M, mutation; MR, mental retardation; NF1, neurofibromatosis type 1; TCS, tube

    www.sciencedirect.comTCS ASD in 4386% of TS patients [6]NF1 Learning disabilities in 3045% of NF1


    ASD Rare variants observed in ASD [31]

    MR, ASD Binding partner of NLGN [32]

    ASD Highly significant association [46]ASD Two independent studies [47]ASD Duplication of 15q is the major CR in ASD [45]ASD Evidence for allelic heterogeneity in ASD [41]ASD Two positive and one negative


    ASD [49]ASD [50]

    ASD [52]TS, ASD Multiorgan dysfunction [55]ASD [51]Disorder Observation Refs

    MR, Rett,ASD

    Girls with autistic features, one malewith ASD



    2040% of boys with FXS have ASD [15,16,18]

    ASD [2123]ASD [2527]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 proteinsAt 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

    MR, ASD Typical autism, Asp [6165]MR, ASD Typical autism, Asp [6165]ASD [70]

    ASD [77]ASD [70]

    ndidates found within a linkage region and/or associated with ASD, but no mutation

    e; Chr, chromosome; CR, chromosomal rearrangement; FXS, fragile X syndrome; L,

    rous sclerosis; TS, Timothy syndrome.


    Review TRENDS in Neurosciences Vol.29 No.7 July 2006352GTP











    CACNA1for 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 transportersConverging 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






    Figure 1. Proteins that are known or suspected to be altered in function or amounts in au

    and HOXA1 are transcription factors. The fragile-X mental retardation protein FMRP is an

    synapse. AGC1 is the mitochondrial aspartateglutamate carrier encoded by the

    neurofibromatosis type 1 (NF1) and the guanosine exchange factor cAMP-GEFII influenc

    the protein kinase PKCb in its two isoforms I and II, both encoded by the same PRKB1

    protein SHANK3 organizes the architecture of the postsynaptic density by binding cytos

    (NRXN) and NrCAM cell-adhesion molecules and the extracellular proteins reelin and

    recognition and/or cell migration. Finally, the serotonin transporter (5-HTT) and GAB

    transmission. GABA receptor subunits and NLGN2 are expressed at GABAergic synap

    preferentially at glutamatergic synapses.



    NLGN2functional 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[3840] 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 15q1113. Chromosomalrearrangements in this region might be the most frequent

    Me Me Me

    Me Me Me



    TRENDS in Neurosciences

    tism spectrum disorders (ASD). MECP2 binds to methylated DNA (Me). ELN, WNT2

    RNA-binding protein involved in transport and translation of specific mRNA at the

    gene SLC25A12. The GTP-activating proteins tuberous sclerosis 1 (TSC1) and

    e the actin cytoskeleton by controlling the rho GTPase cycle. Neurobeachin (NBEA),

    gene, and the Ca2C channel CACNA1 regulate signal transduction. The scaffolding

    keletal, membrane and other scaffolding proteins. The neuroligin (NLGN), neurexin

    laminin b1 (LAMB1) are involved in synapse formation and maintenance, cellcell

    A and glutamate receptor subunits (NMDA and GRIK2) participate in neuronal

    ses, whereas the other illustrated synaptic proteins implicated in ASD are found

  • Review TRENDS in Neurosciences Vol.29 No.7 July 2006 353cytogenetic abnormality in ASD [44]. Extensive genotyp-ing of 14 GABA receptor subunit genes found significantevidence of genegene 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 in

    Box 2. Animal models of autism

    Mice that have genetic diseases associated with autismspectrum disorders Fragile X syndrome. Mice lacking FMRP display the main

    morphological, behavioral and neuroanatomical characteristicsof fragile X syndrome and several features of autism in humans:(i) macroorchidism; (ii) hyperactivity; (iii) mild deficits in spatiallearning (which are especially evident in tasks involving suddenchanges), blunted startle reflex responses and enhancedprepulse inhibition, all of which are reminiscent of the reducedresponsiveness to external stimuli also observed in autism; (iv)significantly decreased seizure threshold [102]; and (v) long,thin and immature dendritic spines with abnormal synapticconnections [103].

    Rett syndrome. Several mouse models exist for Rett syndrome,either lacking MeCP2 [104,105] or carrying one of the truncatingmutations identified in Rett syndrome patients [106]. After the firstsix weeks of postnatal life, heterozygous mutant female micedevelop a progressive neurological phenotype including the mainfeatures of Rett syndrome: tremors, motor impairment, hypoactiv-ity, increased anxiety-related behaviors, seizures, and stereotypicalforelimb motions. They also show microcephaly and a generalreduction in neuronal cell size.

    Tuberous sclerosis complex. Mice carrying an astrocyte-specificinactivation of the TSC1 gene display progressive, age-dependentastrocyte proliferation leading to altered hippocampal cytoarchi-tectonics, seizures and death [107].this category, such as those encoding the NMDA subunitGRIN2A [46], the kainate receptor GluR6/GRIK2 [47], themitochondrial aspartateglutamate 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 systemsA 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

    www.sciencedirect.comimmune 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 by

    Neurofibromatosis type 1. Nf1 mutations have been found to affectvisualspatial learning, attention and motor coordination, leavingunaltered other forms of learning and memory, such as classicalconditioning [108,109]. Increased numbers of astrocytes in thecortex, hippocampus and brainstem, enhanced GABA-mediatedinhibition, and specific deficits in long-term potentiation [110] mightall contribute to these behavioral deficits. Interestingly, only 4060%of the mutant mice are affected, and learning deficits can be rescuedby pharmacological manipulations that decrease Ras function [110].

    Animal models of autism based on animal behaviorSeveral animal models have been proposed exclusively on the basis ofanimal behaviors resembling specific features of human ASD [111].Profound alterations in socio-emotional behavior are present in lesionmodels involving the amygdala in monkeys or rats, the medialcerebellum in neonatal rats, and Borna virus infections in neonatalrats [112,113]. Furthermore, modulation of the oxytocinvasopressinsystem, which is involved in communication, ritual and socialbehaviors, has also produced behaviors resembling autism in rodents[114]. For example, oxytocin-deficient or oxytocin-receptor-deficientmice fail to develop social discrimination and memory, whereas otherforms of learning and memory appear unaffected [115]. Interestingly,levels of oxytocin might be significantly lower than normal in autisticchildren [116], and a positive association between oxytocin receptorgene variants and autism has been reported [49].MADS-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) ApoptosisReduced 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 moleculesNLGNs 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: NLGN1NLGN4X,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

  • ato



    Poor lamination in the anteriorcingulate cortex









    Review TRENDS in Neurosciences Vol.29 No.7 July 2006354sequences of X-linked NLGN3 and NLGN4 wereidentified in individuals with autistic disorder, Aspergersyndrome and mental retardation [6163]. Although

    Cerebellar cortex Decreased Purkinje cell number [11Modest decrease in granule cellcounts


    Deep cerebellar nuclei Increased cell size before age 12 anddecreased cell counts after age 22


    Dysplasia in the dentate nucleus [11Subcortical ectopic gray matter [11

    Inferior olivarynucleus

    Increased cell size before age 12 anddecreased cell size after age 22


    Olivary dysplasia [11Entorhinal cortex Increased cell density and reduced

    neuronal size[11

    Facial nucleus Cell density decreased by 95% [12

    Hippocampus (CA4and subiculum)

    Increased cell density and reducedneuronal size


    Decreased dendritic branchingAmygdala (central,medial, cortical nuclei)

    Increased cell density and reducedneuronal size


    aFor reviews, see Refs [9,10,121].Table 2. Distribution and characteristics of microscopic neuroan

    Brain regions Autistic patients Ref

    Cerebral cortex Increased cell density [11Smaller cortical minicolumns [11Ectopic neurons [11Neuronal disorganization [11Areas of increased cortical thickness [11mutated NLGN genes explain !1% of ASD diagnoses[6165], 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 [6668]. Further-more, the association of NLGNs with scaffoldingproteins seemingly regulates the glutamateGABAbalance [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 moleculesLinkage 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

    www.sciencedirect.comneural migration yielding cytoarchitectonic alterations innumerous brain regions (Table 2), and a behavioralphenotype characterized by action tremor, dystonic

    mical alterations in brains of autistic patients and reelermicea

    Reeler mice Refs

    Inversion of cortical lamination [121,122]Neuronal disorganization [121125]Altered intracortical course of afferent fibers, [124,125]with quantitatively normal thalamocorticaland callosal connections

    9] Decreased Purkinje cell number [126]Purkinje cells are disorganizedEctopic subcortical Purkinje cellsClimbing fibers innervate more than onePurkinje cellDecreased cell counts and dysplasia in lateralnucleus (dentate nucleus in humans)


    Subcortical ectopic gray matter

    Olivary dysplasia [126,127]

    9]Cytoarchitectonic disturbances [128]

    Heterotopic neurons [127,129,130]Less distinct boundariesAltered fiber input from entorhinal cortexCytoarchitectonic disturbances


    [132,133]Cytoarchitectonic disturbances [128]posture 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, encompassingR12 repeats immediately adjacent tothe AUG initiator codon, were shown both in vitro [75] andin vivo [76] to blunt RELN gene expression by 2550%.These same long variants were also found associated withASD in an initial study [77] and in three independentsamples [7880], but not in four others [8184]. 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 geneenviron-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

  • unbalanced excitatoryinhibitory networks in abnormalCNS excitability and function in autism [12,89]. Finally, p


    Review TRENDS in Neurosciences Vol.29 No.7 July 2006 355identical to organophosphates that are routinely used asagricultural pesticides and household insecticides [87].Consequently, a subgroup of vulnerable individualscarrying genetic or epigenetic variants expressing reduced

    = >12 GGC

    = 710 GGC

    = Threshold for correct neuronal migration

    = Exposure to organophosphates



    TRENDS in Neurosciences

    Figure 2. A model of geneenvironment interactions involving the reelin and

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

    organophosphates. RELN variants carrying either normal (710 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.Low PON1 activityHigh PON1 activity


    e ac

    tivityamounts 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[7780]. 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 geneenvironment interactions involving RELN andrelated genes.

    Three paths to ASD: reduced cell migration, excitatoryinhibitory imbalance and abnormal synaptogenesisThrough 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 glutamateGABA equilibrium,and the third encompasses synapse formation andmaintenance, as well as dendritic morphology. On onehand, the evidence surrounding the reelin pathway, in

    www.sciencedirect.comseveral 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 glutamateGABA 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, becausew70% 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 Andre`Goffinet for helpful comments, and all the patients and their familiesconjunction 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 ofparticipating in these studies who have generously contributed to theadvancement of the autism field.

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    Free journals for d

    The WHO and six medical journal publishers have launched theworlds developing countries to gain free access to biomedical

    The science publishers, Blackwell, Elsevier, the Harcourt WorldwSpringer-Verlag and John Wiley, were approached by the WHOjournals will be available for free or at significantly reduced pricein developing countries. The second stage involves extending t

    Gro Harlem Brundtland, director-general for the WHO, said thareducing the health information gap between rich and poor couFor more information, visit http://

    www.sciencedirect.com130 Rossel, M. et al. (2005) Reelin signaling is necessary for a specific stepin the migration of hindbrain efferent neurons. Development 132,11751185

    131 Del R`o, J.A. et al. (1997) A role for Cajal-Retzius cells and reelinin the development of hippocampal connections. Nature 385,7074

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    133 Zhao, S. et al. (2004) Reelin is a positional signal for the lamination ofthe dentate granule cells. Development 131, 51175125

    eloping countries

    ccess to Research Initiative, which enables nearly 70 of therature through the Internet.

    e STM group, Wolters Kluwer International Health and Science,d the British Medical Journal in 2001. Initially, more than 1000o universities, medical schools, research and public institutionsinitiative to institutions in other countries.

    is initiative was perhaps the biggest step ever taken

    Searching for ways out of the autism maze: genetic, epigenetic and environmental cluesIntroductionNeurodevelopmental genes and autism(i) Chromatin remodeling and regulation of transcription(ii) Actin cytoskeleton dynamics(iii) Scaffolding proteins(iv) Neurotransmitter receptors and transporters(v) Second-messenger systems(vi) Apoptosis(vii) Cell adhesion molecules(viii) Secreted molecules

    Three paths to ASD: reduced cell migration, excitatory-inhibitory imbalance and abnormal synaptogenesisAcknowledgementsReferences


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