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PTEN signaling in autism spectrum disorders
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PTEN signaling in autism spectrum disorders Jing Zhou1 and Luis F Parada PTEN germline mutations are found in a small subset of children diagnosed with autism spectrum disorder (ASD) and accompanying macrocephaly. In this review, we discuss recent advances that offer insight into the pathogenesis of this subgroup of autism patients. We provide an overview of how disrupting PTEN function influences neuronal cells, and describe efforts to decipher the cellular mechanisms associated with altered social behaviors. We discuss the PTEN downstream signaling pathways that likely mediate these cellular and behavioral effects. In addition, emerging data suggest that PTEN mutation can synergize with mutations in other autism susceptibility genes to contribute to the development of autistic behaviors. These studies extend our knowledge of PTEN and the PTEN signaling pathway, and offer molecular and cellular clues to better understand the etiology of ASDs. Address Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9133, USA Corresponding author: Parada, Luis F ([email protected]) 1
Current address: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA. Current Opinion in Neurobiology 2012, 22:873–879 This review comes from a themed issue on Neurodevelopment and disease Edited by Joseph Gleeson and Franck Polleux
we discuss recent advances in the field that have led to a better understanding of PTEN-associated neurological disorders, particularly autism. Autism spectrum disorders (ASDs) are complex neurodevelopmental disorders characterized by abnormal social interaction, deficits in verbal and nonverbal communication, and restricted/repetitive behaviors and interests. In the past decade, due to better diagnostic tools and increased awareness, the incidence of autism has increased, to a range of 3–6 children per 1000 [4–6]. Thus, research aimed at identifying the mechanisms underlying ASDs is ever more urgent. However, despite tremendous effort made toward this goal, the etiology of ASDs remains murky. So far, hundreds of genes and multiple chromosome regions have been proposed to be associated with ASDs. Interestingly, reported ASD susceptibility genes function in various intracellular signaling pathways that control multiple aspects of cellular functions, from neuronal growth and migration to synaptic formation [7]. Thus, at present, it is difficult to discern a unifying mechanism or properties underlying ASD development that would explain the diverse clinical manifestations. Clinically, autism is often associated with various other symptoms, including seizures, anxiety, mental retardation, and sleep disorders. Additionally, non-neuronal symptoms, such as gastrointestinal and immunological deficiency are also described [8–12]. These further complicate the study of ASD pathology.
For a complete overview see the Issue and the Editorial Available online 2nd June 2012 0959-4388/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.conb.2012.05.004
Introduction PTEN (phosphatase and tensin homolog deleted on chromosome 10) is an important negative regulator of the PI3K/AKT signaling pathway [1] (Figure 1). Its lipid phosphatase function directly counteracts the kinase function of PI3K, thereby inhibiting the activation of AKT. Given the essential roles of PI3K/AKT signaling in controlling cell growth, cell survival and cell proliferation, it is not surprising that PTEN inactivation is found in a broad spectrum of human cancers. Today, PTEN function as a tumor suppressor is well recognized. However, individuals that harbor PTEN inactivating germline mutations are also prone to develop neurological disorders, including macrocephaly, epilepsy, mental retardation/ developmental delay, and autism [2,3]. In this review, www.sciencedirect.com
PTEN germline mutation and autism One hope for gaining a foothold into the molecular pathways and cells that influence ASD is the study of rare monogenic causes such as mutations in the PTEN tumor suppressor gene. The first study clearly linking PTEN mutation to autism is the 2005 clinical report by Butler et al. [13]. In this study, the authors examined the PTEN gene in 18 individuals with autism and macrocephaly, and found that 3 individuals (17%) carried germline mutations. This study thus suggests a causal role of PTEN mutation in a subset of autism patients. Several lines of pre-existing evidence supported this notion. A number of independent studies noted that a range of autistic children (10–20%) develop macrocephaly [14,15]. Also, it was known that germline PTEN mutations could cause PTEN-hamartoma tumor syndrome (PHTS), which is often associated with macrocephaly [2], and sporadic clinical case studies reported PHTS patients with autistic features [16–18]. To directly test whether Pten mutations could influence ASD-like symptoms in mouse models, Kwon et al. genetically engineered mice in which Pten is ablated in a subset of post-mitotic neurons in the cortex Current Opinion in Neurobiology 2012, 22:873–879
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Figure 1
Growth factors RTK PIP2
IRS RAS
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CRMP2, Tau, MAP1B.......
mTOR
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PTEN signaling pathway. PTEN is a negative regulator of the PI3K/AKT pathway. Inactivation of PTEN leads to over-activation of AKT, which affects multiple cellular processes in neurons, including cell growth, cell survival, and cell migration. Among the AKT downstream effectors, GSK3s have been shown to control neuronal polarity through regulation of microtubule-binding proteins, such as CRMP2, Tau and MAP1B. The TSC/mTORC1 downstream signaling pathway is the key regulator of protein synthesis, therefore, controlling cell growth. However, recent data suggest that the TSC1/2 complex also influences neural polarity. Both PTEN and TSC1/2 mutation are associated with autism.
and hippocampus. These mice developed macrocephaly, and displayed behavioral phenotypes reminiscent of human ASDs, including social behavior deficits and seizures, as well as increased anxiety and learning deficits [19]. In the intervening time, many additional clinical cases of PTEN germline mutations in autistic children have been reported [20–26]. According to these studies, PTEN germline mutations are present in about 1–5% of the total autism patient population. Today, screening for PTEN mutations in autism patients with macrocephaly is strongly recommended. However, it should be pointed out that screening for PTEN mutations is currently performed by sequencing genomic DNA isolated from patient blood samples or saliva, and is mostly limited to the coding sequence; only a few studies have examined the promoter and exon-flanking regions [20]. This could result in an underestimation of PTEN mutation frequency, as mutations in non-coding regions might also perturb PTEN protein levels. Additionally, in most of the above studies, the sample pool size was relatively small compared to other contemporary genomic studies on autism. This might explain the variable results reported Current Opinion in Neurobiology 2012, 22:873–879
by different groups when calculating the proportion of autism patients with PTEN germline mutations. In the future, more comprehensive analyses of PTEN gene status in larger numbers of autism patients should give a clearer picture of the prevalence of PTEN mutation in ASDs. Additionally, these studies exclude possible alternative reduction in PTEN levels either by mutational or epigenetic mechanisms somatically within specific brain regions that would not be observed in peripheral blood sampling. Interestingly, most germline PTEN mutations identified in ASD patients to date are point mutations, including nonsense mutations, missense mutations, and single base insertions. Some of these mutations had been identified earlier in patients that developed PHTS, and were known to severely affect PTEN lipid phosphatase function. For example, the R130X mutation, which is frequently found in both autism and PTEN-associated tumors, resides in the phosphatase catalytic core motif of the protein [3,13,21]. However, many PTEN mutations discovered in ASD patients are novel mutations. Although computer analysis suggests some of these mutations could result in changes in certain features of the PTEN protein, such as www.sciencedirect.com
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cell surface accessibility of the protein, thorough molecular studies on these mutant forms are only now emerging [13]. A recent study by Redfern et al. evaluated one particular PTEN mutant form found in ASDs, H93R PTEN [27]. They found that this point mutation alters PTEN-membrane binding and decreases PTEN phosphatase activity in the U87MG glioblastoma cell line. More recently, a comprehensive functional analysis of PTEN mutations was performed by Pulido and colleagues, who used a humanized yeast bioassay to evaluate a large number of PTEN mutations found in both tumors and in autism patients [28]. Interestingly, the data suggested that most ASD-associated PTEN mutations do not substantially abolish the lipid phosphatase function of PTEN and are less severe mutations compared to tumor-related PTEN mutant forms. This is consistent with the absence of correlation of PTEN-associated ASDs and PTEN-associated cancers. Although these surrogate PTEN assays are informative, they should also be extended to neural cells, including neurons and glia. More importantly, the loss/decrease of PTEN function in the brain of autistic individuals needs to be confirmed. However, this will be difficult due to the paucity of available tissue for study.
Cellular functions of PTEN in the brain Understanding PTEN-associated neurological disorders will require genetic and physiologic studies in the brain. PTEN function in the brain has been examined mainly using mouse models. Earlier studies demonstrated that ablating Pten broadly in the brain during developmental stages causes dramatic anatomical disruption and premature death, often accompanied by development of severe seizure activity [29,30]. Recent studies avoided these broad developmental phenotypes by deleting Pten in limited cell populations/restricted brain regions using conditional knockout technology or by knocking down PTEN expression with shRNAs. The data indicate that PTEN has differential cellular effects in different neuronal cell types. For example, PTEN loss in proliferating neural stem/progenitor cells (NSPCs) results in increased proliferation [31], while loss of PTEN in postmitotic neurons induces cellular hypertrophy [19]. Both cellular effects of PTEN loss, however, could contribute to the development of macrocephaly, by either increasing cell number or cell size. In contrast, a recent study demonstrated that in vivo Pten deletion in mature astrocytes induced neither astrocyte hypertrophy nor hyperproliferation [32]. Although PTEN function in mature astrocytes needs to be further evaluated, these data suggest that PTEN cellular functions in the brain are cell-type specific. Additionally, even within the same cell type, PTEN can regulate multiple aspects of cell activity. For example, in vitro and in vivo studies showed that loss of PTEN in neurons leads to neuronal hypertrophy, with soma, denwww.sciencedirect.com
dritic, and axonal overgrowth [19,33]. The dendritic overgrowth was further reflected by increased dendritic arborization, increased thickness of dendritic caliber, and increased number of dendritic spines. However, the phenotype is not just restricted to ‘overgrowth’. PTEN also controls neuronal polarity; disrupting PTEN function can lead to multiple ectopic axons and loss of proper axonal projections [19,34]. Furthermore, Luikart et al. recently developed a viral-based strategy that allows in vivo knockdown of Pten specifically in mouse hippocampal granule cells [35]; they found that granule cells with Pten knockdown have a preferential increase in excitatory synaptic functions. Thus, attenuating PTEN function in neurons has profound effects on neuronal morphology and circuitries. As another example, an earlier study showed that deleting Pten in embryonic neural stem cells leads to increased cell proliferation capacity [31]. Recently, a study of PTEN function in adult subventricular zone stem cells reached the same conclusion: PTEN loss enhances constitutive neurogenesis [36]. However, in another recent study, Bonaguidi et al. exploited a new strategy to label adult NSPCs in the subgranular zone of dentate gyrus, allowing them to examine the role of PTEN in individual NSPCs [37]. They found that deleting Pten promotes NSPC selfrenewal initially, but ultimately leads to preferential astrocytic terminal differentiation over neuronal differentiation. These data demonstrate that PTEN not only regulates NSPC proliferation, but is also involved in controlling NSPC lineage specification. More importantly, PTEN function in NSPCs may change at different developmental stages and vary in different stem cell populations. These studies provide a clearer picture of the cellular functions of PTEN in the brain and offer a cellular and molecular basis for understanding PTEN-associated neurological disorders.
PTEN downstream effectors and neuronal disorders Currently, most cellular effects of PTEN are believed to be mediated through its lipid phosphatase activity. Consistent with this, many PTEN-null cellular phenotypes could be recapitulated by activation of either PI3K or AKT. As such, the AKT downstream effectors thought to mediate these cellular functions and social behaviors have been evaluated. One AKT downstream effector that needs to be mentioned here is glycogen synthase kinase 3 (GSK3). There are two isoforms of GSK3: GSK3a and GSK3b. It has been well established that AKT can directly phosphorylate both GSK3 isoforms at the N-terminal (Ser21 in GSK3a and Ser9 in GSK3b) and lead to subsequent kinase inactivation [38] (Figure 1). It has also been shown that inactivation of GSK3 by AKT at the tip of neurites is Current Opinion in Neurobiology 2012, 22:873–879
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essential for axon formation, and that this is likely mediated by a group of microtubule-binding proteins that are regulated via GSK3 phosphorylation, including Tau, CRMP2 and MAP1B [34]. This provided a mechanistic explanation for how both over-activation of AKT and global inhibition of GSK3s promoted multiple axon formation. However, GSK3a-Ser21A/GSK3b-Ser9A double knock-in mice, in which GSK3s cannot be phosphorylated by AKT, do not show overt neuronal deficits, and double knock-in neurons showed no polarity defects, either in vivo or in vitro, while isolated double knockin neurons developed multiple axons upon treatment with GSK3 inhibitors [39]. This questions the role of AKT as a major regulator of GSK3s to control neuronal polarity, and suggests that alternative inactivation mechanisms for GSK3s may exist. On the other hand, GSK3s are involved in multiple intracellular signaling pathways besides the PI3K/AKT signaling pathway and mediate various cellular processes, including neurogenesis, neuronal migration, and neuronal polarity [40]. Changes in GSK3 activity have been linked to several neurological disorders, including Alzheimer’s disease, schizophrenia, and autism. However, whether inactivation of GSK3 contributes to the cellular and behavioral abnormalities seen in Pten mutants needs to be further evaluated. Another AKT downstream signaling pathway that has been a focus of study in this context is the TSC/mTORC1 pathway [41] (Figure 1). AKT directly phosphorylates and inhibits the function of the TSC1/TSC2 complex, releasing the inhibitory effects of this complex on mTOR complex 1 (mTORC1). mTORC1 is an essential protein complex that regulates cell growth, mainly by controlling protein synthesis through two major downstream effectors: ribosome protein S6 kinase-1 (S6K1) and eukaryotic initiation 4E-binding protein 1 (4E-BP1). In humans, loss of either the TSC1 or TSC2 gene leads to development of tuberous sclerosis complex (TSC), a multi-system genetic disease that causes benign tumors to grow in the brain and other vital organs, such as the kidney, heart, lungs and skin [42]. TSC patients frequently suffer from mental retardation, seizures, and autism, with autistic behaviors observed in 25–50% of individuals with TSC [43]. Previous studies have shown that loss of TSC1/2 function or overactivation of mTORC1 results in neuronal hypertrophy [44,45]. Interestingly, a recent study demonstrated that lack of either TSC1 or TSC2 in neurons also promotes ectopic axonal formation, indicating loss of neuronal polarity; this function of the TSC1/2 complex is, at least in part, mediated by up-regulation of SAD kinase levels which is mTORC1 dependent (Figure 1). Of note, it has been reported that SAD kinase is required for maintaining neuronal polarity, and recently a de novo mutation in the human BRSK2 gene, which encodes SAD-A kinase, was identified in an autism patient [46,47]. Furthermore, consistent with the Pten deletion phenotype, knocking out TSC1/2 broadly in the brain resulted in macrocephaly and Current Opinion in Neurobiology 2012, 22:873–879
seizures [44,48]. In addition, Tsc2 heterozygous mice with elevated mTORC1 activity in the brain demonstrated deficits in learning and memory [49], and TSC2 dominant-negative transgenic mice showed elevated anxiety [50]. Thus, disrupting TSC1/2 complex function in neurons mimics the cellular effects of PTEN loss, and loss of TSC1/2 in the brain produces similar behavioral consequences as Pten ablation. Together, these data indicated that the TSC signaling pathway is a major downstream pathway mediating the cellular and behavioral changes observed in PTEN mutants. This was further supported by the study showing that treating Pten mutant mice with the mTORC1 inhibitor, rapamycin, effectively prevented and reversed the major cellular changes, and ameliorated PTEN-associated abnormal behaviors [51]. These data not only point to a role for the TSC/mTORC1 pathway in autism patients bearing PTEN mutations, but also suggest a possible treatment strategy for this subset of autism patients.
Cross-talk of PTEN with other autism-related pathways Investigation into the genetic etiologies underlying ASDs, particularly recent genome-wide analysis studies, has led to an ever-growing list of potential ASD susceptibility genes. Of note, the cellular functions of these genes are diverse, making it difficult to pinpoint any one mechanism underlying ASD development. One hypothesis is that there are common biological pathways or brain circuits that give rise to ASDs. If so, investigating the common/shared pathways that involve multiple ASD susceptibility genes might offer clues to the cellular and molecular mechanisms involved in autism development. For example, it has been suggested that overgrowth during early brain development could be a key factor in the pathogenesis of autism [52], highlighting the role of pathways that generally control cell growth. Other studies have demonstrated the role of synaptic proteins in affecting social behaviors, which is supported by the large number of ASD-susceptibility molecules known to be involved in synaptic function, including neuroligin and neurexin [53]. However, some currently known autism-related genes, such as PTEN, TSC1/2, FMRP and MeCP2, are involved in diverse signaling pathways and have multiple cellular functions. It is also possible that mutations in multiple autism-related genes/ pathways in one patient function in synergy to contribute to the development of autism phenotypes. This ‘multi-hit’ model is supported by a recent exome sequencing study on sporadic ASD [54]. This study identified both de novo and inherited gene mutations on known autism-related genes in one sporadic ASD case by comparison with the nonsymptomatic parents, implying that co-existence of these two mutations prompts development of autistic features. Thus, the nature of autism susceptibility genes, along with possible combination of ASD gene mutations could explain the phenotypic diversity among autism patients. These findings highlight the complex nature of autism, a www.sciencedirect.com
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neurological disorder with a range of severity that is often intertwined with other neurodevelopmental disorders. Similar questions remain regarding the small subset of autism patients with PTEN germline mutations. Do they develop autistic features solely because of loss of PTEN function? Or do those patients carry additional mutations that also contribute to the social deficits? The answer may vary for each individual patient. Although we know from mouse models that loss of PTEN in neurons is sufficient to cause social deficits, the relation of mouse behavior to human behavior is tenuous at best and cannot therefore exclude the possibility that other autism-related genes are necessary for the development of the complex autistic phenotype seen in patients. In particular, germline PTEN mutations found using peripheral blood samples of those ASD patients are often heterozygous mutations, and as discussed earlier, there is a lack of information regarding how severely these mutations affect PTEN function in the brain. An interesting study by Sur and colleagues indicated that it is likely that Pten mutation synergizes with mutations in other autism-related genes/pathways to affect social behaviors [55]. The authors found that Pten haploinsufficient (Pten+/) mice develop macrocephaly over time, and this phenotype was exacerbated in Pten+/;Slc6a4+/ mice. Slc6a4 encodes the serotonin transporter, which is also considered to be an autism susceptibility gene. Accordingly, impaired social behavior in female Pten+/ mice was also exacerbated in female Pten+/;Slc6a4+/ mice. Thus, haploinsufficiency for Pten and Serotonin transporter can cooperatively influence brain size and social behavior, supporting the multi-hit model in which PTEN signaling cooperates with other signaling pathways to affect social behavior.
Conclusions PTEN mutation is a recently recognized causative factor in a small subset of autism patients. Extensive studies on the PTEN protein and the effect of PTEN deletion/ mutation on cellular functions in the nervous system indicate that PTEN mutation results in varied cellular effects in different neuronal cell types. In mouse models, cellular changes following PTEN loss in the brain correlate with behavioral phenotypes, including development of social deficits. Dysfunction of PTEN signaling could also couple with alterations in other autism-related genes/pathways to affect social behaviors. These studies offer insight into the role of PTEN in a subset of ASD patients and highlight the complexity of the pathogenesis of ASDs.
Conflict of interest None declared.
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We thank Dr. Rene´e McKay for critical advice during preparation of this manuscript. Grant support: Simons Foundation (LFP). LFP is an American Cancer Society Research Professor.
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drive onto dentate granule cells. J Neurosci 2011, 31:4345-4354. In this study, the authors used in vivo stereotactic injections of lentivirus expressing a short hairpin RNA to knock down Pten in adult dentate granule cells, and assessed the morphology and synaptic physiology of Pten knockdown cells. Data reveals that attenuating PTEN function causes neuronal hypertrophy and a preferential increase in excitatory synaptic functions in granule cells. Thus, Pten knockdown results in an imbalance between excitatory and inhibitory synaptic activity. 36. Gregorian C, Nakashima J, Le Belle J, Ohab J, Kim R, Liu A, Smith KB, Groszer M, Garcia AD, Sofroniew MV et al.: Pten deletion in adult neural stem/progenitor cells enhances constitutive neurogenesis. J Neurosci 2009, 29:1874-1886. This study showed that conditional deletion of Pten in a subpopulation of adult subventricular zone neural stem cells leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. 37. Bonaguidi MA, Wheeler MA, Shapiro JS, Stadel RP, Sun GJ, Ming GL, Song H: In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics. Cell 2011, 145:1142-1155. Using a genetic marking strategy, the authors were able to trace individual neural stem/progenitor cells (NSPCs) in the adult mouse dentate gyrus. Their data demonstrated that deleting Pten in these NSPCs promotes self-renewal, but ultimately leads to terminal astrocytic differentiation and NSPC depletion. 38. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA: Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995, 378:785-789. 39. Gartner A, Huang X, Hall A: Neuronal polarity is regulated by glycogen synthase kinase-3 (GSK-3beta) independently of Akt/PKB serine phosphorylation. J Cell Sci 2006, 119(Pt 19):3927-3934. 40. Hur EM, Zhou FQ: GSK3 signalling in neural development. Nat Rev Neurosci 2010, 11:539-551. 41. Manning BD, Cantley LC: AKT/PKB signaling: navigating downstream. Cell 2007, 129:1261-1274. 42. Kwiatkowski DJ, Manning BD: Tuberous sclerosis: a GAP at the crossroads of multiple signaling pathways. Hum Mol Genet 2005, 14:R251-R258 Spec No. 2. 43. Wiznitzer M: Autism and tuberous sclerosis. J Child Neurol 2004, 19:675-679. 44. Meikle L, Talos DM, Onda H, Pollizzi K, Rotenberg A, Sahin M, Jensen FE, Kwiatkowski DJ: A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival. J Neurosci 2007, 27:5546-5558. 45. Tavazoie SF, Alvarez VA, Ridenour DA, Kwiatkowski DJ, Sabatini BL: Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2. Nat Neurosci 2005, 8:1727-1734. 46. Kishi M, Pan YA, Crump JG, Sanes JR: Mammalian SAD kinases are required for neuronal polarization. Science 2005, 307:929932. 47. O’Roak BJ, Vives L, Girirajan S, Karakoc E, Krumm N, Coe BP, Levy R, Ko A, Lee C, Smith JD et al.: Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 2012, 485:246-250. 48. Zeng LH, Rensing NR, Zhang B, Gutmann DH, Gambello MJ, Wong M: Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of tuberous sclerosis complex. Hum Mol Genet 2011, 20:445-454. 49. Ehninger D, Han S, Shilyansky C, Zhou Y, Li W, Kwiatkowski DJ, Ramesh V, Silva AJ: Reversal of learning deficits in a Tsc2+/S mouse model of tuberous sclerosis. Nat Med 2008, 14:843-848. 50. Ehninger D, Silva AJ: Increased levels of anxiety-related behaviors in a Tsc2 dominant negative transgenic mouse model of tuberous sclerosis. Behav Genet 2011, 41:357-363. 51. Zhou J, Blundell J, Ogawa S, Kwon CH, Zhang W, Sinton C, Powell CM, Parada LF: Pharmacological inhibition of mTORC1 www.sciencedirect.com
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suppresses anatomical, cellular, and behavioral abnormalities in neural-specific Pten knock-out mice. J Neurosci 2009, 29:1773-1783. In this study, the authors applied the mTORC1 inhibitor, rapamycin, to Pten conditional knockout mice, and demonstrated that inhibiting mTORC1 activity could effectively suppress development of macrocephaly and PTEN-associated abnormal behaviors. 52. Courchesne E, Pierce K, Schumann CM, Redcay E, Buckwalter JA, Kennedy DP, Morgan J: Mapping early brain development in autism. Neuron 2007, 56:399-413. 53. Penzes P, Cahill ME, Jones KA, VanLeeuwen JE, Woolfrey KM: Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 2011, 14:285-293.
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54. O’Roak BJ, Deriziotis P, Lee C, Vives L, Schwartz JJ, Girirajan S, Karakoc E, Mackenzie AP, Ng SB, Baker C et al.: Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet 2011, 43:585-589. This study provides the first genetic evidence that supports the multi-hit model for autism development. 55. Page DT, Kuti OJ, Prestia C, Sur M: Haploinsufficiency for Pten and Serotonin transporter cooperatively influences brain size and social behavior. Proc Natl Acad Sci U S A 2009, 106:1989-1994. By using a mouse model, the authors demonstrated that two autism susceptability genes, Pten and Serotonin transporter, can cooperatively influence brain size and social behavior, supporting the multiple hit model for the etiology of autism.
Current Opinion in Neurobiology 2012, 22:873–879
PTEN signaling in autism spectrum disorders Jing Zhou1 and Luis F Parada PTEN germline mutations are found in a small subset of children diagnosed with autism spectrum disorder (ASD) and accompanying macrocephaly. In this review, we discuss recent advances that offer insight into the pathogenesis of this subgroup of autism patients. We provide an overview of how disrupting PTEN function influences neuronal cells, and describe efforts to decipher the cellular mechanisms associated with altered social behaviors. We discuss the PTEN downstream signaling pathways that likely mediate these cellular and behavioral effects. In addition, emerging data suggest that PTEN mutation can synergize with mutations in other autism susceptibility genes to contribute to the development of autistic behaviors. These studies extend our knowledge of PTEN and the PTEN signaling pathway, and offer molecular and cellular clues to better understand the etiology of ASDs. Address Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9133, USA Corresponding author: Parada, Luis F ([email protected]) 1
Current address: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA. Current Opinion in Neurobiology 2012, 22:873–879 This review comes from a themed issue on Neurodevelopment and disease Edited by Joseph Gleeson and Franck Polleux
we discuss recent advances in the field that have led to a better understanding of PTEN-associated neurological disorders, particularly autism. Autism spectrum disorders (ASDs) are complex neurodevelopmental disorders characterized by abnormal social interaction, deficits in verbal and nonverbal communication, and restricted/repetitive behaviors and interests. In the past decade, due to better diagnostic tools and increased awareness, the incidence of autism has increased, to a range of 3–6 children per 1000 [4–6]. Thus, research aimed at identifying the mechanisms underlying ASDs is ever more urgent. However, despite tremendous effort made toward this goal, the etiology of ASDs remains murky. So far, hundreds of genes and multiple chromosome regions have been proposed to be associated with ASDs. Interestingly, reported ASD susceptibility genes function in various intracellular signaling pathways that control multiple aspects of cellular functions, from neuronal growth and migration to synaptic formation [7]. Thus, at present, it is difficult to discern a unifying mechanism or properties underlying ASD development that would explain the diverse clinical manifestations. Clinically, autism is often associated with various other symptoms, including seizures, anxiety, mental retardation, and sleep disorders. Additionally, non-neuronal symptoms, such as gastrointestinal and immunological deficiency are also described [8–12]. These further complicate the study of ASD pathology.
For a complete overview see the Issue and the Editorial Available online 2nd June 2012 0959-4388/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.conb.2012.05.004
Introduction PTEN (phosphatase and tensin homolog deleted on chromosome 10) is an important negative regulator of the PI3K/AKT signaling pathway [1] (Figure 1). Its lipid phosphatase function directly counteracts the kinase function of PI3K, thereby inhibiting the activation of AKT. Given the essential roles of PI3K/AKT signaling in controlling cell growth, cell survival and cell proliferation, it is not surprising that PTEN inactivation is found in a broad spectrum of human cancers. Today, PTEN function as a tumor suppressor is well recognized. However, individuals that harbor PTEN inactivating germline mutations are also prone to develop neurological disorders, including macrocephaly, epilepsy, mental retardation/ developmental delay, and autism [2,3]. In this review, www.sciencedirect.com
PTEN germline mutation and autism One hope for gaining a foothold into the molecular pathways and cells that influence ASD is the study of rare monogenic causes such as mutations in the PTEN tumor suppressor gene. The first study clearly linking PTEN mutation to autism is the 2005 clinical report by Butler et al. [13]. In this study, the authors examined the PTEN gene in 18 individuals with autism and macrocephaly, and found that 3 individuals (17%) carried germline mutations. This study thus suggests a causal role of PTEN mutation in a subset of autism patients. Several lines of pre-existing evidence supported this notion. A number of independent studies noted that a range of autistic children (10–20%) develop macrocephaly [14,15]. Also, it was known that germline PTEN mutations could cause PTEN-hamartoma tumor syndrome (PHTS), which is often associated with macrocephaly [2], and sporadic clinical case studies reported PHTS patients with autistic features [16–18]. To directly test whether Pten mutations could influence ASD-like symptoms in mouse models, Kwon et al. genetically engineered mice in which Pten is ablated in a subset of post-mitotic neurons in the cortex Current Opinion in Neurobiology 2012, 22:873–879
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Figure 1
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PTEN signaling pathway. PTEN is a negative regulator of the PI3K/AKT pathway. Inactivation of PTEN leads to over-activation of AKT, which affects multiple cellular processes in neurons, including cell growth, cell survival, and cell migration. Among the AKT downstream effectors, GSK3s have been shown to control neuronal polarity through regulation of microtubule-binding proteins, such as CRMP2, Tau and MAP1B. The TSC/mTORC1 downstream signaling pathway is the key regulator of protein synthesis, therefore, controlling cell growth. However, recent data suggest that the TSC1/2 complex also influences neural polarity. Both PTEN and TSC1/2 mutation are associated with autism.
and hippocampus. These mice developed macrocephaly, and displayed behavioral phenotypes reminiscent of human ASDs, including social behavior deficits and seizures, as well as increased anxiety and learning deficits [19]. In the intervening time, many additional clinical cases of PTEN germline mutations in autistic children have been reported [20–26]. According to these studies, PTEN germline mutations are present in about 1–5% of the total autism patient population. Today, screening for PTEN mutations in autism patients with macrocephaly is strongly recommended. However, it should be pointed out that screening for PTEN mutations is currently performed by sequencing genomic DNA isolated from patient blood samples or saliva, and is mostly limited to the coding sequence; only a few studies have examined the promoter and exon-flanking regions [20]. This could result in an underestimation of PTEN mutation frequency, as mutations in non-coding regions might also perturb PTEN protein levels. Additionally, in most of the above studies, the sample pool size was relatively small compared to other contemporary genomic studies on autism. This might explain the variable results reported Current Opinion in Neurobiology 2012, 22:873–879
by different groups when calculating the proportion of autism patients with PTEN germline mutations. In the future, more comprehensive analyses of PTEN gene status in larger numbers of autism patients should give a clearer picture of the prevalence of PTEN mutation in ASDs. Additionally, these studies exclude possible alternative reduction in PTEN levels either by mutational or epigenetic mechanisms somatically within specific brain regions that would not be observed in peripheral blood sampling. Interestingly, most germline PTEN mutations identified in ASD patients to date are point mutations, including nonsense mutations, missense mutations, and single base insertions. Some of these mutations had been identified earlier in patients that developed PHTS, and were known to severely affect PTEN lipid phosphatase function. For example, the R130X mutation, which is frequently found in both autism and PTEN-associated tumors, resides in the phosphatase catalytic core motif of the protein [3,13,21]. However, many PTEN mutations discovered in ASD patients are novel mutations. Although computer analysis suggests some of these mutations could result in changes in certain features of the PTEN protein, such as www.sciencedirect.com
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cell surface accessibility of the protein, thorough molecular studies on these mutant forms are only now emerging [13]. A recent study by Redfern et al. evaluated one particular PTEN mutant form found in ASDs, H93R PTEN [27]. They found that this point mutation alters PTEN-membrane binding and decreases PTEN phosphatase activity in the U87MG glioblastoma cell line. More recently, a comprehensive functional analysis of PTEN mutations was performed by Pulido and colleagues, who used a humanized yeast bioassay to evaluate a large number of PTEN mutations found in both tumors and in autism patients [28]. Interestingly, the data suggested that most ASD-associated PTEN mutations do not substantially abolish the lipid phosphatase function of PTEN and are less severe mutations compared to tumor-related PTEN mutant forms. This is consistent with the absence of correlation of PTEN-associated ASDs and PTEN-associated cancers. Although these surrogate PTEN assays are informative, they should also be extended to neural cells, including neurons and glia. More importantly, the loss/decrease of PTEN function in the brain of autistic individuals needs to be confirmed. However, this will be difficult due to the paucity of available tissue for study.
Cellular functions of PTEN in the brain Understanding PTEN-associated neurological disorders will require genetic and physiologic studies in the brain. PTEN function in the brain has been examined mainly using mouse models. Earlier studies demonstrated that ablating Pten broadly in the brain during developmental stages causes dramatic anatomical disruption and premature death, often accompanied by development of severe seizure activity [29,30]. Recent studies avoided these broad developmental phenotypes by deleting Pten in limited cell populations/restricted brain regions using conditional knockout technology or by knocking down PTEN expression with shRNAs. The data indicate that PTEN has differential cellular effects in different neuronal cell types. For example, PTEN loss in proliferating neural stem/progenitor cells (NSPCs) results in increased proliferation [31], while loss of PTEN in postmitotic neurons induces cellular hypertrophy [19]. Both cellular effects of PTEN loss, however, could contribute to the development of macrocephaly, by either increasing cell number or cell size. In contrast, a recent study demonstrated that in vivo Pten deletion in mature astrocytes induced neither astrocyte hypertrophy nor hyperproliferation [32]. Although PTEN function in mature astrocytes needs to be further evaluated, these data suggest that PTEN cellular functions in the brain are cell-type specific. Additionally, even within the same cell type, PTEN can regulate multiple aspects of cell activity. For example, in vitro and in vivo studies showed that loss of PTEN in neurons leads to neuronal hypertrophy, with soma, denwww.sciencedirect.com
dritic, and axonal overgrowth [19,33]. The dendritic overgrowth was further reflected by increased dendritic arborization, increased thickness of dendritic caliber, and increased number of dendritic spines. However, the phenotype is not just restricted to ‘overgrowth’. PTEN also controls neuronal polarity; disrupting PTEN function can lead to multiple ectopic axons and loss of proper axonal projections [19,34]. Furthermore, Luikart et al. recently developed a viral-based strategy that allows in vivo knockdown of Pten specifically in mouse hippocampal granule cells [35]; they found that granule cells with Pten knockdown have a preferential increase in excitatory synaptic functions. Thus, attenuating PTEN function in neurons has profound effects on neuronal morphology and circuitries. As another example, an earlier study showed that deleting Pten in embryonic neural stem cells leads to increased cell proliferation capacity [31]. Recently, a study of PTEN function in adult subventricular zone stem cells reached the same conclusion: PTEN loss enhances constitutive neurogenesis [36]. However, in another recent study, Bonaguidi et al. exploited a new strategy to label adult NSPCs in the subgranular zone of dentate gyrus, allowing them to examine the role of PTEN in individual NSPCs [37]. They found that deleting Pten promotes NSPC selfrenewal initially, but ultimately leads to preferential astrocytic terminal differentiation over neuronal differentiation. These data demonstrate that PTEN not only regulates NSPC proliferation, but is also involved in controlling NSPC lineage specification. More importantly, PTEN function in NSPCs may change at different developmental stages and vary in different stem cell populations. These studies provide a clearer picture of the cellular functions of PTEN in the brain and offer a cellular and molecular basis for understanding PTEN-associated neurological disorders.
PTEN downstream effectors and neuronal disorders Currently, most cellular effects of PTEN are believed to be mediated through its lipid phosphatase activity. Consistent with this, many PTEN-null cellular phenotypes could be recapitulated by activation of either PI3K or AKT. As such, the AKT downstream effectors thought to mediate these cellular functions and social behaviors have been evaluated. One AKT downstream effector that needs to be mentioned here is glycogen synthase kinase 3 (GSK3). There are two isoforms of GSK3: GSK3a and GSK3b. It has been well established that AKT can directly phosphorylate both GSK3 isoforms at the N-terminal (Ser21 in GSK3a and Ser9 in GSK3b) and lead to subsequent kinase inactivation [38] (Figure 1). It has also been shown that inactivation of GSK3 by AKT at the tip of neurites is Current Opinion in Neurobiology 2012, 22:873–879
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essential for axon formation, and that this is likely mediated by a group of microtubule-binding proteins that are regulated via GSK3 phosphorylation, including Tau, CRMP2 and MAP1B [34]. This provided a mechanistic explanation for how both over-activation of AKT and global inhibition of GSK3s promoted multiple axon formation. However, GSK3a-Ser21A/GSK3b-Ser9A double knock-in mice, in which GSK3s cannot be phosphorylated by AKT, do not show overt neuronal deficits, and double knock-in neurons showed no polarity defects, either in vivo or in vitro, while isolated double knockin neurons developed multiple axons upon treatment with GSK3 inhibitors [39]. This questions the role of AKT as a major regulator of GSK3s to control neuronal polarity, and suggests that alternative inactivation mechanisms for GSK3s may exist. On the other hand, GSK3s are involved in multiple intracellular signaling pathways besides the PI3K/AKT signaling pathway and mediate various cellular processes, including neurogenesis, neuronal migration, and neuronal polarity [40]. Changes in GSK3 activity have been linked to several neurological disorders, including Alzheimer’s disease, schizophrenia, and autism. However, whether inactivation of GSK3 contributes to the cellular and behavioral abnormalities seen in Pten mutants needs to be further evaluated. Another AKT downstream signaling pathway that has been a focus of study in this context is the TSC/mTORC1 pathway [41] (Figure 1). AKT directly phosphorylates and inhibits the function of the TSC1/TSC2 complex, releasing the inhibitory effects of this complex on mTOR complex 1 (mTORC1). mTORC1 is an essential protein complex that regulates cell growth, mainly by controlling protein synthesis through two major downstream effectors: ribosome protein S6 kinase-1 (S6K1) and eukaryotic initiation 4E-binding protein 1 (4E-BP1). In humans, loss of either the TSC1 or TSC2 gene leads to development of tuberous sclerosis complex (TSC), a multi-system genetic disease that causes benign tumors to grow in the brain and other vital organs, such as the kidney, heart, lungs and skin [42]. TSC patients frequently suffer from mental retardation, seizures, and autism, with autistic behaviors observed in 25–50% of individuals with TSC [43]. Previous studies have shown that loss of TSC1/2 function or overactivation of mTORC1 results in neuronal hypertrophy [44,45]. Interestingly, a recent study demonstrated that lack of either TSC1 or TSC2 in neurons also promotes ectopic axonal formation, indicating loss of neuronal polarity; this function of the TSC1/2 complex is, at least in part, mediated by up-regulation of SAD kinase levels which is mTORC1 dependent (Figure 1). Of note, it has been reported that SAD kinase is required for maintaining neuronal polarity, and recently a de novo mutation in the human BRSK2 gene, which encodes SAD-A kinase, was identified in an autism patient [46,47]. Furthermore, consistent with the Pten deletion phenotype, knocking out TSC1/2 broadly in the brain resulted in macrocephaly and Current Opinion in Neurobiology 2012, 22:873–879
seizures [44,48]. In addition, Tsc2 heterozygous mice with elevated mTORC1 activity in the brain demonstrated deficits in learning and memory [49], and TSC2 dominant-negative transgenic mice showed elevated anxiety [50]. Thus, disrupting TSC1/2 complex function in neurons mimics the cellular effects of PTEN loss, and loss of TSC1/2 in the brain produces similar behavioral consequences as Pten ablation. Together, these data indicated that the TSC signaling pathway is a major downstream pathway mediating the cellular and behavioral changes observed in PTEN mutants. This was further supported by the study showing that treating Pten mutant mice with the mTORC1 inhibitor, rapamycin, effectively prevented and reversed the major cellular changes, and ameliorated PTEN-associated abnormal behaviors [51]. These data not only point to a role for the TSC/mTORC1 pathway in autism patients bearing PTEN mutations, but also suggest a possible treatment strategy for this subset of autism patients.
Cross-talk of PTEN with other autism-related pathways Investigation into the genetic etiologies underlying ASDs, particularly recent genome-wide analysis studies, has led to an ever-growing list of potential ASD susceptibility genes. Of note, the cellular functions of these genes are diverse, making it difficult to pinpoint any one mechanism underlying ASD development. One hypothesis is that there are common biological pathways or brain circuits that give rise to ASDs. If so, investigating the common/shared pathways that involve multiple ASD susceptibility genes might offer clues to the cellular and molecular mechanisms involved in autism development. For example, it has been suggested that overgrowth during early brain development could be a key factor in the pathogenesis of autism [52], highlighting the role of pathways that generally control cell growth. Other studies have demonstrated the role of synaptic proteins in affecting social behaviors, which is supported by the large number of ASD-susceptibility molecules known to be involved in synaptic function, including neuroligin and neurexin [53]. However, some currently known autism-related genes, such as PTEN, TSC1/2, FMRP and MeCP2, are involved in diverse signaling pathways and have multiple cellular functions. It is also possible that mutations in multiple autism-related genes/ pathways in one patient function in synergy to contribute to the development of autism phenotypes. This ‘multi-hit’ model is supported by a recent exome sequencing study on sporadic ASD [54]. This study identified both de novo and inherited gene mutations on known autism-related genes in one sporadic ASD case by comparison with the nonsymptomatic parents, implying that co-existence of these two mutations prompts development of autistic features. Thus, the nature of autism susceptibility genes, along with possible combination of ASD gene mutations could explain the phenotypic diversity among autism patients. These findings highlight the complex nature of autism, a www.sciencedirect.com
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neurological disorder with a range of severity that is often intertwined with other neurodevelopmental disorders. Similar questions remain regarding the small subset of autism patients with PTEN germline mutations. Do they develop autistic features solely because of loss of PTEN function? Or do those patients carry additional mutations that also contribute to the social deficits? The answer may vary for each individual patient. Although we know from mouse models that loss of PTEN in neurons is sufficient to cause social deficits, the relation of mouse behavior to human behavior is tenuous at best and cannot therefore exclude the possibility that other autism-related genes are necessary for the development of the complex autistic phenotype seen in patients. In particular, germline PTEN mutations found using peripheral blood samples of those ASD patients are often heterozygous mutations, and as discussed earlier, there is a lack of information regarding how severely these mutations affect PTEN function in the brain. An interesting study by Sur and colleagues indicated that it is likely that Pten mutation synergizes with mutations in other autism-related genes/pathways to affect social behaviors [55]. The authors found that Pten haploinsufficient (Pten+/) mice develop macrocephaly over time, and this phenotype was exacerbated in Pten+/;Slc6a4+/ mice. Slc6a4 encodes the serotonin transporter, which is also considered to be an autism susceptibility gene. Accordingly, impaired social behavior in female Pten+/ mice was also exacerbated in female Pten+/;Slc6a4+/ mice. Thus, haploinsufficiency for Pten and Serotonin transporter can cooperatively influence brain size and social behavior, supporting the multi-hit model in which PTEN signaling cooperates with other signaling pathways to affect social behavior.
Conclusions PTEN mutation is a recently recognized causative factor in a small subset of autism patients. Extensive studies on the PTEN protein and the effect of PTEN deletion/ mutation on cellular functions in the nervous system indicate that PTEN mutation results in varied cellular effects in different neuronal cell types. In mouse models, cellular changes following PTEN loss in the brain correlate with behavioral phenotypes, including development of social deficits. Dysfunction of PTEN signaling could also couple with alterations in other autism-related genes/pathways to affect social behaviors. These studies offer insight into the role of PTEN in a subset of ASD patients and highlight the complexity of the pathogenesis of ASDs.
Conflict of interest None declared.
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Acknowledgements
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We thank Dr. Rene´e McKay for critical advice during preparation of this manuscript. Grant support: Simons Foundation (LFP). LFP is an American Cancer Society Research Professor.
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drive onto dentate granule cells. J Neurosci 2011, 31:4345-4354. In this study, the authors used in vivo stereotactic injections of lentivirus expressing a short hairpin RNA to knock down Pten in adult dentate granule cells, and assessed the morphology and synaptic physiology of Pten knockdown cells. Data reveals that attenuating PTEN function causes neuronal hypertrophy and a preferential increase in excitatory synaptic functions in granule cells. Thus, Pten knockdown results in an imbalance between excitatory and inhibitory synaptic activity. 36. Gregorian C, Nakashima J, Le Belle J, Ohab J, Kim R, Liu A, Smith KB, Groszer M, Garcia AD, Sofroniew MV et al.: Pten deletion in adult neural stem/progenitor cells enhances constitutive neurogenesis. J Neurosci 2009, 29:1874-1886. This study showed that conditional deletion of Pten in a subpopulation of adult subventricular zone neural stem cells leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. 37. Bonaguidi MA, Wheeler MA, Shapiro JS, Stadel RP, Sun GJ, Ming GL, Song H: In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics. Cell 2011, 145:1142-1155. Using a genetic marking strategy, the authors were able to trace individual neural stem/progenitor cells (NSPCs) in the adult mouse dentate gyrus. Their data demonstrated that deleting Pten in these NSPCs promotes self-renewal, but ultimately leads to terminal astrocytic differentiation and NSPC depletion. 38. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA: Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995, 378:785-789. 39. Gartner A, Huang X, Hall A: Neuronal polarity is regulated by glycogen synthase kinase-3 (GSK-3beta) independently of Akt/PKB serine phosphorylation. J Cell Sci 2006, 119(Pt 19):3927-3934. 40. Hur EM, Zhou FQ: GSK3 signalling in neural development. Nat Rev Neurosci 2010, 11:539-551. 41. Manning BD, Cantley LC: AKT/PKB signaling: navigating downstream. Cell 2007, 129:1261-1274. 42. Kwiatkowski DJ, Manning BD: Tuberous sclerosis: a GAP at the crossroads of multiple signaling pathways. Hum Mol Genet 2005, 14:R251-R258 Spec No. 2. 43. Wiznitzer M: Autism and tuberous sclerosis. J Child Neurol 2004, 19:675-679. 44. Meikle L, Talos DM, Onda H, Pollizzi K, Rotenberg A, Sahin M, Jensen FE, Kwiatkowski DJ: A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival. J Neurosci 2007, 27:5546-5558. 45. Tavazoie SF, Alvarez VA, Ridenour DA, Kwiatkowski DJ, Sabatini BL: Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2. Nat Neurosci 2005, 8:1727-1734. 46. Kishi M, Pan YA, Crump JG, Sanes JR: Mammalian SAD kinases are required for neuronal polarization. Science 2005, 307:929932. 47. O’Roak BJ, Vives L, Girirajan S, Karakoc E, Krumm N, Coe BP, Levy R, Ko A, Lee C, Smith JD et al.: Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 2012, 485:246-250. 48. Zeng LH, Rensing NR, Zhang B, Gutmann DH, Gambello MJ, Wong M: Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of tuberous sclerosis complex. Hum Mol Genet 2011, 20:445-454. 49. Ehninger D, Han S, Shilyansky C, Zhou Y, Li W, Kwiatkowski DJ, Ramesh V, Silva AJ: Reversal of learning deficits in a Tsc2+/S mouse model of tuberous sclerosis. Nat Med 2008, 14:843-848. 50. Ehninger D, Silva AJ: Increased levels of anxiety-related behaviors in a Tsc2 dominant negative transgenic mouse model of tuberous sclerosis. Behav Genet 2011, 41:357-363. 51. Zhou J, Blundell J, Ogawa S, Kwon CH, Zhang W, Sinton C, Powell CM, Parada LF: Pharmacological inhibition of mTORC1 www.sciencedirect.com
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suppresses anatomical, cellular, and behavioral abnormalities in neural-specific Pten knock-out mice. J Neurosci 2009, 29:1773-1783. In this study, the authors applied the mTORC1 inhibitor, rapamycin, to Pten conditional knockout mice, and demonstrated that inhibiting mTORC1 activity could effectively suppress development of macrocephaly and PTEN-associated abnormal behaviors. 52. Courchesne E, Pierce K, Schumann CM, Redcay E, Buckwalter JA, Kennedy DP, Morgan J: Mapping early brain development in autism. Neuron 2007, 56:399-413. 53. Penzes P, Cahill ME, Jones KA, VanLeeuwen JE, Woolfrey KM: Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 2011, 14:285-293.
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54. O’Roak BJ, Deriziotis P, Lee C, Vives L, Schwartz JJ, Girirajan S, Karakoc E, Mackenzie AP, Ng SB, Baker C et al.: Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet 2011, 43:585-589. This study provides the first genetic evidence that supports the multi-hit model for autism development. 55. Page DT, Kuti OJ, Prestia C, Sur M: Haploinsufficiency for Pten and Serotonin transporter cooperatively influences brain size and social behavior. Proc Natl Acad Sci U S A 2009, 106:1989-1994. By using a mouse model, the authors demonstrated that two autism susceptability genes, Pten and Serotonin transporter, can cooperatively influence brain size and social behavior, supporting the multiple hit model for the etiology of autism.
Current Opinion in Neurobiology 2012, 22:873–879