A blueprint for research on Shankopathies: a view from research on autism spectrum disorder.

A Blueprint for Research on Shankopathies: A View From Research on Autism Spectrum Disorder Salvatore Carbonetto Centre for Research in Neuroscience, Department of Neurology, McGill University Health Centre, Montreal, Quebec H3G1A4, Canada Received 7 October 2013; accepted 6 November 2013

ABSTRACT: Autism spectrum disorders (ASD) are associated with mutations in a host of genes including a number that function in synaptic transmission. Phelan McDermid syndrome involves mutations in SHANK3 which encodes a protein that forms a scaffold for glutamate receptors at the synapse. SHANK3 is one of the genes that underpins the synaptic hypothesis for ASD. We discuss this hypothesis with a view to the

“There is from the start an extreme autistic aloneness that, whenever possible, disregards, ignores, shuts out anything that comes to the child from the outside. Direct physical contact. . .is either treated as if it weren’t there or . . .resented painfully as a distressing interference” L. Kanner (1943) Nerv. Child 2:217–250.

AUTISM SPECTRUM DISORDERS: A BRIEF INTRODUCTION In his classic study, Kanner (1943) described children with “autistic disturbances of affective contact.” Autism is now considered a spectrum of neurodevelopmental disorders (autism spectrum disorders, or ASD), whose hallmarks are defined by the following behavioral symptoms: (1) persistent deficits in social communication and social interaction, (2) restricted, Correspondence to: S. Carbonetto ([email protected]). Contract grant sponsors: CIHR and NSERC. Ó 2013 Wiley Periodicals, Inc. Published online 12 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/dneu.22150

broader context of ASD and with special emphasis on highly penetrant genetic disorders including Shankopathies. We propose a blueprint for near and longer-term goals for fundamental and translational research on Shankopathies. VC 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 85–112, 2014

Keywords: Shank3; autism; synaptic genes; neurodevelopmental disorders

repetitive patterns of behavior, interests, or activities. These core ASD symptoms may be accompanied by others such as dysmorphic features and intellectual deficiency (e.g., Phelan McDermid syndrome), or the disorder may be more benign, with deficits only in social interaction. ASD occurs with a frequency of about 1% in the general population (Lord, 2011). The hallmarks of ASD typically appear during the first 3 years of life. One of the more disturbing aspects is that affected infants may develop normally, learn to speak, and reach other developmental milestones, only to regress within days to the extent that they are no longer responsive to verbal cues and fail to make eye contact (e.g., Rett Syndrome). Because these children require care for life, the personal and economic burden for families can be enormous. There is a large genetic component to ASD and many genes that contribute more or less strongly to the multiple types of ASD (Betancur, 2011). In this article, we present an overview of ASD, with special emphasis on rare types that involve a single, highly penetrant gene i.e. ASD is found in a large proportion of those carrying the mutant gene. While all forms of ASD have a neurodevelopmental basis, a surprising number of relatively well-characterized forms 85

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involve genes that function in synaptic transmission (Zoghbi, 2003; Auerbach et al., 2011). Much recent research has focused on characterizing these rare forms. Research on a single target gene allows one to study genetically well-characterized patients and to generate invaluable animal and cell models to probe the pathophysiology and explore therapeutics in preclinical studies. It also helps focus research in the face of a flood of information and speculation on ASD including claims in the media that are premature and, in some instances, determined to be unfounded, but only at considerable effort. This article was prompted by a conference organized last year by the Phelan McDermid Syndrome Foundation. The conference brought together basic and clinician scientists as well as families of children with Phelan McDermid Syndrome (PMS). Discussions at the conference were wide-ranging and touched whether diseases caused by mutations in SHANK3 (“Shankopathies”) are synonymous with PMS, fundamental research on animal models of ASD, and how to mobilize industrial interest in research on ASD. In this blueprint, we describe the principle targets for cellular and molecular research that will advance our understanding of ASD, and whose resolution is critical for translational approaches. These include priorities for both collective and individual projects. The primary discussion here centers on Shankopathies, but a good deal of it pertains to other forms of ASD, especially monogenic ones that are highly penetrant.

ENVIRONMENTAL INFLUENCES ON ASD Kanner wrote that children with autism “come into the world with innate inability to form the usual, biologically provided affective contact with people.” He also noted that the parents of the 11 children he studied were themselves “limited in genuine interest in people.” He speculated that parental–infant interactions had some impact on the genesis of autism. As a result, parental neglect remained a prominent theory for autism for some time and was popularized in terms such as the “refrigerator mother.” Yet Kanner also concluded “aloneness from the beginning of life makes it difficult to attribute the whole picture exclusively to the type of early parental relations.” Seventy years later there is clear evidence that most forms of ASD have a genetic basis (Sebat et al., 2007; Abrahams and Geschwind, 2008). There has been much attention to the role of environmental factors during prenatal life and early infancy as causative in ASD. Vaccination has been discussed as an environmental factor that might be Developmental Neurobiology

thought to explain the increased occurrence of ASD. However, research implicating vaccination has been challenged and the publications retracted (DeStefano and Thompson, 2004). Moreover, it now seems that at least part of the increased incidence of ASD is due to increased awareness and broader diagnostic criteria leading to increased reporting (Hertz-Picciotto and Delwiche, 2009). More convincing cases have been made for an involvement of prenatal viral or bacterial infections as well as for inflammation (Pardo et al., 2005), including data from post mortem studies (Vargas et al., 2005), epidemiological studies, and animal models (Patterson, 2002, 2011). Parental age is also a risk factor for ASD. Genome wide sequencing has shown that fathers transmit almost four times more de novo germ line mutations than their mates. The number of these mutations increases with age (Kong et al., 2012), increasing the risk of ASD for offspring of older fathers. Other data have implicated zinc deficiency in ASD (Grabrucker, 2014), and there has been discussion of chemically induced ASD following prenatal exposure to a variety of medications, pesticides, and drugs. Valproic acid is an antiepileptic sometimes used during pregnancy (Roullet et al., 2013) and a recent study indicates that valproic acid increases the incidence of ASD (Christensen et al., 2013). The FDA has warned about the use of valproic acid by pregnant mothers. While environmental influences may directly affect many molecular events, valproic acid has been implicated in pathways involved in epigenetic modification of gene expression. Epigenetics entails changes in gene expression resulting not from alteration in nucleotide sequence, but from covalent modification (e.g., methylation, acetylation) of nucleotides or modification of proteins, called histones, that organize DNA into chromosomes. Valproic acid is an inhibitor of histone deacetylase and has been used to induce ASD-like features in animal models (Roullet et al., 2013). Perturbation of epigenetic pathways has been implicated in a variety of diseases including ones that overlap with ASD (Millan, 2013). Most convincingly, children with Rett Syndrome (see Pizzorusso, this volume) often have behavioral and molecular features of ASD (Hogart et al., 2007). Rett syndrome results from mutations in the MECP2 gene which encodes a Methyl-CpG binding protein that binds to methylated cytosines and can regulate gene expression epigenetically. Recent work demonstrates that MeCp2 can be phosphorylated in response to neural activity (Ebert et al., 2013) and a point mutation in MeCP2 associated with ASD interferes with this phosphorylation and leads to ASD-like behavior in mice. This makes epigenetic modification an interesting candidate

A Blueprint for Research on Shank3 and Autism

pathway. Epigenetic changes have been implicated in maternal care in mouse models (Zhang and Meaney, 2010). Here attentive maternal grooming of pups by their mothers makes them more likely to be nurturing mothers and this involves covalent modification of the genome (Bagot et al., 2012). There is a worldwide effort to identify epigenetic pathways in many areas including psychiatric disorders and this is expanding as the definition of epigenesis expands (Millan, 2013).

GENETICS OF ASD There is a large genetic contribution to ASD estimated from studies of families and, especially identical and fraternal twins. The concordance rate for monozygotic (identical) twins reaches 90% whereas for dizygotic (fraternal) twins it is 10–20% (Bailey et al., 1995; Hallmayer et al., 2011). A fundamental challenge facing geneticists is simply to identify the many genes that contribute to ASD directly or predispose to environmental influences. This task has proven far from straightforward. Research on ASD faces many of the same challenges of other complex disorders (McClellan and King, 2010). These are suspected to include: (1) a large number of rare mutations, (2) variants with small individual contributions, (3) many functional/regulatory variants on individual genes in unrelated individuals (“allelic heterogeneity”), (4) genetic background effects where the same mutation gives distinct symptoms in different individuals, and (5) interaction of many genes functioning in common pathways whose dysregulation yields similar symptoms. For ASD, this is further complicated by the lack of objective biomarkers for what are a spectrum of psychiatric disorders, and an ill-defined contribution of environmental factors. There has also been much discussion about whether ASD is caused by mutations within the genome that give rise to a relatively small number of common variants, or from rare or de novo variants that are individually more penetrant (Geschwind and Levitt, 2007). There has, however, been considerable success in demonstrating that ASD can arise de novo from Copy Number Variations (CNVs) that result from deletion or duplication of relatively large regions of a chromosome (Huguet et al., 2013), though ASD may involve a combination of de novo and inherited CNVs (Leblond et al., 2012). These and other issues bear strongly on the genetic approach one takes to studying such complex traits (Reich and Lander, 2001) and have been reviewed in detail elsewhere (Miles, 2011; State and Levitt, 2011). Others have argued that even with rapid sequencing of entire exomes or entire genomes one is still

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confronted with validating variants that are predicted to be damaging (Karayiorgou et al., 2012). Thus there is a growing emphasis on studying rare, highly penetrant variants in order for research to move forward to the translational level and to a deeper understanding of brain connectivity disorders. A successful example of this is research on Shankopathies (discussed below) that result from mutations in the gene SHANK3. Research on this monogenic disorder has led to the development of animal models essential to elucidating molecular pathways, cellular events, and changes in brain connectivity as well as for development of therapeutics. In other areas, such as research on muscular dystrophies, there has been much progress in understanding a diverse set of diseases by the combination of human genetics and relevant animal models. This work has led to identification of proteins (Cote et al., 1999) that interact with dystrophin, the Duchenne Muscular Dystrophy gene (Kunkel et al., 1986), and that function in maintaining muscle integrity (Cohn and Campbell, 2000). As a result, we now have a rather detailed description of this molecular pathway, a mechanistic model for its role in muscle function, and animal models for a number of muscular dystrophies. This work has brought coherence to diagnosis of muscular dystrophies in this pathway, as well as approaches to therapy. While muscular dystrophy is much simpler than ASD, and has quantifiable biomarkers for validation, work in this area makes a strong case for how genetic research can uncover a unifying pathway involved in disease. Focusing on a subset of rare, Mendelian forms of ASD may provide a similar benefit for genes that function in synaptic transmission (Karayiorgou et al., 2012).

CURRENT HYPOTHESES FOR ASD A number of interesting hypotheses have been proposed for ASD. These range from psychiatric to molecular (Geschwind and Levitt, 2007; Kelleher and Bear, 2008); as such, they are not mutually exclusive. The “synaptic hypothesis” has arisen largely from the research on monogenic disorders, such as fragile X syndrome, PMS, Tuberous sclerosis complex (TSC), and Rett Syndrome, where penetrance is high. Genomic studies on these disorders have revealed a cluster of genes including: synaptic cell adhesion molecules, regulators of protein translation, and synaptic scaffolding proteins (Fig. 1) which contribute to the hypothesis that dysfunction in synaptic plasticity causes ASD (Zoghbi, 2003). The data supporting this hypothesis are particularly satisfying because genetic discovery makes no Developmental Neurobiology

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Figure 1 The synaptic machinery underlying many ASD. A glutamatergic synapse (left) is illustrated in proximity to a GABAergic synapse (right). The former consists of a nerve terminal on a dendritic spine and within the spine are illustrated the major proteins, discussed in the text, that have been implicated in ASD; notably the scaffolding protein Shank, which anchors glutamate receptors within the postsynaptic density on the spine head. The nerve terminal releases neurotransmitters but also BDNF and IGF-1. Within the synaptic cleft is an extracellular matrix that is important for pre- and postsynaptic differentiation. Glutamatergic synapses (excitatory) and GABAergic synapses are homeostatically regulated and this plasticity is important in the sequence of events following changes in synaptic transmission that occur with mutations that lead to ASD.

assumptions about etiology or pathophysiology. For example, fragile X syndrome was found to originate from a trinucleotide expansion in the 50 end of the gene FMR1 (See Cook et al., this edition). The syndrome includes mental deficiency and is associated with a high incidence of ASD. The fragile X mental retardation protein (FMRP) is an RNA binding protein and negative regulator of protein synthesis (Fig. 1). Loss of FMRP expression is accompanied by an increase in the synthesis of synaptic proteins and excessive production of dendritic spines. Mouse models of fragile X syndrome display enhanced long-term depression and behavior that mimics some aspects of ASD. The same protein translational machinery that is inhibited by FMRP is activated in spines by metabotropic glutamate receptors via the mammalian target of rapamycin (mTORC1), an important intermediate in the mTOR pathway Developmental Neurobiology

(Hoeffer and Klann, 2010) and in regulation of the number of ionotropic glutamate receptors which are critical to long-term changes in synaptic efficacy. Together, these results not only have helped elaborate the synaptic hypothesis but have revealed new targets (mGluR5, mTORC, FMRP) for therapy (Dolen et al., 2007) that are being tested in the clinic (discussed below). Glutamate, GABA (Chao et al., 2010), endocannabinoid (Foldy et al., 2013), and serotonin neurotransmission have all been implicated in ASD (Carlson, 2012). Identification of synaptic genes as risk factors has raised the challenge of explaining the pathophysiological consequences leading to ASD. In this respect, categorization of ASD genes based on neurotransmitter systems should be viewed as a way station on the route to a deeper understanding. Research on major depression has been stifled in translational and


other efforts by intense focus on drugs that regulate these broad classes of neurotransmission. There has been relatively little progress in targeting the circuits critical to psychiatric disorders in which these neurotransmitters function. Rather than identifying neurotransmitter systems, we need to answer the more important question of how genes map onto the brain and how they affect social interactivity and other hallmarks. Another hypothesis suggests that ASD is a disorder of brain circuitry or neural activity within those circuits (Shepherd, 2013). Rubenstein and Merzenich (2003) proposed that neuronal connectivity deficits arise as a result of an imbalance of excitation and inhibition in select circuits important to ASD core symptoms. This imbalance may result in abnormally high endogenous activity that obscures synaptic inputs and hence the normal responsiveness of circuits to sensory inputs (Dani et al., 2005; Hines et al., 2008). Experiments on mouse models of ASD reveal evidence for excitatory/inhibitory imbalance (Dani et al., 2005). In recent studies of TSC, where mTOR signaling is affected, excitatory–inhibitory balance is disrupted leading to hyperexcitability of hippocampal neurons (Bateup et al., 2013). Others have focused on directly identifying neural circuits that underlie the behavioral features of ASD. Koster-Hale et al. (2013) studied a region of the temporo–parietal junction responsible for discerning the intentions of others, a feature previously shown to be impaired in patients with ASD. They have found abnormal responses in activity of the temporo–parietal junction that are detectable by functional magnetic resonance imaging in patients given the task of judging intent (Koster-Hale et al., 2013). Others speculate that “mirror neurons” which are activated by performance of a movement or by observing another individual doing the same task (Oberman et al., 2005) are part of circuits that malfunction in ASD (Ramachandran and Oberman, 2006). ASD research impinges on our understanding of the mind and brain. In essence, it asks us to resolve the brain–mind duality problem, not generally, but for a set of genes that affect how patients with ASD interpret social interactions, etc. This will test some of our current assumptions about brain function and connectivity, such as the idea that unique behaviors are served by a dedicated network of neurons; an idea that has proven extremely fruitful in animal studies of circuit formation and function. Implicit in this notion for ASD is the premise that the stereotyped behaviors that characterize ASD arise from disruption of specific circuits. However, our understanding of brain circuitry is still quite primitive. We currently speak of structural vs. functional connectivity (Horwitz, 2003), which harkens

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back to the mind–brain duality proposed by Descartes in the 17th century. This gap in our understanding of brain connectivity is a major obstacle to progress on psychiatric disorders. Previous work has revealed that distinct psychiatric disorders such as ASD or schizophrenia may arise from different mutations in the same genes (e.g., Gauthier et al., 2010). On the other hand, polymorphisms in a number of common genes underlie multiple psychiatric disorders (Levine, 2013). In the latter instance it is parsimonious to assume that this results largely from multiple genes acting in the same pathway; the assumption made in genome-wide association studies of gene pathways (Sullivan, 2012). The former instances are a profound challenge not only to studies of ASD, but also in attempts to understand psychiatric disease broadly and how the genome maps onto the mind (Croning et al., 2009). Other observations indicate that inborn errors of metabolism (discussed below), which are manifest in all cells, result in psychiatric disorders including ASD (Manzi et al., 2008). Smith–Lemli–Optiz Syndrome is a disorder of cholesterol biosynthesis and up to 86% of children with this syndrome have core features of ASD (Sikora et al., 2006). A conservative interpretation of this finding might suggest that the specific circuits serving behaviors of ASD are vulnerable to disruption of cholesterol metabolism. A more problematic one is that functional circuits that underlie these behaviors may be very highly distributed. Fortunately, we have entered a promising period of new developments in functional and structural mapping of brain circuits (Amunts et al., 2013; Cai et al., 2013; Chung and Deisseroth, 2013; Chung et al., 2013). This has been reinforced by the commitment of considerable funding from public and public–private consortia in launching several large-scale efforts to generate a brain map (discussed below). In addition, progress continues in mapping circuits that mediate behaviors relevant to ASD, such as anxiety (Kim et al., 2013). The challenge is formidable and so will be the rewards. Our brains evolved to react to rapid changes in its environment and this likely entails small, molar changes in expression, post-translational modifications, etc, that result in major, long-term changes in behavior. Plasticity in the brain is most robust during early development just when ASD is becoming manifest. Some have suggested that ASD is a disorder of brain “critical periods” when circuits are being fine-tuned by neural activity to attain their mature connectivity (LeBlanc and Fagiolini, 2011). Plasticity is expressed both in structural alterations, such as increases in dendrite complexity, and axonal Developmental Neurobiology

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pruning or as a neuron changes “within its skin,” for example by inserting glutamate receptors into established synapses to enhance synaptic efficacy. Synaptic plasticity is normally beneficial and essential for learning, memory, neural regeneration, etc. However, the same mechanisms of plasticity that maintain functional homeostasis within brain circuits may be particularly vulnerable in contributing to aberrant connectivity and brain dysfunction (Toro et al., 2010; Bateup et al., 2013). Also, primary changes initiated in the brain by loss (PMS) or gain of function (Fragile X syndrome) may trigger a host of secondary plastic changes that amplify the initial disruption. They would also make it difficult to detect the impact of the primary defect. Thus, while mapping gene mutations onto neural circuits, we also need to understand the structural and functional dynamics that ensue. It is worth noting again that children with ASD may develop normally and then regress suddenly. Other studies suggest that in a significant number of instances the core symptoms of ASD dissipate (Fein et al., 2013). We need to know whether this waxing and waning of symptoms is related to primary or secondary changes in brain connectivity and the mechanisms responsible. The enthusiasm around the synaptic hypothesis of ASD prompts a reminder that not all ASD are caused by primary defects in synaptic function. There are many genes postulated to be risk factors for ASD (gene.sfari.org). Some of these are not expressed at synapses and others are expressed throughout the body and more likely to affect synapse function indirectly. The gene for trimethyllysine hydroxylase epsilon that encodes the first enzyme in carnitine biosynthesis has been suggested as a risk factor for ASD (Celestino-Soper et al., 2012). Carnitine is required for transport of fatty acids into mitochondria. Exogenous carnitine can regulate synapse formation by modulating mitochondrial function (Merrill et al., 2011), but it is by no means clear that this is a primary defect important to ASD (CelestinoSoper et al., 2012). In another example, Novarino et al. (2012) have linked to ASD a point mutation in branch-chained keto acid dehydrogenase kinase. This leads to excessive degradation of branch-chained amino acids like leucine, isoleucine, and valine and it seems likely that this has global effects not limited to synaptic function (Beaudet, 2012). Events such as neuronal migration, stem cell replication etc. that are important to development but distant from synapse form or function may indirectly affect connectivity and result in ASD. Indeed, carnitine affects neuronal proliferation and differentiation (Nakamichi et al., 2012). Developmental Neurobiology

SHANKOPATHIES Shank3 is protein that is a critical component of the postsynaptic density which is arguably the most intricate protein super complex in our bodies (Grabrucker et al., 2011). The discovery that SHANK3 is linked to PMS is one of the important findings that underpins the synaptic hypothesis and has prompted efforts to characterize the role of the Shank3 protein in development of the brain, as well as to understand how deletion or mutation of this gene in Shankopathies affects behavior. These developments have led to a more precise understanding of how DNA alterations can induce PMS-related behaviors, and has initiated efforts to develop treatments. In this section, we briefly review research on Shankopathies, and in the next section we discuss the remaining challenges in translating the basic science of SHANK3 to better treatments for PMS. We also note how these developments relate to insights from research on other forms of ASD. More than 50% of the children with PMS have ASD that may be accompanied by dysmorphic features, intellectual deficiencies, and delayed or severely impaired speech. SHANK3 was implicated in the ASD by karyotyping of cells from a PMS patient with a balanced translocation between chromosomes 12 and 22. In this instance, the breakpoint for the translocation occurred within exon 21 of the gene for SHANK3 (Bonaglia et al., 2001). Subsequent work identified a sequence of 15 base pairs in SHANK3 that is associated with recurrent deletions and PMS (Bonaglia et al., 2006). Several studies of ASD patients, broadly defined (Durand et al., 2007; Moessner et al., 2007; Gauthier et al., 2009), identified SHANK3 mutations that were not found in controls (Waga et al., 2011), including cases where there was evidence that the mutations were damaging to cell or protein function (Gauthier et al., 2009, 2010; Durand et al., 2012). These and other observations strongly indicate that SHANK3 is important in the etiology of ASD in PMS (Wilson et al., 2003; Bonaglia et al., 2006; Durand et al., 2007; Phelan and McDermid, 2012). As a result, PMS is the most common Shankopathy. It remains to be seen whether other genes in the 22q 13.3 region contribute to the ASD (Aldinger et al., 2013). Synaptic transmission in the brain is typically rapid and requires both quick neurotransmitter release and a high density of neurotransmitter receptors in the postsynaptic membrane to respond effectively. Neurotransmitter receptors are membrane proteins that can diffuse throughout the plasma membrane. Cells have evolved Shank3 and other scaffolding


proteins (Fig. 1) that: (a) tether receptors to achieve precise localization beneath the nerve terminal, as well as a high density in the subsynaptic membrane, (b) bring together signaling complexes into these scaffolds that function downstream of receptor activation, and (c) recruit molecules that function transsynaptically to affect nerve terminal form and function. Shank3 is among the first proteins to be expressed during differentiation of the postsynaptic density (PSD; Boeckers et al., 2002). There it helps anchor (Fig. 1) other subcomplexes centered on PSD 95, SAPAP, AKAP (Grabrucker et al., 2011; Ting et al., 2012) and especially Homer (Hayashi et al., 2009). Each of these subcomplexes acts cooperatively within the PSD contributing distinct features (Ting et al., 2012). Shank3 has multiple functional domains (Jiang and Ehlers, 2013), notably an SH3 domain, ankyrin repeats, a PDZ domain, a Homer binding region, a cortactin binding domain, and a sterile alpha motif (SAM). The SAM domain has been implicated in synaptic localization and assembly of Shank3 into multimolecular sheets (Baron et al., 2006). Other domains mediate interaction with ionotropic (AMPA, NMDA) and metabotropic (G-protein) glutamate receptors (mGluR5), via PSD 95, SAPAP, and Homer (Verpelli et al., 2011). Still other domains tether the PSD to the cytoskeleton (ankyrin repeats), and signaling complexes (Schuetz et al., 2004; Grabrucker et al., 2011; Jiang and Ehlers, 2013; Kunde et al., 2013). The Shank scaffold is not only important for the PSD and concentration of glutamate receptors on the heads of dendritic spines (Roussignol et al., 2005; Verpelli et al., 2011), but Shank3 mutant mice have smaller and reduced numbers of spines as well as thinner PSDs (Bozdagi et al., 2010), suggesting a central role for Shank3 in the formation, maturation, and stabilization of the entire spine (Sala et al., 2001; Roussignol et al., 2005). Shank3 also interacts directly and indirectly via PSD95 (Prange et al., 2004) with the synaptic adhesion molecule neuroligin (Sudhof, 2008). Neuroligin binds across the synaptic cleft to neurexin (Fig. 1) (Sudhof, 2008; Arons et al., 2012). Thus, changes in Shank in the PSD may affect presynaptic function. While in PMS there is loss of one copy of SHANK3 (haploinsufficiency), several Shankopathies have been described that are associated with mutations within coding regions and result in truncated proteins. In one instance, this is has been linked to schizophrenia rather than ASD (Gauthier et al., 2010). Continued studies of the structure and function of human mutations in SHANK3, and the interactions of Shank3 domains, will be essential to our understanding of

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ASD as well as other mental disorders (Chao and Zoghbi, 2012). Activation of NMDA receptors requires the proximity of AMPA receptors. This special combination allows long-term changes in synaptic transmission that underlie learning and memory mediated by coincident activation of the two types of receptors. In long-term potentiation (LTP), increased synaptic efficacy results from an increase in the number of AMPA receptors due to insertion into the surface (Granger et al., 2013) and/or diffusion of extrasynaptic AMPARs into the postsynaptic density and anchoring to the subsynaptic scaffold. The changes in synaptic currents are initially restricted to the synapse and tap into local pathways in the spine (e.g., protein synthesis) and more distant ones in the cell body (gene transcription) that may alter synaptic structure. A key feature of the early events that result in LTP and LTD (long-term depression) is that the PSD is remodeled, while retaining its essential integrity. These synaptic dynamics are perturbed by disruption of Shanks (Verpelli et al., 2011). The biosynthesis of Shank3 is itself regulated by neural activity (Miletic et al., 2010), and Shank3 is ubiquitinated in response to neural activity to hasten its internalization and degradation (Ehlers, 2003). Nerve terminals also release Zn12 ions, which have the potential to stimulate the assembly of Shank monomers into oligomeric sheets to expand or stabilize the PSD matrix (Grabrucker, this edition). Individual synapses have remarkable potential for plasticity, but a postsynaptic neuron integrates activity from many thousands of excitatory and inhibitory synapses. In the near term, this determines whether the cell will generate an action potential. In the longer-term, this activity may result in global reorganization of synapses by the postsynaptic neuron to maintain a balance of excitatory and inhibitory inputs (Fig. 1). Such homeostatic plasticity occurs (Ting et al., 2012) when reduced excitatory inputs trigger a reduction in inhibitory synaptic efficacy, making the cell better able to respond to reduced levels of excitation (Turrigiano, 2012). Similarly, increased excitatory activity can homeostatically upregulate inhibitory synaptic efficacy. A potentially critical issue is how the developing brain responds to reduced glutamatergic function. The limits of homeostatic and other forms of plasticity in the context of Shankopathies are largely unknown. Recent data suggest that in animal models of Rett Syndrome, where patients have a high incidence of ASD, homeostatic plasticity is significantly compromised (Blackman et al., 2012). This may well contribute to the imbalance of excitation and inhibition in cortical circuits Developmental Neurobiology

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that is thought to be important in the etiology of ASD (Dani et al., 2005). Transgenic animal models have been indispensable in demonstrating effects of SHANK mutations on PMS-related traits. Generally, data from multiple SHANK mutant mice have revealed deficits in synaptic transmission, structure, and social behaviors. However SHANK3 has 22 exons (Jiang and Ehlers, 2013) and encodes multiple isoforms as a result of alternative splicing (Lim et al., 1999), as well as multiple promoters within the gene. Of particular importance is identification of the regulatory elements for SHANK3 expression (Lennertz et al., 2012), not only for understanding the impact of mutations in these regions but also for therapeutic efforts (discussed below). SHANK3, like a number of other ASD genes (Chao and Zoghbi, 2012), is tightly regulated. Reduced expression (Schmeisser et al., 2012) causes ASD whereas increased expression, due to duplication of terminal 22q region, leads to Asperger’s Syndrome (Durand et al., 2007) or hyperkinesis (Han et al., 2013). Furthermore, in mice and humans intragenic SHANK3 promoter activity is regulated epigenetically by DNA methylation to the extent that alternative transcripts may be differentially expressed in the same cell type from different brain regions (Ching et al., 2005). The period of most active methylation corresponds to the developmental period of active synapse formation (Uchino et al., 2006). In view of the phenotypic variability seen in SHANK mutants, and the phenotypic heterogeneity in humans with Shankopathies, characterizing SHANK transcripts their localization in the adult and developing brain, how they change with neural activity (Wang et al., 2011) and their epigenetic modifications will likely be important for understanding how mutations that affect various transcripts of SHANK3 have distinct behavioral consequences. While the bulk of research to date has been on SHANK3, humans have two other SHANK genes 1 and 2 that have been implicated in Shankopathies. All three SHANKs are expressed rather broadly in the brain, including in regions critical for cognition and all three genes function, as synaptic scaffolds with many of the same protein–protein interactions. There are a number of reports of ASD and related psychiatric disorders associated with mutations in SHANKs1, 2 (Berkel et al., 2010; Pinto et al., 2010; Sato et al., 2012b). There are, however, clear differences resulting from mutation of these genes. Recent work has revealed that SHANK1 mutations in humans have the mildest phenotype in patients with ASD, SHANK2 are intermediate, and SHANK3 the most severe and includes intellectual deficits (Leblond et al., unpubDevelopmental Neurobiology

lished). These authors speculate that SHANK1 mutations lead to “immature neuronal networks” with reduced dendritic spine heads. These issues can be tackled in mutant mice models with high resolution imaging of synapses, as well as correlative physiology. Several mouse models mutant in SHANKS 1–3 have been generated (Jiang and Ehlers, 2013). These mice have deficits in synaptic proteins, including glutamate receptors and spine structure that may vary with brain region (Jiang and Ehlers, 2013). Mice mutant in SHANK3 (Bozdagi et al., 2010; Peca et al., 2011) have deficits in social interactions, including repetitive and abnormal vocalization, though the behaviors differ somewhat depending on the mutation (Jiang and Ehlers, 2013). Mice mutants in the SHANK1 PDZ domain have smaller PSD, smaller dendritic spines, and reduced synaptic transmission. Behaviorally, these mice have increased anxiety but perform better than controls in spatial learning. SHANK2 mutant mice (Schmeisser et al., 2012) display behaviors that mimic ASD in humans. It will be important to our understanding of synaptic and connectivity dysfunction and the behavioral consequences to make these mouse models available to confirm these differences and determine genetic bases.

BLUEPRINT FOR RESEARCH ON SHANKOPATHIES There is no bona fide treatment for ASD. Behavioral therapy can improve social skills, mitigate intellectual deficits, and improve verbal communication but it must be applied intensively (Oono et al., 2013). There are a number of new computer-assisted approaches available and under development to help with interpersonal instruction and delivery of behavioral therapy (Ramdoss et al., 2012). Drugs currently used for ASD, such as risperidone and aripiprazole, are antipsychotics for secondary symptoms. They have substantial side effects that make them a special concern for use in infants and young children. Future breakthroughs will profit greatly from the application of evidence-based approaches to therapy. In this regard, studies of monogenic ASD have much to offer with regard to detailed characterization of a subset of ASD. With this in mind, we describe in the following sections a blueprint for research on Shankopathies. Our aim is to help develop a consensus within the community on research to mitigate the core symptoms of Shankopathies. The goals are centered on the synaptic and connectivity hypotheses of ASD. In the following text and Table 1 we prioritize near- and long-term goals to strengthen fundamental


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Table 1 Targets for Research on Shankopathies Basic Research—Near Term Describing the natural history of Shankopathies (PMS) with a special attention to regression. Restoring SHANK3 expression in animal models. Does restoration block or reverse synaptic and behavioral deficits during development? Is there a critical period during development for normalizing SHANK3 expression? Can Shankopathies be reversed in adult mice? Modulating synaptic efficacy, increasing synapse number in Shankopathy models. Genomic studies to identify mutations in SHANKS and that correlate structure with clinical presentation.

Translational Research—Near Term

Translational Research-Longer Term

Clinical—Databases for better gene structure-symptom correlation

Better models: Expanding research on genetics, physiology, circuit analysis and behavior of non-human primates.

High-throughput drug screens to discover means of increasing genetic expression of SHANK3, increasing protein translation or reducing degradation. IPS cell core facility for Shankopathies.

Defining the circuits in social behavior the effects of reduced Shank on connectivity.

Collaborative efforts and sharing mouse models Best Practices for analysis new mutant models

Correlation of genes with circuit function in animal models and in humans (fMRI)

understanding, and to develop tools for translational efforts, for treatment of Shankopathies. We propose a combination of team efforts, some of which are largely service-oriented, as well as efforts by smaller groups or individual labs. Human genetics will remain a mainstay at levels from continued gene structure– function analysis of SHANK3 to clinical trials. It is unclear to what extent research on Shankopathies will generalize to other ASD. This will be dictated in large part by the pathways that we discover. There has been considerable progress, and what seemed an intractable problem for any ASD not long ago, is yielding to research efforts that have spawned a host of clinical trials that are currently underway.

CAN SHANKOPATHIES BE REVERSED? There are a number of anecdotal reports and, more recently, systematic evidence that core symptoms of ASD can dissipate with time, to the extent that patients have normal social interactions and communication skills (Fein et al., 2013). It will be important to confirm and extend this work since it implies that ASD is reversible (Delorme et al., 2013). Research on mouse models of ASD suggest that reversibility is possible even outside of the developmental period when brain plasticity is most robust and symptoms first become evident (Herbert, 2011). For example, mice engineered to be reversibly silenced for MeCP2

display disrupted synaptic plasticity (LTP in the hippocampus) as well as behavioral traits reminiscent of Rett Syndrome (Giacometti et al., 2007; Guy et al., 2007). Induction of MeCP2 expression under control of its own promoter, in mature or immature mice, results in rescue of brain and behavioral deficits (Guy et al., 2007). In fact, deletion of MeCP2 in adult mice resulted in a Rett-like phenotype indicating that ongoing expression of MeCP2 is necessary for maintenance of normal functional circuitry outside of the neurodevelopmental period. In both studies (Giacometti et al., 2007; Guy et al., 2007), the level of expression was viewed as critical, and was generally believed to be at levels seen in normal brains. Nevertheless, it is revealing for therapeutic considerations that the endogenous promoter produced a more complete rescue (Guy et al. 2007; c.f. Giacometti et al., 2007), although even in this case when the onset of expression was precipitous it was lethal. This is a reminder that MeCP2 must be carefully controlled quantitatively and temporally, or its expression, intended to mitigate the original symptoms, may introduce new ones (Collins et al., 2004).

WHAT IS THE NATURAL HISTORY OF SHANKOPATHIES? A recent report describes the PMS in a small cohort of patients aged 1–45 years (Soorya et al., 2013). The Developmental Neurobiology

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study includes neurological, psychological, and genetic data that will be helpful in future clinical studies of Shankopathies. Additional and more detailed studies of the natural history of Shankopathies will detail the incidence of remission, relevant circumstances for onset, as well as help define any regression of symptoms in infants. These data will be useful in counseling, patient management, and in generating baseline data for future clinical trials. They will also be important for future studies in defining whether there is a developmental window during which Shankopathies can be best treated. To help in this effort the PMS Foundation has designed a questionnaire related to the clinical and developmental features of PMS, as well as genetic data curated from reports submitted by families (http://pmsiregistry.patientcrossroads.org). It has over 500 registrants.

tor) and BDNF (Brain derived nerve growth factor). In the following sections, we discuss these and other routes to treating the synaptic defects in Shankopathies. Overexpression of SHANK3 in mice has deleterious effects (Okamoto et al., 2007), including maniclike behavior (Han et al., 2013). Thus in efforts to restore expression it may well be necessary to tightly control SHANK3 to achieve wild-type levels. For experimental purposes this can be achieved by homologous recombination to silence SHANK3 expression, followed by reactivation of the gene by inducible Cre-mediated recombination. This would leave the expression of SHANK3 under control of its endogenous promoter. This experiment should be given high priority for its value as proof-of-principle in future efforts at therapeutically upregulating expression.

Restoration of SHANK3 Expression Shankopathies result from haploinsufficiency and data from mouse models show that haploinsufficiency of SHANK3 leads to a 50% reduction in the expression of Shank3 protein (Schmeisser et al., 2012). This should be confirmed in post mortem human brain tissue or, at least, in induced pluripotent stem (IPS) cells derived from patients with a genetically validated Shankopathy. A priority for preclinical studies in animal models would be to: (a) restore expression of SHANK3 and assess its ability to reverse or mitigate structural, physiological, and behavioral phenotypes related to ASD; (b) determine whether there is a critical period during development for the rescue, and whether rescue can occur in mature mice; (c) determine whether increasing expression of SHANK1 and 2 can compensate SHANK3 loss of function; and (d) determine whether re-expression of SHANK3 rescues ASD-like symptoms without introducing any additional deficits. Increased expression of SHANK3 is an obvious route to therapy and could be accomplished by: (a) gene therapy to add an extra copy of SHANK3; (b) increasing expression from the unaffected copy of SHANK3; (c) increasing Shank3 translation, assembly, synaptic targeting, or recycling; and (d) reducing Shank3 degradation. Also SHANK3 expression is regulated epigenetically, and it may be possible to block methylation to increase expression. SHANK3 mutation results in fewer synapses and synaptic spines, so increasing the efficacy of remaining synapses may help with synaptic and behavioral deficits. Alternatively, one could stimulate the formation of new spines or protect the old preformed ones with growth factors such as IGF1 (Insulin like growth FacDevelopmental Neurobiology

Gene Therapy for Shankpathies Gene therapy allows one to add an extra copy of SHANK3 under the control of a promoter that may yield relatively precise regulation of expression levels, and even some selectivity as to location of expression (Gray, 2013; Simonato et al., 2013). There are a number of impediments that have slowed the implementation of gene therapy generally, and for psychiatric disorders there are secondary complications. For example, it may be necessary to restore expression within a narrow developmental window, for a precise duration, and possibly only in those circuits that underlie the disorder. Even if one leaves aside these secondary considerations, employing viral vectors to obtain expression broadly, with no regard for localization, is a major undertaking (Gray, 2013) and a goal best be tackled by the larger neuroscience community. Consider that it is difficult to obtain: (a) viral vectors that allow incorporation of the transgene into the host genome, but are selective for the cell type of interest and are not immunogenic or oncogenic; (b) long-term expression in non-dividing neural cells; and (c) access to the brain and effective transduction. The blood–brain barrier will prohibit entry of viral vectors injected into the blood. Moreover, the dense parenchyma of the brain will impede their spread if they are injected directly into the brain. This is particularly problematic for (cell autonomous) disorders where the replacement gene must be expressed directly in the affected cells, as in Shankopathies. There are disorders where the replacement gene may, for example, encode a secreted protein that can be expressed in unaffected cells but has the potential to diffuse within the extracellular space of


the brain to rescue affected ones. A further problem is that vector injections generate a decreasing gradient, and hence expression, away from the injection site. There is also the question of what SHANK3 isoforms to express. While CNVs most commonly are associated with Shankopathies, frame shift, missense, splice site, and other sequence changes in SHANK3 have also been identified in patients Jiang and Ehlers, (2013) nicely describe the reported repertoire of these mutations, their location in the SHANK3 sequence, and the heterogeneity in symptomology among these mutations. Despite these constraints there has been clear progress on gene therapy. Recent successes have used lentiviral vectors to transduce autologous hematopoietic stem cells (HSC). The HSC cells have the capacity to directly cure hematopoietic disorders or serve as “Trojan Horses” since they give rise to cells (microglia) that transit to the brain. In a striking example this strategy was used to cure metachromatic leukodystrophy which results from a deficiency in the enzyme arylsulpatase. Patients with this disorder have major cognitive and motor deficiencies and die early in life due to intolerable levels of toxic metabolites. Biffi et al. (2013) reasoned correctly that movement to the brain of HSC derived cells carrying the missing gene might be sufficient to treat the disease. This may be relevant to ASD in Rett Syndrome where studies in mouse models have implicated microglia derived from the hematopoietic system in the disorder. In one report, bone marrow transplantation into MECP2 mutant mice was found to cure ASD symptoms (Derecki et al., 2012). Similar transplants may mitigate any ASD that are found to be caused by or exacerbated by inflammation (discussed above).

Increasing SHANK Expression The most expeditious way to restore SHANK3 expression in PMS would be to increase expression from the unaffected copy of SHANK3. In most cases the mutated copy is completely deleted so that one would not hazard the expression of a mutant and potentially interfering protein. High priority should be given to establishing methods for identifying molecules that increase the expression from the unaffected copy of SHANK3. This entails the development of cell and animal models to test candidate molecules, as well as high throughput, “unbiased” drug screens. Just such an approach to drug discovery was reported for Angelman Syndrome, a disorder that is associated with a high risk of ASD (Huang et al., 2012). Angelman Syndrome is

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caused by mutation of the maternal allele for ubiquitin ligase (UBE3A) which is involved in protein degradation (Fig. 1); the paternal allele is silenced by methylation. A large, unbiased screen aimed at activating the dormant allele evaluated more than 2000 small molecules. An inhibitor of DNA topoisomerase was found to increase Ube3A in neurons in culture, and subsequently in vivo. As if to emphasize the value of unbiased drug screens, the drug does not appear to function by altering the methylation of UBE3A and might well have not been included in a screen for candidate drugs where one makes an educated guess as to therapeutic potential. Thus, in addition to identifying a potentially valuable molecule, this study has uncovered a new pathway that may yield additional insights and targets (King et al., 2013). Carrying out a similar screen for drugs that upregulate the expression of SHANK3 might entail knocking GFP (green fluorescent protein) into the coding regions of SHANK3 so that GFP is expressed under control of the SHANK3 promoter. Showing that the expression pattern of the GFP matches the expression pattern of the endogenous Shank3 protein would help to confirm that no regulatory elements for the gene have been disrupted. Cultured neurons from these mice could be used for automated large-scale screens to identify drugs that increase SHANK3 (GFP) expression. Work in the same mouse model could determine: (a) the precise level of upregulation of expression with drug dose, (b) whether the drug can pass through the blood–brain barrier, (C) which cells in the brain are affected, (D) the persistence of expression, and (E) the effects on mice of different ages, etc. Similar studies could be done to screen for drugs that upregulate SHANKs1, 2. In SHANK2 mutant mice, SHANK3 expression is increased (Schmeisser et al., 2012) and may be compensatory. Indeed, SHANK3 mutant mice seem to have more severe phenotypes than mice with mutations in SHANK1, 2. Recent work in humans is consistent with this interpretation and may also reflect compensation by SHANK3 in other instances (Leblond et al, unpublished). Crossing mice mutant in the various SHANKs to produce double or triple knockouts would ascertain whether mice mutant in more than one SHANK have exaggerated deficits, consistent with the notion that there may be cross compensation among the three SHANKs. Drugs that increase the expression of SHANK1,2 may not only be valuable to patients with mutations in these genes, but also to compensate for other Shankopathies. IPS cells (discussed below) may prove invaluable to help validate drugs that upregulated SHANK3 Developmental Neurobiology

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expression in mouse cells. IPS cells are derived from the cells of patients and have the ASD causal mutation and their genetic background (discussed below). Recent work describes methods for producing large numbers of human neurons from IPS cells (Zhang et al., 2013). Thus it is now possible to directly test drugs for increased expression in human neurons mutant in SHANKS, rather than use mouse models (Shcheglovitov et al., 2013). One could also screen for drugs that might affect expression of SHANKs epigenetically. For example, 5-Aza-20 -deoxycytidine, an FDA approved drug that inhibits DNA methylation, has been reported to increase SHANK3 expression in mouse neurons (Beri et al., 2007).

Regulation of Protein Synthesis The post-transcriptional life of Shank3 protein begins with its translation, followed by targeting, assembly into synaptic scaffolds (Boeckers et al., 2005; Tsuriel et al., 2006), and its eventual degradation. The balance between protein synthesis (Kelleher and Bear, 2008) and degradation (Bingol and Sheng, 2011; Tsai et al., 2012) is critical for maintaining synapse structure and function. Synaptic cell adhesion molecules, synaptic scaffolding proteins, neurotransmitter receptors, etc. are synthesized in the dendrite (Miyashiro et al., 2003), where translation is regulated by neural activity. In the brain, the machinery of protein synthesis, common to all cells, has been elaborated to allow encoding of synaptic activity into structural changes that underlie the establishment of proper connectivity during development and experiential change (Ebert and Greenberg, 2013). A consequence of disruption of protein synthesis is evident in fragile X syndrome with its high incidence of ASD (Kelleher et al., 2012). FMRP is an inhibitor of protein synthesis (discussed by Cooke et al., in this edition). Mice null for FMR1, which encodes FMRP, have enhanced protein synthesis, and an increased number of dendritic spines (Bagni and Greenough, 2005), as well as hypersensitivity to sensory stimulation in LTD (Chen and Toth, 2001). In another example of regulation of protein synthesis, patients with TSC are at high risk to develop ASD. TSC is a neurodevelopmental disorder that results from a mutation in genes that encode the proteins hamartin (TSC1) or tuberin (TSC2). Loss of either of these proteins increases activation of the mTOR (mammalian target of rapamycin) pathway of protein synthesis indicating that TSC1, 2 are negative regulators of protein synthesis via mTOR (Fig. 1). Rapamycin inhibits mTORC1 and reverses ASD in TSC mutant mice (Sato et al., 2012a) and ASD-related symptoms in Developmental Neurobiology

other mouse models (e.g., Zhou et al., 2009; Tsai et al., 2012; Zhou and Parada, 2012). In a recent example (Gkogkas et al., 2013), the machinery responsible for initiation of protein translation was found to selectively regulate the synthesis of the synaptic adhesion molecule neuroligin (Fig. 1). Genetic deletion in mice (Gkogkas et al., 2013) of the gene for the translation initiation factor 4E-binding protein 2 (4E-BP2) results in excitatory–inhibitory imbalance as well as disrupted social interactions. Pharmacological inhibition of protein initiation corrected the physiological and behavioral deficits. This pathway functions downstream of mTOR and the report (Gkogkas et al., 2013) describes a potential therapeutic, which may be more selective than rapamycin that disrupts immune, endocrine, and other systems in the body. Metabotropic glutamate receptors are a nexus between synaptic activity and protein synthesis (Fig. 1). They signal intracellularly to stimulate protein synthesis, necessary for synaptic plasticity (Santoro et al., 2012). In studies relevant to ASD, the drug MPEP, which inhibits mGlurR5, reduced anxiety in a mouse model of fragile X syndrome (Yan et al., 2005). This pathway of mGluR regulation of protein synthesis has also been implicated as a risk factor in Shankopathies (Kelleher et al., 2012). Shank binds to Homer forming a scaffold for mGluRs, cell adhesion molecules, and signaling complexes at the cell surface. Shank3 also integrates the PSD 95 subcomplex, including NMDARs, with Homer and mGluRs in the postsynaptic density (Tu et al., 1999). Knockdown of Shank3 levels in cultured neurons reduces mGluR5 at synapses, alters spine number, and compromises synaptic transmission (Verpelli et al., 2011). NMDARs are reduced at synapses in SHANK2 mutant mice whose deficits in social behavior are improved by treatment with a positive allosteric modulator of mGluR5 function (Won et al., 2012). Drugs that target mGluR 5, mTORC1, and other intermediates in the protein translation pathway have emerged as key targets for Shankopathies, TSC, fragile X syndrome, and other ASD (Sahin, 2012). There is much interest in developing new and more selective inhibitors of this pathway (Ehninger, 2013). A recent article describes a screen that successfully identified a small molecule that increased protein synthesis by inhibiting EIF2a phosphorylation and results in improved memory in mice (Sidrauski et al., 2013). Everolimus is a second-generation mTOR inhibitor that has been used effectively in TSC (Krueger et al., 2010), and a clinical trial has been launched to examine its efficacy for ASD (Clinical Trials.gov; NCT01289912). Fenobam was developed


as a selective negative modulator of mGluR5 tested in animal models (Ballard et al., 2005). Patients with fragile X syndrome treated with fenobam (Porter et al., 2005) were found to have lower levels of anxiety. Other inhibitors (acomproasate, arbaclofen, STX107, AFQ056, and RP4917523) are in various stages of preclinical testing or clinical trials.

Inhibiting Protein Degradation The level of synaptic proteins can be regulated rapidly by internalization and degradation in response to neural activity (Colledge et al., 2003; Greer and Greenberg, 2008; Greer et al., 2010). Degradation regulates not only the level of glutamate receptors at synapses (Snyder et al., 2001; Yin et al., 2011), but also the number of synapses during development. Synapse elimination is necessary in the nervous system to prune the excessive number of synaptic contacts that form initially to establish proper brain circuitry. Neural activity is critical to this pruning and, for excitatory synapses, acts via the transcription factor MEF2 and FMRP. Tsai et al. (2012) describe how these collaborate with additional intermediates to suppress degradation of synaptic proteins and contribute to the increased number of dendritic spines in mouse models of fragile X syndrome. They report that MEF2 and FMRP coordinately regulate the level of protocadherin 10. MEF 2 also controls expression of an E3 ubiquitin ligase (Mdm2) that ubiquitinates PSD-95 that is then targeted to the proteasome for degradation via its interaction with protocadherin 10. Surprisingly, EIF1a, a translational elongation factor whose synthesis is inhibited by FMRP, is key to these events. EIFa is expressed at high levels in FMR1 null mice and sequesters Mdm2 inhibiting PSD 95 degradation synapse disassembly. MEF2, protocadherin, and FMRP have all been implicated in ASD (Tsai et al., 2012). Other genes involved directly or indirectly in proteasomal degradation have been linked to ASD (Glessner et al., 2009; King et al., 2013); notably, UBE3A, which (Fig. 1), is deleted in Angelman Syndrome and controls the degradation of Arc protein that helps mediate activity-dependent internalization of AMPAR (Greer et al., 2010). Perturbing Ube3A function results in decreased AMPA receptors at synapses, a condition found in ASD patients (Purcell et al., 2001). Also Uba6-null mice have dramatically abnormal social interactions similar to mice with mutations in SHANK3 (Peca et al., 2011). UBA6, functions in proteasomal degradation but in a pathway distinct from the classical (Uba1-E2-E3) pathway (Lee et al., 2013). Mice null for UBA6 reveal

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that this gene inhibits spine formation/stabilization in the amygdala, and is associated with altered social behavior. Shank3 levels are increased in Uba6 deficient mice, consistent with the increase in spine density. Thus scaffold proteins Shank3 and PSD 95 are tightly regulated in the brain and either abnormally high or low degradation leads to ASD. But the ASD are different, or appear to be so. Encouragingly, peptide disruption of degradation, in the case of PSD95, can rescue the synaptic and behavioral deficits in mice (Tsai et al., 2012). Clearly we need to know more about these new degradative pathways that will reveal additional targets to manipulate degradation in Shankopathies and other ASD.

MODULATING SYNAPTIC EFFICACY TO MITIGATE ASD In view of the evidence that glutamatergic synapses are disrupted in Shankopathies and other forms of ASD (Berkel et al., 2010; Berkel et al., 2012; Etherton et al., 2011; Wang et al., 2011) there has been a concerted attempt to identify drugs that positively modulate ionotropic and metabotropic glutamate receptors to increase the efficacy of the residual transmission. Neurotransmitter systems for GABA, endocannabinoids, and serotonin (Costa et al., 2012) have also been implicated in ASD and are under study. Still other approaches are aimed at observations that growth factors, such as BDNF and IGF-1, stimulate dendritic spine formation and may be of potential value in those ASD where spines numbers are reduced. Finally, oxytocin is being tested as a therapeutic because of its ability to improve social behavior in ASD patients. We discuss each of these topics briefly below. AMPAkines are positive allosteric modulators of AMPAR that increase glutamatergic transmission and LTP without interfering with the binding of glutamate. In addition to facilitating neurotransmission, AMPAkines stimulate the transcription and translation of BDNF (Jourdi et al., 2009a,b) that is an important synaptic modulator (discussed below). AMPAR are also important for maintenance of spine integrity (Chang et al., 2012). CX 1739 and CX 1837 are AMPAkines that improve social behavior and cognitive performance in a mouse model (BTBR) of ASD, and are effective even in adult mice (Silverman et al., 2013). Beneficial effects of AMPAkines on LTP and learning have also been reported in an animal model of Angleman Syndrome where they increase actin polymerization in spines (Baudry et al., 2012). In studies relevant to Shankopathies a Developmental Neurobiology

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recent report shows that Shank3, in addition to anchoring AMPAR, interacts with proteins that mediate recycling of synaptic proteins and is important for the targeting of AMPAR and other PSD proteins to the synapse (Raynaud et al., 2013). This likely contributes to the reduced levels of AMPAR and disrupted LTP in SHANK3 mutant mice (Bozdagi et al., 2010) so that Shankopathies may benefit therapeutically from application of AMPAkines. Drugs that modulate the function of metabotropic glutamate receptors have received special attention because of their role in transducing glutamate directly into pathways critical for synaptic plasticity. Bear et al. (2004) proposed inhibitors of mGluR 5 as potential therapeutics. Studies in FMR1 mutants and other mouse models have borne out this prediction, revealing improvements in synaptic and behavioral parameters (Dolen et al., 2007; Auerbach et al., 2011; Carlson, 2012; Silverman et al., 2012). There have also been some positive results in patients with fragile X syndrome, though these are preliminary, and prompt larger studies with better-defined populations of patients (Jacquemont et al., 2011; Krueger and Bear, 2011). For Shankopathies, it is noteworthy that Shank3 together with Homer can cause mGlurR5 to aggregate at a high density in the membrane and integrate into the larger PSD (Tu et al., 1999). Decreased Shank3 expression results in reduced mGluR5 levels and signaling. A positive allosteric modulator of mGluR5 reversed the signaling and synaptic defects in cell culture (Verpelli et al., 2011). Future work in vivo may validate drugs that positively modulate mGluR5 function for PMS similar to results in TSC (Auerbach et al., 2011). However, the experiments of Auerbach et al. (2011) raise concerns about generalizing too widely results from one monogenic ASD to another. They studied mice mutant in FMR1 or TSC2, both of which have ASD-like behaviors. And though these mice also have defects in mGlurR5 signaling and synaptic protein synthesis they are opposite to one another. In TSC 2 mutants, allosteric activators of mGlurR5 reversed, decreased LTD, decreased synaptic protein synthesis and ASD behavior. In Fmr1 mutants, allosteric inhibitors of mGluR5 reversed the increased LTD, increased synaptic protein synthesis and behavioral deficits. The implications for therapy in humans are substantial since in the clinic the major or only determinant of treatment for ASD may be phenotypic. This would be clearly insufficient in the experimental work noted above (Auerbach et al., 2011). SHANK2 mutant mice also have reduced NMDAR function and deficits in social behavior; both Developmental Neurobiology

improved following application of D-cycloserine, an agonist of NMDAR (Won et al., 2012). NMDAR are decreased in Fmr1 null mice and in MECP2 mutant mice indicating that fragile X syndrome and Rett Syndrome might also benefit from enhancing NMDAR function. Clinical studies have employed amantadine, an NMDA antagonist, or positive allosteric modulators of NMDAR. The results to date are ambiguous with both classes of drugs having had modest effects (King et al., 2001; Niederhofer, 2007). While glutamate synapses have been the major focus of research on ASD, there is increasing interest in GABA (Han et al., 2013), endocannabinoids (Jung et al., 2012; Busquets-Garcia et al., 2013), serotonin (Costa et al., 2012), and other neurotransmitter systems. GABA synapses are major inhibitory synapses in the brain. Reduction in GABA synapse related proteins have been found in brain tissue from patients with ASD (Oblak et al., 2010, 2011). Activating metabotropic GABA receptors in FMR1-null mice with the drug STX209 normalized the high level of AMPA receptor internalization as well as the high level of protein synthesis and spine density (Henderson et al., 2012), suggesting a homeostatic interaction between GABA and glutamate synapses. In FMR1null mice (ionotropic), GABA A receptor subunits are down regulated along with gephyrin in the inhibitory synaptic scaffold (Fig. 1). These changes are correlated with substantial dysfunction of GABA transmission (Paluszkiewicz et al., 2011). In a mouse model of Rett Syndrome, conditional knockout of MECP2 in forebrain GABAergic neurons resulted in reduced amplitude of spontaneous inhibitory currents (quantal size), reduced GABA and its synthesizing enzymes (GAD1, 2), as well as ASD-like behaviors. These ASD-like behaviors overlapped with those seen when MECP2 is knocked out in the entire brain (Chao et al., 2010). Thus forebrain neuron defects appear sufficient to account for the behavioral deficits in this model. These data not only reveal that defects in GABA neurons may contribute directly to ASD, but they also help localize neurons vulnerable to loss of MECP2 mutations (Samaco et al., 2009). Foldy et al. (2013) engineered a mouse to replace the normal NLGN 3 gene (Fig. 1) with a mutant gene (R451C). This mutant knock-in was compared to the knockout of this same gene. Both mice mutants had been previously shown to have ASD-like behaviors (Krueger and Brose, 2013), but in this study there were dramatic differences in the physiology of GABAergic transmission in the two models revealed by studying pyramidal neurons in the hippocampus driven by activation of two different inputs. Their results suggest that the distinct pathways are involved


in these different mutants and harken to human genetic studies where different mutations near SHANK3 were associated with distinct psychiatric disorders, such as schizophrenia vs. ASD (Gauthier et al., 2010), although behavior in the mouse models were similarly perturbed (Foldy et al., 2013). In this same report (Foldy et al., 2013), the authors found increased GABA release in both knockout and R451C mutants. This was due to reduced presynaptic effects of endocannabinoid signaling that normally inhibits GABA release. These data do not prove that perturbed endocannabinoid signaling results in ASD behavior, but together with other findings (Jung et al., 2012; Busquets-Garcia et al., 2013) they support the involvement of endocannabinoid signaling in ASD. Moreover, these detailed cellular and circuit studies reveal the level of complexity that we face in trying to tackle the many genetic disorders that are labeled ASD. Here GABAergic synapses on the same postsynaptic cell may be differentially perturbed in two mutations of the same gene. Since ASD appears to involve the contribution of rare genetic variants (discussed above), this complicates the prospect of finding common pathways for ASD. It also emphasizes the need to define ASD-relevant circuits as well as neurotransmitter systems.

Synaptogenic Molecules in ASD Nerve terminals release more than neurotransmitters. They also release soluble growth factors, such as BDNF and IGF-1 (Nagahara and Tuszynski, 2011), as well as extracellular matrix proteins important for plasticity (Matsumoto-Miyai et al., 2009). The latter include heparan sulfate proteoglycan, which binds to neuregulin a ligand for ErbB4. Neuregulin-ErbB4 proteins modulate spine formation (Krivosheya et al., 2008; Mei and Xiong, 2008) and they have been implicated in ASD (Irie et al., 2012) and other psychiatric disorders (Krivosheya et al., 2008; Mei and Xiong, 2008). Zn12 is also released presynaptically and is important for PSD assembly (Grabrucker, this edition). BDNF is a member of the neurotrophin family of growth factors (i.e., NGF, BDNF, Neurotrophin 3 and 4) and binds to the receptor tyrosine kinase TrkB. BDNF functions at multiple levels from neuronal survival to differentiation, including synapse formation and plasticity (Lu et al., 2013; Park and Poo, 2013) in the human brain (Kleim et al., 2006). The pathways for BDNF in synaptic function and psychiatric disorders (Castren and Castren, 2013) are still emerging. In recent reports, endosomal acidification was shown to interfere with BDNF/TrkB signaling that had been

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implicated in ASD (Ouyang et al., 2013). BDNF/ TrkB also regulates protein translation and, cytoskeletal changes via, CYFIP1, which has been implicated in ASD (De Rubeis et al., 2013). Mutations in the CAPS2 gene are also a risk factor for ASD and mice null for this gene have reduced release of BDNF, reduced dendrite spine density as well as increased anxiety (Sadakata et al., 2012). In another example, Cao et al. (2013) studied a mouse model of Angelman syndrome (mutant in Ube3a). They found that the level of the cytoskeletal accessory protein, Arc, which binds to PSD95 and is important in synaptic plasticity, is abnormally high, and LTP in the hippocampus is compromised, as is learning. Interestingly, TrkB signaling is disrupted in this mouse because it must bind to PSD 95, and excessive levels of Arc inhibit this interaction. With this information in hand, the authors designed a peptide that successfully interfered with the interaction of PSD 95 and Arc. It rescued Trk B signaling and LTP. All of these findings from endosomal acidification to PSD 95 interaction offer new insights into synapse proximal pathways of BDNF signaling that are related to degradation of synaptic proteins (discussed above). A number of potential therapeutics have been developed to target BDNF/TrkB signaling (Lu et al., 2013) that could prove useful for ASD if warranted by preclinical data in mouse models (Scattoni et al., 2013). Indeed, genetically reducing BDNF expression in FMR1-null mice improves hyperactivity and sensorimotor defects, although it also exacerbates learning deficits (Uutela et al., 2012). BDNF application improves synaptic transmission in mouse models of Rett syndrome (Kline et al., 2010). BDNF has been reported to affect assembly of Shank3 into the synaptic scaffold (Iki et al., 2005). A better understanding of BDNF/TrkB and its effects on the Shank life cycle would add important details to our knowledge of BDNF function at synapses. That BDNF is targeted to axons by its interaction with Caps2 (Sadakata et al., 2012) and released from nerve terminals with neural activity is relevant to observations that BDNF can be released with transcranial stimulation (Fritsch et al., 2010) of the motor cortex. Transcranial stimulation is both non-invasive and can activate, somewhat restricted brain regions. This could be coupled with agents that modulate the BDNF pathway, including Caps2, to increase release. The potential for regional activation, though crude, offers some selectivity with regard to therapeutically targeting circuits involved in ASD. IGF1 signals intracellularly via its receptor tyrosine kinase, IGF1R. Its signaling pathways overlap somewhat with BDNF (Yoshii and Constantine-Paton, Developmental Neurobiology

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2007; Banerjee et al., 2012; Deak and Sonntag, 2012). IGF1 shares with BDNF the ability to stimulate synapse formation but, unlike BDNF, IGF1 is permeable to the blood–brain barrier. In Shankopathies the entire structure of the spine is affected (Durand et al., 2012). Shank expression is sufficient to drive dendritic spine maturation and this stimulates nerve terminal differentiation and synaptic function (Sala et al., 2001). In several other models of ASD, synaptic transmission is perturbed and there are also changes in the structure of dendritic spines (Penzes et al., 2011) as well as evidence of spine disruption in humans with ASD (Hutsler and Zhang, 2010). In mouse Shankopathies IGF 1 reversed defects in LTP and behavior (Bozdagi et al., 2013). Also intraperitoneal injection of IGF1 increased dendritic spine density, reversed deficits in synaptic transmission, and improved motor skills in a mouse model of Rett Syndrome (Belichenko et al., 2009). Perhaps the relatively broad effects of IGF 1 and BDNF (Lai and Ip, 2013) on the many molecules that comprise spines make them applicable to a variety of ASD where synapse and spine number are compromised. Indeed, in a recent report Shcheglovitov et al. (2013) found that neurons derived from IPS cells from patients with PMS had significant deficits in glutamatergic vs. GABAergic transmission consistent with the reduced levels of Shank3 expression. The deficits could be significantly reversed by overexpression SHANK3 as well as by treatment of cultures with IGF1. Surprisingly, IGF1 did not increase Shank3 levels but actually reduced them. It did increase expression of PSD95 that is thought to be compensatory. In a pilot study of IGF1, six young girls diagnosed with Rett Syndrome were treated with IGF1 intraperitoneally. There were no reported risks associated with this treatment, setting the stage for a larger trial (Pini et al., 2012). Clinical trials on PMS patients have similarly begun to test the safety and efficacy of IGF-1 treatment (Clinicaltrial.gov NCT01525901). As noted previously, extracellular matrix proteins important for synapse formation are released from nerve terminals with neural activity. A graphic example of this occurs at neuromuscular synapses where the effect of the neurotransmitter acetylcholine on synapses is opposed by the extracellular matrix protein agrin that is released with activity (Misgeld et al., 2005). In this instance, the neural activity produced by acetylcholine disperses developing synapses, while agrin stabilizes them. These opposing effects help localize synapses (Kummer et al., 2006). This may be more than a pointed example, since agrin release from nerve terminals has also been implicated in synaptic plasticity in the hippocampus Developmental Neurobiology

(Matsumoto-Miyai et al., 2009). In another, vivid instance, the protein cerebellin is deposited in the synaptic extracellular matrix to mediate adhesion of pre- and postsynaptic elements and trigger synapse formation onto parallel fibers in the cerebellum (Matsuda et al., 2010; Uemura et al., 2010). Synapse formation in cerebellin-null mice is stalled but can be completed when exogenous cerebellin is supplied, even in adult animals (Ito-Ishida et al., 2008). It is likely that the repertoire of synaptogenic, extracellular matrix molecules will increase. Irie et al. (2012) reported recently that mice deficient in heparan sulfate proteoglycan, an extracellular matrix protein, have behavioral deficits reminiscent of ASD. Heparan sulfate proteoglycan can binds to neuregulin, a ligand for the ErbB4 receptor tyrosine kinases at synapses (Jo et al., 1995). Both neuregulin and Erbb4 have been linked to schizophrenia (Stefansson et al., 2002; Hahn et al., 2006; Bennett et al., 2012). Since the proteins discussed above are all released with neural activity it will be important in the future to differentiate the effects of neurotransmitters from these co-released proteins to help identify new and potentially therapeutic synaptogenic molecules.

Oxytocin and ASD It is instructive to note that IGF 1 and BDNF evolved as potential therapeutics from consideration of their cellular and physiological effects. Oxytocin, on the other hand, was implicated as a possible therapeutic due to its effects on behavior. Oxytocin is released by the hypothalamus and into the bloodstream via the pituitary gland, but it also functions as a neurotransmitter in the brain (Owen et al., 2013). In animal models, oxytocin enhances social interactions by reducing anxiety, and increasing attentiveness to social cues. Transgenic mice lacking oxytocin are aggressive and have difficulty with memory of social interactions, deficits that can be reversed by application of oxytocin (Ferguson et al., 2000). There are now many reports in humans and nonhuman primates (Ebitz et al., 2013) of the positive effects of oxytocin on social interactions and alleviation of social stress (Canitano, 2013; Modi and Young, 2012; Yamasue et al., 2012). Also, genomic studies have associated polymorphisms in the gene for the oxytocin receptor with susceptibility to psychiatric disorders and deficits in social behavior (Campbell et al., 2011; Yamasue et al., 2012; c.f. Tansey et al., 2010). Application of oxytocin makes normal subjects more trusting (Kosfeld et al., 2005), altruistic (De Dreu et al., 2010), and increases their social awareness. Studies on patients with ASD


treated with a single dose of oxytocin report statistically significant but small (Bartz et al., 2011) improvements in social behavior (Hollander et al., 2007; Guastella et al., 2010). Oxytocin administered intranasally in one study improved the ability of ASD patients to recognize emotional responses in others (Guastella et al., 2010; Canitano, 2013). Preliminary data on the distribution of oxytocin receptors in the brain (Loup et al., 1991) make it seem unlikely that oxytocin specifically affects a single behavior, even one as general complex as social behavior. Experimental work has revealed untoward effects of oxytocin on pair bonding and reproductive efficacy in animal models (Bales and Perkeybile, 2012; Bales et al., 2013). In humans, similar negative effects have also been reported (Shamay-Tsoory et al., 2009; De Dreu et al., 2011; Stallen et al., 2012). These drawbacks seem minor compared with deficits in social interactions in some children with ASD. Nevertheless, there is wide agreement that we must better understand the mechanism of oxytocin action at molecular, cellular, and circuit levels (Yamasue et al., 2012). Recent work shows that oxytocin functions in the nucleus accumbens, eliciting LTD in collaboration with serotonin receptors (Dolen et al., 2013). It also acts to increase the signal to noise ratio and improve the fidelity of information transfer in the hippocampus (Owen et al., 2013). As discussed above, this would be predicted to enhance functional connectivity and may mitigate symptoms of ASD (Rubenstein and Merzenich, 2003). At present we know little of how oxytocin might affect synapse structure or function in Shankopathies hence it would be important to extend these and other studies of oxytocin to relevant animal and cellular models of ASD linked to synaptic genes. Many fundamental questions remain, including the precise distribution of oxytocin receptors in the brain, their relative function on the brain vs. peripheral nervous system, and whether and why oxytocin improves social interactions in ASD patients who produce normal amounts of oxytocin. In addition, more needs to be done to establish oxytocin pharmacodynamics (Zhong et al., 2012), method and frequency of treatment (Veening and Olivier, 2013), age of application, as well as better defining behavioral effects on ASD subjects. The dramatic affect of this peptide on behavior relevant to ASD, together with the ease of nasal delivery, has led to a host of clinical trials www.clinicaltrials.com for ASD and other psychiatric disorders. Oxytocin is available as a nasal spray on-line without prescription and it seems has the potential be used ad hoc because of its promise and the lack of better alternatives for ASD. Currently,

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there appears to be caution among ASD families and few reports of self-medication. Nevertheless, it might be wise for public and private agencies to remain attuned to this issue and to monitor reports of any changes in use of oxytocin.

LONG-TERM TARGETS AND GROUP EFFORTS For the foreseeable future, mouse models of ASD will continue to be the mainstay for fundamental and preclinical studies primarily because of our ability to generate mice that harbor mutations similar to those in patients with ASD. There is wide agreement that at the molecular and cellular levels the mouse brain is similar to that of humans. This also obtains to a significant extent in Zebra fish, fruit flies, and round worms (C. elegans), so that the forward genetics and many powerful techniques developed for these models offer a valuable adjunct to the identification of molecular pathways relevant to understanding ASD genes. A good example of this can be taken from studies of Sonenberg and coworkers on the regulation of protein translation by eIF4E (Fig. 1). This work matured from biochemistry, to genetic studies in flies and mice, and eventually in humans to identify a gene involved in human cognition and ASD (Cho et al., 2005; Hershey et al., 2012; Gkogkas et al., 2013; Sidrauski et al., 2013). When we have even a modest understanding of the relationship of brain connectivity to behavior in mice we will have learned a good deal about the human brain. We are in the midst of a revolution in working toward that goal (Alivisatos et al., 2013b). The development of groundbreaking methods to: (a) histologically map connections in an entire brain (Cai et al., 2013; Chung and Deisseroth, 2013; Morgan and Lichtman, 2013); (b) functionally map circuits “optogenetically” by activating or inactivating subsets of neurons (Reiner et al., 2013), (c) visualize synapses and other structures in live brains at high resolution by two photon microscopy (Trachtenberg et al., 2002), and (d) the ability to record neural activity simultaneously from many neurons in live brains (Muller et al., 2009; Alivisatos et al., 2013a) offers an unprecedented repertoire of new methods for elucidating connectivity. The histological methods have been developed in animal models and will require scaling for application to postmortem human brains. This will complement ongoing work on assembling structural maps of brain connectivity in humans (Amunts et al., 2013). Correlating brain maps with functional, genomic, and patient data will not only be Developmental Neurobiology

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critical for understanding ASD, but will generate deep insights that will allow us to understand how distinct psychiatric disorders such as ASD and schizophrenia have common genetic roots (Smoller et al., 2013) and many other issues. There are many ASD-associated genes (Aldinger et al., 2011), and for Shankopathies we have a number of mouse models (Jiang and Ehlers, 2013). Study of the many additional mouse models that will become available for ASD will require highly integrated efforts by multiple labs. Coordination of research among the various groups working in this area would significantly accelerate progress while conserving increasingly scarce funds. Among those labs generating mouse models it will be important to have free exchange of the various lines for confirmation of published results and to identify particularly useful models. Jiang and Ehlers (2013) discuss several apparent discrepancies in mouse models of Shankopathies that will be resolved only following exchange among labs. Others (Landis et al., 2012) have discussed this in some detail and outlined criteria for improving preclinical research to maximize chances of its translation into the clinic. Current attempts to guarantee sharing of animal models by research journals are not always effective and it continues to be a high priority that funding agencies rigorously enforce this policy. Also there has been discussion of standards for development and study of genetic models that will help reduce the incidence of discrepancies (Karayiorgou et al., 2012). For Shankopathies it would also be helpful to have a common understanding of which SHANK mutations and lines are of proximate interest, how many lines should be generated, on what genetic backgrounds, etc. (Karayiorgou et al., 2012), as well as standards for physiological and behavioral assays. Increasingly clever assays (Silverman et al., 2010) have revealed insights into complex mouse behavior so that articles in the press sometimes refer to “empathy” in mice. Despite this, there has been much discussion of the relevance of mouse behavior to psychiatric disorders, potential pitfalls, and criteria for carrying out behavioral studies (e.g. Silverman et al., 2010). It would be highly desirable to be able to study animals with closer genetic and behavioral proximity to humans. There is a history of neurophysiological and behavioral research on non-human primates, though they have not been introduced extensively into psychiatric studies. This may be changing. Recent work describes the striatum as a locus of social interactions in macaques (Klein and Platt, 2013). Research on monkeys (macaques) typically involves few animals in deference to concerns about using such advanced species, as well as their high Developmental Neurobiology

cost. Nevertheless, methods are available for genetic engineering of macaques that have been applied to research on neurological disease (Yang et al., 2008). Marmosets (new world monkeys), breed more rapidly than macaques, are easier to maintain, and less costly. They also can be engineered to express transgenes (Sasaki et al., 2009). Marmosets are emerging as models for research on social communication relevant to ASD and, in this regard, far outstrip the meager behavior of mice (Schneider et al., 2012a,b). It’s likely that broadening research to non-human primates would face significant societal objection. It would be worthwhile to begin a discussion of their value and the potential for humane approaches especially in non-invasive studies of behavior, genetics, etc. Parr et al. (2013) illustrate the value of research on non-human primates in parametric studies of oxytocin affects on behavior, and their potential to significantly improve our confidence in moving from preclinical to clinical studies of ASD therapies. Primary cultures of neurons have proven valuable models for identifying synaptic and signaling pathways that are relevant to ASD (discussed above). IPS cells (Farra et al., 2012; Takahashi and Yamanaka, 2013) derived from ASD patients (Dolmetsch and Geschwind, 2011) are emerging as a major advance in attempt to expand these efforts (Pasca et al., 2011). IPS cells can be artificially derived from a nonpluripotent cell, such as a fibroblast by forced expression of specific genes (Kondo et al., 2013; Takahashi and Yamanaka, 2013; Zhang et al., 2013). In principle, stem cells can differentiate into any cell type, and there are protocols tailored to neurons, and glia. They have the advantage of harboring the disease causing mutation found in patients, along with the full genetic background necessary for emergence of disease. They also allow study of disease-causing deficits without first identifying the mutation and then creating a mouse model and, as such, have the potential to be an invaluable complement to mouse models of ASD. In IPS cells, one can detail perturbations in autonomous aspects of cell differentiation, intracellular signaling, as well as non-autonomous functions such as synapse formation and cell adhesion. IPS cells are particularly well-suited for studies of highly penetrant mutations such as Shankopathies (Kim et al., 2012) which allow one to rescue the cellular or biochemical deficits to confirm that mutations in SHANK are responsible for the deficits under study in the distantly derived IPS cell lines. To cure Shankopathies it may be necessary to reintroduce expression of SHANK3 in the brain at normal levels, at the correct time in development, and in the circuits affected. Expression levels are critical, and a recent report


(Shcheglovitov et al., 2013) illustrates the potential of IPS cells for study of Shankopathies. It should be high priority to develop IPS cells for high-throughput screening of small molecules, cDNAs, etc, that increase SHANK expression. There are a number of libraries of small molecules, and even drugs that have been clinically approved, that could be tested for effects on SHANK3 expression. Beyond this, IPS cells would be valuable in determining whether candidate drugs mitigate synaptic defects (e.g., spine formation, synaptic transmission) that can be assayed in culture and then tested further in mouse models of Shankopathies. In this regard it is noteworthy that for neurological disorders mouse models have been rather disappointing, and approaches that lead to significant amelioration in Alzheimer’s and other disorders mice have largely proven ineffective in clinical trials (Malenka and Malinow, 2011). This may be even more problematic in mouse models of ASD where behavioral assays are a key aspect of the evaluation. It’s plausible that preclinical outcomes could be improved by correlating mouse studies with molecular and cellular studies on IPS cells. It is now possible to produce large numbers of neurons from human IPS cells which, until recently, was an impediment to biochemistry and large scale efforts at drug screening (Zhang et al., 2013). There remain complications resulting from the fact that neurons from different cell lines differentiated by identical methods may have very different characteristics (Wu et al., 2007) and those characteristics may be unstable with time in culture (Mekhoubad et al., 2012). Some encouraging preliminary data suggest methods to help with problems of variability among different IPS lines (Zhang et al., 2013). There is a currently a large, well funded effort in Europe to establish banks of undifferentiated IPS cells. These include an industrial/academic consortium led by Roche (StemBANCC), and the European Innovative Medicine Initiative. The sources and amounts of this funding reflect the fact that granting agencies consider development of these procedures as “precompetitive research,” but that their potential for high-throughput drug screening and disease research makes support compelling. The NIMH is establishing a bank of blood and fibroblasts from patients with PMS and is generating IPS cells (Stem Cell Center NIMH). Supporting this and other banks is a high priority.

CONCLUSIONS It is our hope that this brief overview and blueprint will be helpful to members of the ASD community

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both as an informational tool, to understand currents in research, as well as in obtaining a perspective on future directions. We have discussed research on genetic, cellular, and molecular aspects of ASD, with an emphasis on Shankopathies. Current research suggests that there are a subset of ASD, including PMS, that devolve from mutations in genes encoding proteins that function in synaptic transmission. This is likely to expand to include more genes and more forms of ASD, and extend to other mental disorders. Not all genetic forms of ASD will relate back to primary defects in synaptic function, but this focus offers an important entree into these heterogeneous disorders that could motivate future iterations on other ASD pathways. A recent document discussing preclinical studies for Rett Syndrome (Katz et al., 2012) has a similar purpose of bringing coherence, driven by the internal logic of current research, toward future goals and standards of research. Readers may not share the priorities proposed here (Table 1) and they are welcome to extend the discussion publically. There are many important topics that we have not discussed, from accurate diagnosis of ASD, to the state of brain imaging, to the lack of biomarkers for ASD. We also need to know what families and patients with ASD see as their priorities short of a complete cure. Is the epilepsy that often accompanies some forms of ASD as much of a difficulty as the core symptoms and how successfully is it treated by current drugs? Brain science is on the cusp of major breakthroughs and calls for large, collective efforts are being implemented worldwide (Insel et al., 2004). These include collaborative efforts at high resolution mapping of the structure of the human brain, as well as neural circuits in mice. Consortia in Europe and North America have launched initiatives such as the Brain Activity Map, Blue Brain, and The Human Connectome Project. Other efforts will exploit recently developed methods for direct 3-dimensional imaging of brains as well as advances in functional imaging of human brains by fMRI and PET. They will also expand valuable databases such as the Allen Brain Map and include databases that share genomic data (GENMANIA, DAVID, and for ASD, SFARI and AUTISMKB) with annotation of genes into presumptive pathways. This is big science and group efforts are absolutely necessary. There are, however, systemic constraints to deep collaboration. Research is both highly competitive and individualistic. Scientists may be loath to participate in a project if credit is widely shared. Granting agencies and academic institutions often give little recognition of “supporting” researchers, even for high-profile Developmental Neurobiology

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publications. While it is clear that this individualism generates much energy and creativity there is a dire need for the synergy of team efforts in brain science. The teams should form as they are needed to tackle a problem and remain fluid. This would require institutional recognition of contributions made by all, strict criteria for authorship, and more transparent description of individual contributions. In genomics, these constraints seem less severe, as group efforts are relatively common. It should be possible in other areas as well. A major concern in the ASD community is the need to reverse the decline in research on psychiatric disorders by the pharmaceutical industry. It is highly unlikely that there will be much progress made on treatments for ASD by modification of existing compounds. On the other hand, the free market is averse to high-risk endeavors that may require a different business model. The European Union Innovative Medicine Initiative launched in 2012 is a collaboration between government and large pharmaceutical firms to help fill this need. We need more initiatives of this sort because the answers we are seeking will be transformational for understanding psychiatric disorders and the human mind. Author thanks Ms. Geraldine Bliss of the Phelan McDermid Syndrome Foundation for organizing the meeting that prompted this review and colleagues and the families who attended and from whom the author learned much about PMS. Author also thanks Drs. Peter Carbonetto and YongHui Jiang for their helpful comments on this manuscript.

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