Accepted Manuscript

Dendritic spine in autism spectrum disorder

Merja Joensuu, Vanessa Lanoue, Pirta Hotulainen

PII: S0278-5846(17)30414-1 DOI: doi: 10.1016/j.pnpbp.2017.08.023 Reference: PNP 9212 To appear in: Progress in Neuropsychopharmacology & Biological Psychiatry Received date: 31 May 2017 Revised date: 21 August 2017 Accepted date: 30 August 2017

Please cite this article as: Merja Joensuu, Vanessa Lanoue, Pirta Hotulainen , Dendritic spine actin cytoskeleton in autism spectrum disorder. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Pnp(2017), doi: 10.1016/j.pnpbp.2017.08.023

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Dendritic spine actin cytoskeleton in autism spectrum disorder Merja Joensuu1,2,3, Vanessa Lanoue2,3, Pirta Hotulainen1,*

1 Minerva Foundation Institute for Medical Research, 00290 Helsinki, Finland 2 Clem Jones Centre for Ageing Dementia Research, The University of Queensland, Brisbane, Queensland 4072, Australia 3 Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia * Correspondence should be addressed to P.H (email: [email protected], address: BIOMEDICUM Helsinki 2U, Tukholmankatu 8, 00290 HELSINKI, Finland)

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Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses. Changes in the shape and size of dendritic spines correlate with the functional changes in excitatory synapses and are heavily dependent on the remodeling of the underlying actin cytoskeleton. Recent evidence implicates synapses at dendritic spines as important substrates of pathogenesis in neuropsychiatric disorders, including autism spectrum disorder (ASD). Although synaptic perturbations are not the only alterations relevant for these diseases, understanding the molecular underpinnings of the spine and synapse pathology may provide insight into their etiologies and could reveal new drug targets. In this review, we will discuss recent findings of defective actin regulation in dendritic spines associated with ASD.

Keywords: Actin dynamics, Rho GTPases, dendritic spines, postsynapse Abbreviations: Abi-1, Abelson interacting 1; Abp1, actin binding protein 1; ACTN4, α--4; ADF, actin- depolymerizing factor; ADHD, attention deficit/hyperactivity disorder; ADNP, activity-dependent neuroprotective protein; ANK, ; ARHGEF9/PEM2, RhoGTPase exchange factor 9/posterior end mark 2; ASD, autism spectrum disorder; Atf3, stress-induced transcription factor-3; BAR, Bin1/amphiphysin/Rvs167; CaMKII, calcium-calmodulin kinase II; CH1/2, calponin homology; CNS, central nervous system; CNV, copy-number variants; CSWSS, continuous spike wave in slow-wave sleep; CTTNBP2, cortactin-binding protein 2; CYFIP1, Cytoplasmic FMRP-interacting protein 1; Dbl, diffuse B-cell lymphoma; DH, Dbl homology; DIAPH3, diaphanous homolog 3, DISC1, disrupted in schizophrenia; DLGAP2, Disk large-associated protein 2; DMD, ; Dp427, full-length dystrophin containing actin binding domain, E, embryonic day; FDR, false discovery rate; FMRP, fragile X mental retardation protein; FRAP, fluorescence recovery after photobleaching; FXS, fragile X syndrome; GAP, GTPase activating protein; GEF, guanine-exchange factor; GRAF, GTPase regulator associated with focal adhesion kinase; GSN, ; IRSp53/BAIAP2, insulin receptor substrate p53/Brain-specific angiogenesis inhibitor 1-associated protein; Kal7, kalirin-7; LGD, likely -disrupting; LKS, Landau- Kleffner syndrome; LTP, long-term potentiation; MAPK, Mitogen-activated protein kinases; mDia2, mammalian Diaphanous-related formin 2; mEPSC, miniature excitatory postsynaptic currents; mGluR, metabotropic glut amate receptors; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; MYH4/MyHC-IIb, heavy chain IIb; MYO16, Myosin XVI; MYO9B, myosin IXb; NL, Neuroligin; NMD, Nonsense-mediated mRNA decay; NMDA(R), N-methyl-D-aspartate (receptor); NMJ, neuromuscular junction; OPHN1/ARHGAP41, Oligophrenin1/RhoGTPase activating protein 41; PAK, p21-activated kinase; PH, pleckstrin homology; PI3K, phosphatidylinositol 3-kinase; PSD, postsynaptic density; Rac1, Ras-related C3 botulinum toxin substrate 1; ROCK, Rho-associated coiled-coil containing protein kinase; SAM, sterile alpha motif; SAPAP, PSD-95-associated protein; SH3, Src homology 3; SHANK3, SH3 and multiple ankyrin repeat domains; sLTP, structural LTP; SR1-4, repeats; SRA1, specific Rac1-activated; STRN, striatin; STXBP5, syntaxin binding protein 5; SynGAP1, synaptic GTPase activating protein 1; SV, ; TADA, transmission and de novo association; TCGAP, TC10β/Cdc42-GAP; TSC, tuberous sclerosis complex; WRC, WAVE regulatory complexACCEPTED; Zn, zinc; α-Pix/Cool2, α-Pak interactiveMANUSCRIPT exchange factor/Cloned out of library 2.

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1. Synaptic deficiency in autism spectrum disorder

Autism spectrum disorder (ASD) comprises a range of neurological conditions that affect the ability of individuals to communicate and interact with others. ASD is characterized by impaired social interactions, communication deficits, and repetitive behaviors. ASD is typically diagnosed during the first 3 years of life, a period of extensive neurite formation, synaptogenesis and refinement1-4. Family and twin studies have revealed that ASD has a strong genetic component, with numerous being affected. In addition to mutations inherited from parents, many ASD- associated mutations are rare protein disrupting de novo mutations that have arisen in the germline. Mutations can be copy-number variants (CNVs) or single-base-pair mutations5. By definition, CNVs are deleted or duplicated segments of DNA (>1,000 basepairs) that are thought to be involved in the pathogenesis of a wide range of human diseases, including ASD. At least six recurrent CNVs are among the most frequently identified genetic contributors to ASD6. Overall, there seems to be an enrichment in ‘likely gene-disrupting’ mutations (LGDs; nonsense, frameshift and splice site mutations that often result in the production of truncated ) in individuals with ASD as compared to their healthy relatives or to other unaffected individuals5. Multiple ASD susceptibility genes converge on cellular pathways that intersect at the postsynaptic site of glutamatergic synapses7,8, the development and maturation of synaptic contacts9 or synaptic transmission10. The majority of excitatory glutamatergic synapses are located in small dendritic protrusions known as spines. The formation, maturation and elimination of dendritic spines lie at the core of synaptic transmission and memory formation11,12. Many of the ASD risk genes encode synaptic scaffolding proteins, receptors, cell adhesion molecules or proteins that control actin cytoskeleton dynamics, all of which directly affect synaptic strength and number and, ultimately, neuronal connectivity in the brain. In addition, ASD risk genes encode proteins that are involved in chromatin remodeling, transcription, and protein synthesis or degradation, all of which can act as upstream controllers of the expression levels of synaptic proteins.ACCEPTED Changes in any of MANUSCRIPT these proteins can affect dendritic spine density in the brain. When deleterious mutations occur, an individual’s risk for ASD is further affected by how well the defects of a single mutation can be compensated in the brain7. Increased spine density has been observed in the frontal, temporal, and parietal lobes of human ASD brains13 and recent studies indicate a defect in dendritic spine pruning from 13-18 years of age14. Tang et al. (2014) proposed that this pruning deficit may contribute to abnormalities in the cognitive functions that humans acquire in their late childhood, teenage, or early adult years, including the acquisition of executive skills such as reasoning, motivation, judgment, language,

ACCEPTED MANUSCRIPT and abstract thinking15. Many children diagnosed with ASD reach adolescence and adulthood with functional disability in these skills, in addition to social and communication deficits16. Although human ASD studies have reported increased dendritic spine density13,14, only a few genetic ASD animal models display this effect, making it unclear whether the increased spine density causes ASD-like behavior or whether any change in the number or function of synapse leads to atypical brain connectivity and symptoms of ASD7. One of the mouse models that does mimic the spine-pruning defect seen in humans has a constitutively overactive mammalian target of rapamycin (mTOR)14. In this model, the mechanism behind the defective spine pruning is an autophagy deficiency in synapses. Autophagy is an evolutionarily conserved cellular process that provides nutrients during starvation and eliminates defective proteins and organelles via lysosomal degradation. Hyperactive mTOR inhibits autophagy at an early step in autophagosome formation17. Inhibition of neuronal autophagy produces ASD-like inhibition of normal developmental spine depletion, without affecting the rate of spine formation, leading to increased spine density and ASD-like behaviors14. This model offers one mechanism underlying increased spine density and ASD-type behavior, but considering the heterogeneity of ASD genetics, it is likely that other mechanisms affecting spine pruning are defective in different ASD patients. Interestingly, it has been reported that mutations of five autism-risk genes with diversified molecular functions all lead to a similar ASD phenotype of behavioral inflexibility, indicated by impaired reversal-learning in Drosophila18. These reversal-learning defects result from an inability to activate Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent forgetting18. In mammalian neurons, Rac1 affects spine structure, regulates synaptic plasticity in the hippocampus and is required for proper hippocampus-dependent spatial learning19,20. Active Rac1, when targeted to potentiated spines, is able to deplete targeted synapses21. Rac1 is one of the main regulators of the actin cytoskeleton and, therefore, one of the possible mechanisms underlying the defective spine pruning is deficient regulation of the synaptic actin cytoskeleton. It is also possible that spine pruning is normal, but spine formation is overactive,ACCEPTED leading to increased MANUSCRIPT spine density. Although ASD is considered one of the most heritable neurodevelopmental disorders22,23 and majority of the research on ASD has focused on the genetics of the disorders24,25, single causative gene anomalies account for only a small proportion of ASD cases26,27. To date, inheritance of multiple gene variants, rare de novo single gene mutations and copy number variants have been proposed to be liable for ASD. In addition, a multitude of environmental risk factors have been proposed to contribute to the development of ASD26. Prenatal environmental conditions, such as maternal infections during pregnancy, have been linked to social communication difficulties in

ACCEPTED MANUSCRIPT children with ASD-associated CNVs28. Other environmental factors that have been proposed to interact with risk genes include preterm birth, folate deficiency, and exposure to certain toxins28. One of these risk factors is lead exposure, which can cause dose-dependent changes in DNA methylation29, and among the hypomethylated genes caused by lead exposure, some affect actin cytoskeleton30. Genes associated with autism are listed at publicly accessible websites SFARI Gene (https://gene.sfari.org/database/human-gene/) and AutismKB (http://autismkb.cbi.pku.edu.cn/ index.php). In this review, we evaluate the current literature from SFARI listed genes, which encode proteins that regulate actin dynamics and have been linked with autism (Table I and II). The review aims to discuss the current literature to unravel whether the actin regulators encoded by ASD-associated genes regulate synaptic structure and function, and if yes, how? Additionally, the review aims to identify any common pathways on synaptic or actin regulation among these ASD-associated actin regulators.

2. The role of actin cytoskeleton and actin binding proteins on the imbalanced spine formation and plasticity in ASD

Although a wide range of autism-associated genes has been identified, it is becoming increasingly evident that many of these genes converge into common cellular pathways associated with neurite outgrowth, synaptogenesis, synaptic plasticity and spine stability, which together regulate the structural stability of neurons. Actin is the building block of cells and regulates the shape and density of dendritic spines31. Perturbing actin cytoskeleton and its regulators can lead to an imbalance in cytoskeleton dynamics, which in turn, can cause alterations in dendritic spine size, shape and number, as well as neural arborization. The correct morphology of synapses is key to the formation of functional neuronal circuitry and deficits in the structural stability of dendriticACCEPTED spines have been reported MANUSCRIPT in several neurological disorders, including fragile X syndrome (FXS)32, schizophrenia33,34 and autism35.

2.1 Regulation of F-actin polymerization and depolymerization

The dynamic remodeling of the actin cytoskeleton is a critical part of most cellular activities, and the transition between two forms of actin, monomeric globular (G)-actin and filamentous (F)- actin, is tightly regulated in time and space by a large number of signaling, scaffolding and actin-

ACCEPTED MANUSCRIPT binding proteins. Most of these proteins integrate actin binding, protein-protein interaction, membrane binding, and signaling domains. In response to extracellular signals, often mediated by Rho family GTPases, actin-binding proteins control the cellular actin cytoskeleton by regulating actin filament nucleation, elongation (=polymerization) and treadmilling, maintenance, severing, capping, and depolymerization.

Cortactin-binding protein 2 (CTTNBP2) is highly expressed in dendritic spines where it locally interacts with proteins to control the formation and maintenance of the spines. The dendritic spine head typically contains a dense network of short, cross-linked, branched F-actin31, and CTTNBP2 has been shown to regulate the mobility and distribution of cortactin which, in turn, controls dendritic spine morphogenesis via F-actin branching and stabilization36-38 (Figure 1). Cortactin expression supression39 and CTTNBP2 knockdown in neurons37 reduce the spine density39, indicating their importance for the formation and maintenance of the spines. Cortactin acts downstream of CTTNBP2 in spine morphogenesis, as the defects caused by CTTNBP2 knockdown can be rescued by cortactin overexpression but not by overexpressing a CTTNBP2 mutant protein that cannot bind cortactin37. Furthermore, CTTNBP2 oligomerization has been shown to promote dendritic arborization through bundling40. Several mutations of CTTNBP2 gene have been reported in ASD cases41-43, indicating the importance of correct dendritic spine structure and density for proper brain function. In addition, CTTNBP2 is also found to regulate the synaptic distribution of striatin 4 (STRN4) and zinedin, which are the regulatory B subunits of protein phosphatase 2A (PP2A), suggesting that CTTNBP2 targets the PP2A complex to dendritic spines, and together with cortactin regulate spine morphogenesis and synaptic signaling36,44. The involvement of these proteins in autism has been speculated45, and in support of this, the human STRN4 gene is located in 19q13.32, which is the for a reported deletion in ASD46.

Cytoplasmic FMRPACCEPTED-interacting protein 1MANUSCRIPT (CYFIP1) engages in two distinct molecular complexes where it coordinates mRNA translation and actin cytoskeleton remodeling to ensure correct dendritic spine formation47. CYFIP1, also known as specific Rac1-activated (SRA1) protein48, interacts with fragile X mental retardation protein (FMRP)49-53, which regulates the dendritic targeting of mRNAs54 and controls mRNA decay and protein synthesis in the neuronal soma and at spines50. FMRP tethers specific mRNAs to CYFIP1, which then sequesters the cap- binding protein eIF4E and mediates translational repression55 (Figure 1). Translation of target mRNAs, such as Arc/Arg3.1 which are important for synaptic structure and function47, is

ACCEPTED MANUSCRIPT triggered when group I metabotropic glutamate receptors (mGluRs) or the brain-derived neurotrophic factor (BDNF)/NT-3 growth factor receptor (TrkB) are activated and CYFIP1 is released from eIF4E and shifted to the WAVE regulatory complex (WRC)55. The equilibrium of the two complexes is controlled by small GTPase Rac1, which alters the conformation of CYFIP147,48,53. The heteropentameric WRC, consisting of the proteins WAVE1/2/3, ABI1/2, NCKAP1, HPSC300 and CYFIP1 or CYFIP2, is one of the critical modulators of actin filament reorganization in neurons56. The WRC triggers N-WASP-dependent activation of actin-related protein (Arp)-2/3-dependent actin nucleation57,58, which controls actin filament rearrangements connected to dendritic spine formation, retraction, motility, structure and stability59. Disturbing WRC function by genetic ablation of the WRC components therefore alters dendritic spine structure and affects excitability60-62. CYFIP1 is highly enriched at synapses and its expression level is important for correct dendritic arborization and neuronal morphological complexity. Overexpression of CYFIP1 in hippocampal neurons in vitro has been shown to cause altered dendritic spine morphology and lead to an increase in dendritic length and complexity63. Neurons derived from Cyfip1+/− animals, on the other hand, show an increased mobility of F-actin, reduced dendritic complexity and an increase in immature dendritic spines both in vitro and in vivo63. Cyfip1 and Cyfip2 are members of a highly conserved gene family52, and dysregulation of Cyfip1 expression levels leads to pathological changes in neuronal maturation and connectivity, both of which may contribute to the development of the neurological symptoms observed in ASD and schizophrenia63. A CNV in the 15q11.2 region of the has been implicated in several neurological and neuropsychiatric conditions64-70 and a CYFIP1 CNV within 15q11.2 has been linked to ASD71, with an upregulation of CYFIP1 mRNA being demonstrated in genome- wide expression profiling of ASD patients with 15q11–q13 duplication72. A rare CYFIP1 deletion has also been reported in a patient with a mild intellectual disability and a pervasive developmental disorder not otherwise specified71. CYFIP1 therefore represents a link between Rac1, the WAVE complex and FMRP, orchestrating two molecular pathways, translational control and actin ACCEPTED polymerization, both of whichMANUSCRIPT are necessary for correct spinogenesis in neurons. ASD shares common brain circuit alterations and brain abnormalities, such as defects in synaptic transmission and dendritic spine morphology with intellectual disability e.g. FXS. In contrast to the wide heterogeneity of autism-associated genes, FXS is caused by the silencing of FMR-1 that codes for FMRP73. Although most cases of autism are not associated with FXS, the prevalence of autism in FXS is estimated to range from 5% to 60%74. FXS is the most common form of inherited intellectual disability, with the patients displaying dendritic spine defects75,

ACCEPTED MANUSCRIPT neurodevelopmental delay, and autistic-like characteristics76. FXS is a frequent monogenic cause of ASD74,76-78 and the behavioral overlap between autism and FXS patients suggests overlapping neuronal network mechanisms that increase susceptibility to ASD. The regulatory function of CYFIP1 in the context of FMRP- and WAVE-containing complexes and subsequent actin remodeling may explain the convergence of these two neurodevelopmental disorders.

The diaphanous homolog 3 (DIAPH3) gene encodes the actin polymerization factor formin DRF3 (called mDia2 in mice). The formin family of proteins are known to polymerize straight, non-branched actin filaments79 (Figure 1). The small GTPase Rif, and its downstream effector mDia2, play a central role in regulating actin dynamics during dendritic filopodia elongation80. Depletion of mDia2 in cultured neurons resulted in shorter dendritic protrusions whereas over- expression of active mDia2 led to long dendritic filopodia80. In addition to the regulation of spine morphogenesis, mDia2/DFR3 has a significant role in neurite extension81. A maternally inherited deletion and a paternally inherited functional single-nucleotide mutation in a highly-conserved nucleotide sequence of DIAPH3 have been reported in a normally intelligent patient with an autistic disorder, suggesting a homozygous loss of function in the DRF382. Rare genomic alterations in DIAPH3 have also been found in rare epileptic encephalopathies such as the continuous spike and waves during slow-wave sleep (CSWSS) syndrome and Landau-Kleffner syndrome (LKS)83. CSWSS and LKS patients can display behavioral manifestations that overlap those of ASD, implying a possible clinical link between the disorders83.

Gelsolin (GSN) is a Ca2+-sensitive actin binding protein that is widely expressed throughout the adult mammalian brain. Gelsolin severs F-actin filaments by breaking the noncovalent bonds between actin monomers, and remains bound to the barbed ends of the filaments, inhibiting their extension84,85 (Figure 1). Dendritic spines are motile structures that contain high concentrations of filamentous actin,ACCEPTED and based on a fluorescence MANUSCRIPT recovery after photobleaching (FRAP) experiment in hippocampal neurons, the great majority (around 85%) of the F-actin in the spines is dynamic86. The induction of long-term depression (LTD) has been shown to decrease the amount of dynamic actin in the spine, increasing the portion of stable F-actin86, the effect of which is impaired in Gsn-deficient mice86. This actin stabilization was shown to depend on Ca2+ influx86 and N-methyl-D-aspartate (NMDA) receptors86, which have previously been implicated in actin-signaling pathways in spines87. Gelsolin has been shown to have a role in neurogenesis: In the adult mammalian brain, neurons are continually generated in two distinct brain regions, the

ACCEPTED MANUSCRIPT dentate gyrus, where newly generated cells remain relatively static88, and the olfactory bulb, where migratory neuroblasts differentiate into interneurons89. The migration of newly generated neuroblasts into the olfactory bulb has been shown to decrease in Gsn−/− mice while the neurogenesis in the hippocampus increases90. Moreover, neural progenitor cells derived from Gsn−/− mice show reduced migratory capacity in vitro whereas neuronal differentiation and proliferation kinetics are not affected90. A de novo substitution variant in an intron of the GSN gene has been identified in a proband with Asperger syndrome from a deep resequencing dataset generated from a cohort of ASD and schizophrenia cases91. Further support on the involvement of gelsolin and dendritic spine morphology on ASD comes from a study on a neurological disorder called tuberous sclerosis complex (TSC). 30%–50% of TSC patients are affected by ASD92-95. The stress-induced activating transcription factor-3 (Atf3)-gelsolin cascade, following mTOR hyperactivation, was shown to underlie dendritic spine deficits in mice with loss-of-function mutations in tuberous sclerosis complex 1 (Tsc1) or Tsc2 genes96.

2.2 Actin cross-linking proteins

When a spine matures, a pool of F-actin is stabilized to form a relatively stable core97. This core actin is a result the action of F-actin cross-linking proteins, which connect the actin filaments together into a thicker actin bundle. These bundles can be either contractile or non-contractile, depending on whether they contain active myosin II. In addition to creating bundles, actin cross- linkers often protect filaments from depolymerization or at least slow down the depolymerization rate. The actin cross-linkers which have been shown to play a role in morphological dendritic spine maturation are non-muscle myosin IIb97-99, calcium calmodulin kinase II (CaMKII100, drebrin101-103, α-actinin-2104 and α-actinin-4105. In addition to these functions, actin cross-linkers often provide a scaffolding platform for protein interaction and many of them link synaptic receptors to the actinACCEPTED cytoskeleton. From these MANUSCRIPT proteins, de novo mutations in α-actinin-4 and myosin IIb, have been reported in ASD106. However, myosin IIb is not listed in SFARI and is therefore excluded from this review.

Alpha(α)-Actinin-4 (ACTN4) is a member of the evolutionarily conserved family of α- (α-actinin-1-4)107. All isoforms, except α-actinin-3, are found in the brain108, with α-actinin-4 being expressed in the hippocampus, cortex, and cerebellum105. The full-length human α-actinin- 4 protein is divided into three functional subdomains: The N-terminal actin-binding domain,

ACCEPTED MANUSCRIPT which is composed of two calponin homology domains (CH1 and CH2), the C-terminal EF-hands (helix-loop-helix structural motif), and the central rod domain, which is composed of four spectrin repeats (SR1-SR4)109,110. Antiparallel dimerization of the molecules results in the formation of a dimer with an actin-binding domain located at both ends. Dimer formation is required for its normal function as a cross-linker of the actin cytoskeleton111 (Figure 2). In addition to binding to actin filaments, actinins interact with membrane receptors112-115, signaling and adhesion proteins, and phosphoinositides116, and their positioning at the interface between the plasma membrane and cortical actin meshwork may afford a functional link between receptor activation and modification of the actin cytoskeleton. α-Actinin-4 is enriched at excitatory synapses, it colocalizes with group 1 metabotropic glutamate receptors (mGluRs)105 and it binds CaMKII117. It regulates dendritic protrusion dynamics and morphogenesis and is required for the mGluR-induced dynamic remodeling of dendritic protrusions105. Activation of group 1 mGluRs leads to CaMKII phosphorylation and weakening of the α-actinin-4/CaMKII interaction. However, synaptic transmission is not significantly affected by down-regulation of α-actinin-4, indicating that synapses are still formed normally105. The ACTN4 gene was originally identified as an ASD candidate gene based on its enrichment in an autism-associated protein interaction module (=synaptic transmission); sequencing of post- mortem brain tissue from 25 ASD cases resulted in the identification of significant non- synonymous variants in this gene10. Furthermore, a de novo missense mutation in the ACTN4 gene was found by whole exome sequencing118. Iossifov and colleagues (2014) found a missense M554V (methionine to valine) point mutation in SR3. Spectrin repeats form a platform for protein-protein interactions but experimental analysis is needed to validate the effect of this mutation.

CaMKII (CAMK2A) and CaMKII are central regulators of synaptic plasticity. Compared to CaMKII, CaMKII localizes poorly to dendritic spines119, and it was thought that it did not bind actin but was recruitedACCEPTED to spines through oligomerization MANUSCRIPT with CaMKII100. However, another study used single-molecule tracking to show that CamKII does in fact bind to actin, although this binding was threefold weaker than that observed for CaMKIIβ120. A de novo missense variant (E183V, glutamate to valine) in the CAMK2A gene was identified in an ASD proband from the Simons Simplex Collection118. Additional functional characterization demonstrated that this de novo E183V mutation in the CaMKII catalytic domain decreases both CaMKII substrate phosphorylation and regulatory autophosphorylation on Thr286, and that the mutated kinase acts in a dominant-negative manner to reduce CaMKII-wildtype

ACCEPTED MANUSCRIPT autophosphorylation121. The mutation also reduced the protein stability and the number of dendritic spines, induced synaptic deficits and caused ASD-related behavioral alterations in mice121. Furthermore, E183V mutation reduced CaMKII binding to established ASD-linked proteins, such as Shank3 and subunits of L-type calcium channels and NMDA receptors, and increased CaMKII turnover in intact cells121. This example demonstrates how a seemingly non- damaging single amino acid change can lead to multiple defects in protein function and several cellular and behavioral defects.

The VIL 1 gene encodes a Villin 1 protein. Villins are Ca2+-regulated actin-binding proteins, which function in the cytoplasm by severing, capping, nucleating, and bundling actin filaments122 (Figure 2). Villins are best known for their role in the assembly and maintenance of microvilli in polarized epithelial cells. Villin also has a regulated nuclear localization and its function in the nucleus may play an important role in tissue homeostasis. The VIL1 gene was identified in an ASD whole-exome sequencing study and subsequent TADA (transmission and de novo association) analysis as a gene strongly enriched for variants likely to affect ASD risk43. However, the expression and function of Villin 1 in neurons is currently unknown.

2.3 Actin-binding myosin motors

Myosins are a large family of ATP-dependent motor proteins that use actin filaments as their tracks. There are 18 different myosin classes currently known. Some are expressed only in muscles, whereas others are expressed solely in non-muscle cells. The best-known function of myosins is their role in muscle contraction. However, they are also involved in cargo transport, actin filament contraction in non-muscle cells, stereocilia formation and cell division. Synapses undergo continuous molecular and structural changes and myosins contribute to these processes by mediating actin cytoskeleton rearrangement and cargo transport123. In dendritic spines, non- muscle myosin IIb ACCEPTED drives actin cytoskeleton MANUSCRIPT dynamics in response to synaptic stimulation97,124. Myosin Va plays a critical role in spine and synapse development by trafficking the α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors into dendritic spines125. Mutations in MYO5A cause severe neurological defects, including intellectual disability and seizures126,127.

Myosin XVI (MYO16) is an unconventional actin-based molecular motor with ATPase activity (Figure 3). The expression and phosphorylation of myosin XVI (Myr8 or NYAP) in the cortex

ACCEPTED MANUSCRIPT and cerebellum peak during early development, having a role in regulating neuronal migration and neurite extension128. Myosin XVI binds directly to F-actin and also regulates stress fiber remodeling in fibroblasts and neurite outgrowth in neurons via its interaction with phosphatidylinositol 3-kinase (PI3K) and the WAVE complex129. Genetic association between the MYO16 gene and autism has been found in two large cohorts (AGRE and ACC) of European ancestry and replicated in two other major cohorts (CAP and CART)130, as well as in other studies131-134.

The full-length human myosin IXb (MYO9B) protein contains a motor head domain, four IQ calmodulin-binding motifs in the neck area and a Zn2+-binding domain and RhoGAP domain in the tail135. This motorized RhoGAP is mostly studied in cancer cell lines, where it localizes to the sites of actin polymerization136 (Figure 3). However, myosin IXb is also expressed in the central nervous system (CNS) and in cortical neurons, where it has been shown to control RhoA activity, thereby regulating the growth and branching of dendritic processes137. Knockdown of myosin IXb in cultured cortical neurons or in the developing cortex results in decreased dendrite length and number137. The role of myosin IXb in dendritic spine morphogenesis is unknown but as RhoA seems to inhibit dendritic spine growth138, a protein inhibiting RhoA should have a positive effect on spine formation and maturation. Mutations in the MYO9B gene are associated with autoimmune diseases such as celiac disease139 as well as schizophrenia140. The MYO9B gene was also identified in an ASD whole-exome sequencing study and subsequent TADA analysis as a gene strongly enriched for variants likely to affect ASD risk43. Whole exome sequencing applied to families with an ASD child revealed de novo missense mutations in MYO9B leading to an amino acid change in the RhoGAP domain118.

Although the myosin heavy chain IIb isoform (MyHC-IIb) (MYH4) is the predominant in most skeletal muscles of rats and mice, the mRNA for this isoform is only expressed in 141 a very small subsetACCEPTED of specialized muscles in MANUSCRIPT adult large mammals, including humans . MyHC- IIb expression is typically associated with high forces of contraction combined with rapid contractile characteristics, and it has been suggested that these contractile characteristics may be incompatible with the biomechanical constraints of larger muscles142. Considering that its predominant role is in skeletal muscles, it is rather surprising that two families with children having ASD showed a loss of MYH4 inherited from a healthy mother143. However, the Allen brain atlas shows relatively strong expression of MYH4 gene throughout the brain, including in pyramidal neurons in the hippocampus and Purkinje cells in the cerebellum. MyHC-IIb null mice

ACCEPTED MANUSCRIPT are viable, probably due to compensation by other skeletal muscle fast isoforms144. Further studies are required to elucidate how this muscle myosin contributes to neuronal function.

2.4 Cohesive proteins, linking F-actin to other support proteins and extracellular matrix

The distinctive shape of a cell is dependent not only on the organization of F-actin but also on proteins that connect the filaments to the membrane. A subset of actin-binding proteins, discussed below, provides a link between the intracellular actin cytoskeleton and the extracellular matrix, and functions to maintain the integrity of the plasma membrane.

Dystrophin (DMD) is best known as a gene affected in Duchenne muscular dystrophy. This X- linked recessive degenerative neuromuscular disorder is often associated with CNS disorders such as intellectual disability, learning deficits, particularly in relation to language, and ASD145- 147. Large out-of-frame gene deletions or duplications of the DMD gene account for two thirds of the mutations in these patients, and gene microdeletions, insertions, or point mutations occur in the remaining third148. Through two CH domains in tandem at its N-termini, dystrophin provides a link between the intracellular actin cytoskeleton and the extracellular matrix (laminin), which stabilizes the plasma membrane. Dystrophin is most abundant in skeletal and cardiac muscle149. The only non-muscle tissue that expresses substantial quantities of dystrophin is the brain149, where it is found at approximately 10% of the level found in skeletal muscle. The DMD gene encodes at least seven distinct proteins. Although it was initially thought that full-length dystrophin was associated with excitatory synapses150, several independent studies indicate that the main function of dystrophin is the regulation of inhibitory synapses151 (Figure 4).

Utrophin (UTRN) is the closest homolog of dystrophin and functions in a similar way. Studies in -deficient mice have revealed a reduction in the number of postsynaptic-membrane folds 152,153 and acetylcholine receptorsACCEPTED at the neuromuscular MANUSCRIPT junction (NMJ) (Figure 4). These mice are viable and show no sign of muscular weakness and no compensation by dystrophin expression. The pattern of utrophin expression around the soma of neurons is different from the punctate expression of dystrophin in synaptic structures, suggesting that utrophin provides structural support for neuronal membranes, whereas dystrophin is a component of inhibitory synapses154. Interestingly, high utrophin immunoreactivity was reported several weeks after the induction of morphogenic changes in granule cells of the dentate gyrus in an experimental model of temporal lobe epilepsy, suggesting that utrophin contributes to protect CNS neurons against pathological

ACCEPTED MANUSCRIPT insults via long-term structural stabilization of their membranes154. Utrophin was originally identified as an ASD candidate based on its enrichment in an autism-associated protein interaction module. Sequencing of post-mortem brain tissue from 25 ASD cases resulted in the identification of significant non-synonymous variants in this gene, confirming the involvement of this module in autism; this finding was further validated by exome sequencing of an independent cohort of 505 ASD cases and 491 controls10.

2.5 Synaptic scaffolding proteins and synaptic vesicle regulators affecting the actin cytoskeleton

Postsynaptic scaffolding proteins cluster cell adhesion proteins, ion channels, receptors and cytoskeletal regulators to confined synaptic sites, supporting the connection between synaptic structure and signaling. Scaffolding proteins are highly abundant in the postsynaptic density (PSD), in terms of both protein copy numbers and the distinct protein types155,156. The role of these scaffolding proteins is connected to neuronal function, and when disrupted, neuronal morphology and synaptic plasticity are subsequently affected, contributing to the etiology of diverse human psychiatric disorders such as schizophrenia, ASD, and obsessive- compulsive spectrum disorders157.

The SH3 and multiple ankyrin repeat domains (SHANK) proteins 1-3, coded by the SHANK1-3 genes, constitute a scaffolding protein family that is highly enriched in the PSD158. SHANKs (also known as ProSAPs) contain several discrete domains including ankyrin (ANK) repeat domains, Src homology (SH) 3 and PDZ domains, a proline-rich region, and a sterile alpha motif (SAM) domain159,160. These protein interaction domains enable SHANK proteins to interact with a multitude of postsynaptic proteins, linking SHANK proteins to ionotropic glutamate receptors via PSD-95 and PSD-95-associated protein (SAPAP) interactions160, intracellular Ca2+ 161 160 signaling via HOMERACCEPTED interactions , and, MANUSCRIPTactin cytoskeleton regulation via cortactin , α- fodrin162, Rac1163 and actin binding protein 1 (Abp1)164 interactions, among others (Figure 5). Cortactin, Abi-1 (Abelson interacting protein 1), and IRSp53 (insulin receptor substrate p53) bind to the SHANK3 proline-rich domain. Abi-1 regulates the actin cytoskeleton via the Arp2/3 complex, and its knockdown increases dendritic branching and the number of immature spines, but reduces synapse density165. Fodrin stabilizes actin filaments and serves as a calmodulin- binding protein166, linking SHANK proteins indirectly to Ca2+ sensing. Different point mutations identified in the ANK domain of SHANK proteins prevent alpha-fodrin and SHARPIN167

ACCEPTED MANUSCRIPT binding. Loss of fodrin binding may lead to labile actin filaments leading to alterations in spine density and spine maturation168. In non-neuronal cells SHARPIN is known to inhibit integrin activation169. Interesting new evidence from non-neuronal cells show that SHANKS can also inhibit integrin activation. However, this integrin activation inhibition was not mediated through SHARPIN but by sequestering active Rap1 and R-Ras from the plasma membrane170. Furthermore, autism-linked mutations (R12C, arginine to cysteine; L68P, leucine to proline)168,171,172 in SHANK3 impaired its integrin inhibitory function in non-neuronal cells170 prompting a new intriguing proposal that at least some of the SHANK ASD-associated mutations alter the integrin activation in neurons. In general, SHANK1 promotes dendritic spine head enlargement, and SHANK2 and 3 promote dendritic spine formation, with SHANK3 also inhibiting dendritic spine arborization by binding to Densin-180173-177. Shank3 knockout mice suffer from a loss of cortical actin filaments, in association with reduced Rac1/p21-activated kinase (PAK) activity together with increased activity of cofilin178. Overexpression of Shank3 has been found to enhance actin polymerization168 and increase F-actin levels179. The SHANK proteins can regulate Rac1 activity through RhoGEF beta-PIX163 and RhoGAP RICH2180. The altered synaptic actin filament regulation could be connected to the loss of synaptic NMDA receptors observed in these animals, which is proposed to contribute to their autism-like behavior. Shank1-/- mice provide an animal model for human shankopathies, displaying alterations in synaptic protein composition and impairments in dendritic spine morphology, with the spines being thinner and smaller, and the spine number decreased175,181,182. Shank2-/- mice, which exhibit almost all the core features of ASD such as increased stereotypic behavior, impairment in social interaction, hyperactivity and anxiety show a similar reduction in the number of dendritic spines and a decrease basal synaptic transmission with depletion of NMDA receptors183. Several mouse models have been generated to investigate the role of Shank3 and its splice variants in ASD. A complete knockout of Shank3 in mice resulted in defective corticostriatal circuitry and fewer and 184 thinner PSDs . TACCEPTEDhe autistic mouse model alsoMANUSCRIPT showed impaired function of striatal synapses and abnormalities in brain morphology184. CNVs and truncating mutations of SHANKs have been identified in almost 1% of ASD patients185, making the SHANK family of proteins central to the pathophysiology of autism. Altered gene function of all SHANKs has been associated with syndromic and idiopathic ASD186. Shank mutations have very high penetrance at the phenotypic level, and any form of SHANK3 haploinsufficiency is sufficient to cause behavioral changes187. Given that the SHANK proteins are modulators of dendritic spine morphogenesis, it is plausible that related mutations or

ACCEPTED MANUSCRIPT deletions lead to alterations in spine homeostasis. In support of this, deficiency or autism-related mutations of Shank3, the most prevalent of the three, result in altered cortactin, Abp1, cofilin and Rac1 expression or activity, increased integrin activation and a reduction in dendritic spines and synaptic dystrophy168,170,178,188. Furthermore, rare de novo CNVs, deletions and duplications in genes encoding SHANK interacting proteins such as Disk large-associated protein 2 (DLGAP2) have been associated with ASD64,189-192. In line with SHANK mice models, Dlgap2-/- mice show reduced spine density and a downregulation of synaptic proteins such as HOMER1193.

Another postsynaptic density scaffolding protein, linking regulation of the synaptic actin cytoskeleton to ASD, is DISC1 (disrupted in schizophrenia 1) (DISC1). DISC1 plays a major role in early brain development by regulating a number of essential neurodevelopmental events including cell proliferation, neurite outgrowth, synapse formation and neuronal migration194,195. DISC1 maintains spine morphology and function by regulating Rac-1 activator, Kalirin-7 (Kal- 7), access to Rac1196 (Figure 5). Kal-7/Rac1 cascade and Rac-1 activation in response to NMDA receptor activation in both neuronal cultures and rat brain in vivo196 are important for the activity– dependent spine morphology and functional plasticity197. In addition to schizophrenia, genetic association and rare variants in DISC1 have been associated with autism and Asperger’s syndrome198.

IRSp53 (also known as brain-specific angiogenesis inhibitor 1-associated protein 2, BAIAP2) is a multi-domain scaffolding and adaptor protein that has been implicated in the regulation of membrane and actin dynamics in dendritic spines, filopodia and lamellipodia. Accumulating evidence indicates that IRSp53 is an abundant component of multi-protein complexes on the postsynaptic side of excitatory synapses199 and directly binds to PSD-95 and Shank/ProSAP200-202 (Figure 5). IRSp53 has been suggested to form a platform-like structure that organizes synaptic signaling to regulate dendritic spines through its ability to interact with F-actin and actin 155,200,203,204 regulatory proteins ACCEPTED. MANUSCRIPT Both genetic association between the BAIAP2 gene and autism205 and a rare deletion in the BAIAP2 has been identified in association with autism206. IRSp53 has been implicated also in other psychiatric disorders, including ADHD (attention deficit/hyperactivity disorder)207,208 and schizophrenia209,210. Overexpression of IRSp53 in cultured neurons increases the density of dendritic spines without affecting spine length or width. Conversely, short-interfering RNA- mediated knockdown of IRSp53 reduces spine density, length, and width200. In agreement with this, and indications from other in vitro studies199-201,211, IRSp53−/− mice have fewer dendritic

ACCEPTED MANUSCRIPT spines and perforated PSDs (a measure of maturity) in the hippocampus212. Hippocampal neurons from IRSp53−/− mice have been reported to exhibit stabilized F-actin and suppressed basal cofilin activity (i.e. increased phosphorylation as a measure of inactivity)212. These animals also display enhanced NMDA receptor function, as well as social and cognitive deficits similar to those observed in psychiatric disorders212. Pharmacological suppression of NMDA receptor function can improve these behavioral deficits212, prompting the hypothesis that defective actin/membrane modulation in IRSp53-deficient dendritic spines could lead to social and cognitive deficits through NMDA receptor dysfunction.

Although this review focuses on ASD-associated mutations that affect the actin cytoskeleton in dendritic spines, it is important to note that there are some indications that postsynaptic proteins may indirectly affect the presynaptic actin cytoskeleton and presynaptic function. The presynaptic terminal is a subcellular axonal compartment dedicated to releasing upon neuronal activation through synaptic vesicle (SV) exocytosis. The fusion of SVs with the active zone membrane is followed by SV recycling through endocytosis213,214. Actin is abundant in the presynapses and performs active roles in vesicle trafficking and the development of presynaptic terminals203,215. Actin interaction with synapsin, which is phosphorylated upon neuronal activity is crucial for the organization of the SV pools within the presynaptic nerve terminal216. In particular, despite being a postsynaptic protein, SHANK3 may indirectly affect the presynaptic actin-synapsin signaling pathway through trans-synaptic activity involving neurexin- neuroligin (NRXN-NL) protein complexes217: ASD-associated mutations in Shank3 disrupt postsynaptic AMPA and NMDA receptor signaling and also interfere with the ability of Shank3 to signal across the synapse to alter presynaptic structure and function via the NRXN-NL2 trans- synaptic pathway. Additionally, NL3 is a postsynaptic protein that interacts with NRXN in the presynaptic neuron that also regulates synaptic morphogenesis. Mutations in the genes encoding NRXNs and NLs have further been linked to ASD218. These reports indicate that ASD-associated mutations in a subsetACCEPTED of synaptic proteins may MANUSCRIPT target core cellular pathways that coordinate the pre- and postsynaptic matching and maturation of excitatory synapses in the CNS. Interestingly, variants and mutations in other genes coding presynaptic proteins connected to actin, such as PRICKLE1 and syntaxin binding protein 5 (STXBP5, also known as tomosyn), have also been associated with ASD43,192,219-222. For example, Prickle1+/- mice exhibit autism-like behavior, which might result from disrupted interaction with synapsin and dysregulation of SVs221.

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Table I. Summary of autism-associated actin-regulatory human genes. Gene Protein name/ Role Chr. Genetic Category*, References Symbol Aliases band Gene Score (GS)#

Actin polymerization and depolymerization

CTTNBP2 Cortactin binding Regulates formation and 7q31. Rare single gene variant, 36,37,41,43,223 protein 2 maintenance of dendritic spines via 31 GS3 cortactin function on F-actin branching and stabilization. CYFIP1 Cytoplasmic Regulates actin cytoskeleton 15q11 Multigenic CNV, 47,71,224 FMR1 remodeling connected with .2 rare single gene variant, interacting dendritic spine formation. genetic association protein 1 DIAPH3 Diaphanous- Regulates actin dynamics during 13q21 Rare single gene variant, 80,82,83,225 related formin 3, dendritic spine elongation together .2 GS5 DRF3, mDia2 with other actin regulatory proteins. GSN Gelsolin Severs F-actin filaments and 9q33. Rare single gene variant 84,85,91 regulates neurogenesis and neuronal 2 migration.

Actin cross-linking

ACTN4 -actinin-4 F-actin cross-linking to remodel 19q13.2 Rare single gene variant 10,105,118 dendritic protrusions and regulate dendritic morphogenesis. CAMK2A Calcium/calmod May stabilize and maintain the 5q32 Rare single gene variant, 118,120,121 ulin dependent dendritic spine structure together GS4 protein kinase II with CaMKIIβ through actin alpha bundling and by inhibiting the binding of other actin regulators. VIL1 Villin 1 Caps, severs, and bundles actin 2q35 Rare single gene variant, 43 filaments. Function in neurons GS3 unknown. Actin-binding myosin motors MYO16 Myosin XVI Neuronal migration during 13q33.3 Genetic association, 129-132 development and neurite rare single gene variant, ACCEPTEDoutgrowth MANUSCRIPTGS4

MYO9B Myosin IXB, Role in dendritic spine 19p13.1 Rare single gene variant, 43,118,137 myr5 morphogenesis is unknown, but 1 GS3 may augment spine formation and maturation. MYH4 Myosin heavy Predominant role in skeletal 17p13.1 Rare single gene variant, chain IIb isoform muscles. Function in neurons GS4 143,226 unknown. Cohesive proteins, linking F-actin to other support proteins

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DMD Dystrophin Bridges F-actin and the extra- Xp21.2- Syndromic, cellular matrix and may form a p21.1 Genetic 146 component of inhibitory association, synapses. Rare single gene variant UTRN Utrophin Structural support for dendritic 6q24.2 Rare single gene variant 10 spine membranes. Synaptic scaffolding proteins linked to the actin cytoskeleton SHANK1 SH3 and Structural and functional 19q13.3 Rare single gene variant, 223,227 multiple ankyrin organization of the dendritic spine 3 GS3 repeat domains 1 and synaptic junction SHANK2 SH3 and Controls dendritic spine volume 11q13.3 Rare single gene variant, 64,228 multiple ankyrin and branching and synaptic -q13.4 GS2 repeat domains 2 transmission. SHANK3 SH3 and Controls dendritic spine 22q13.3 Rare single gene variant, 91,168,173,174,229 multiple ankyrin formation and maturation. 3 genetic association, repeat domains 3 syndromic, Multigenic CNV

DISC1 Disrupted in Controls early brain development 1q42.2 Rare Single Gene 194,195,198 Schizophrenia 1, by regulating neurite outgrowth, Mutation, Syndromic, DISC1 synapse formation and neuronal Genetic Association migration BAIAP2 BAI1-associated Regulates membrane and actin 17q25.3 Rare single gene variant, 205,206 protein 2, dynamics in dendritic spines. genetic association, GS5 IRSp53 Trans-synaptic pathway and links to presynaptic actin cytoskeleton NRXN1 Neurexin 1 Cell adhesion and receptors. 2p16.3 Rare single gene variant, genetic association, 230-233 syndromic NRXN2 Neurexin 2 Cell adhesion and receptors. 11q13.1 Rare single gene variant, 234 GS4 NRXN3 Neurexin 3 Cell adhesion and receptors. 14q24.3 Rare single gene variant, 235 -q31.1 genetic association, GS3

NLGN1-3 Neuroligin 1-3 Regulates spines and synaptic 3q26.31, Rare single gene variant, 217,236-238 plasticity. 17p13.1, genetic association, ACCEPTED MANUSCRIPTXq13.1 GS2/4 PRICKLE1 Prickle homolog Involved in axonal and dendritic 12q12 Syndromic, rare single 192,221,222 1 (Drosophila) formation gene variant, GS3 STXBP5 Syntaxin binding Associates with F-actin and is 6q24.3 Rare single gene variant, protein 5 involved in synaptic vesicles GS3 43,192,220 exocytosis *Rare single gene variants indicate disruptions/mutations, and submicroscopic deletions/duplications directly linked to ASD. Syndromic indicates genes implicated in syndromes in which a significant subpopulation develops symptoms of autism (for example in Fragile X Syndrome). Association indicates small risk-conferring common polymorphisms identified from genetic association studies in idiopathic ASD.

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#GS= SFARI Gene Score gives an estimation of the strength of the evidence indicating that a gene is relevant for ASD. GS1, High confidence; GS2, strong candidate; GS3, suggestive evidence; GS4, minimal evidence; GS5, hypothesized but untested; GS6, evidence does not support a role. The table is modified from the SFARI gene collection (https://sfari.org/).

2.6 RhoGTPase regulation

The Rho family of GTPases is a family of small signaling G-proteins that are central regulators of intracellular actin dynamics. The most extensively studied members of the family are Cdc42, RhoA and Rac1, which in fibroblasts control filopodia and microspike formation, mediate focal adhesion and stress fiber formation, and induce membrane ruffling and the formation of lamellipodia, respectively239. Through their role in controlling F-actin dynamics, small RhoGTPases and their regulators, GTPase-activating proteins (GAPs) and guanine-exchange factors (GEFs), are critical mediators of dendritic arborization, spine morphogenesis, growth cone development, axon guidance240-245 and neuronal survival246. Mutations in Rho family GTPase regulators in particular are frequently associated with neurological diseases241,247-249.

SLIT-ROBO Rho GTPase-activating protein 3 (SrGAP3) (SRGAP3) is ubiquitously expressed in the developing nervous system250,251. The full-length human SrGAP3 protein contains an iF- Bin1/amphiphysin/Rvs167 (BAR) domain (involved in membrane binding), a Rac1 GAP domain and an SH3 domain interacting with Robo1/2, WAVE1 and lamellipodin. SrGAP3 binds WAVE1 and downregulates Rac1 signaling252-254 (Figure 6). Knocking out SrGAP3 decreases the number of dendritic filopodia in early development, and it is therefore plausible that SRGAP3 deficiency also leads to decreased dendritic spine density in human patients255. Two de novo missense variants in the SRGAP3 gene (one predicted to be probably damaging, and the other predicted to be possibly damaging) were identified in ASD probands from the Simons ACCEPTED Simplex Collection118. In MANUSCRIPT addition, the authors reported a de novo point mutation E469K (glutamic acid to lysine), which changed negatively charged glutamate to positively charge lysine at the end of the iF-BAR domain.

Oligophrenin1 (OPHN1), also referred as RhoGTPase activating protein 41 ARHGAP41, has been identified on the X chromosome region q12256. Ophn1 is expressed during early development, from embryonic day 10 (E10) in the mouse257. In situ hybridizations on mouse embryos have revealed a strong signal in the neural tube and the developing brain. In the adult,

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Ophn1 is widely expressed throughout the brain but is concentrated in the olfactory bulb, the hippocampus and the cerebellum. In young cultured neurons, oligophrenin1 colocalizes with the actin cytoskeleton in the growing tip of dendrites257, and is detected in the axon, dendrites and dendritic spines of mature neurons258. In vitro, the use of RNA interference methods has revealed the important role of oligophrenin1 in the regulation of dendrite morphogenesis and the regulation of dendritic spine maturation258. Notably, the downregulation of oligophrenin1 expression in rat hippocampal slices leads to a decrease in spine length without affecting spine density258. Although oligophrenin1 has the capacity to interact with the three RhoGTPases, its action on spine morphogenesis depends on its negative regulation of RhoA, leading to the activation of Rho-associated coiled-coil containing protein kinase (ROCK) and spine growth258. The role of oligophrenin1 has been confirmed in vivo; knockout Ophn1-/y mice present a decreased complexity of the dendritic tree together with a reduction in the density of mature dendritic spines259. These animals also exhibit a decreased adult neurogenesis. Treatment of Ophn1-/y cultured brain slices with an inhibitor of ROCK/PKA, Fasudil, restores dendritic spine morphology and adult neurogenesis260. At the postsynapse, oligophrenin1 colocalizes and interacts with the scaffolding protein HOMER1b/c256,261 (Figure 6). The disruption of this interaction by the expression of mutated forms of one or the other partner causes the mispositioning of the endocytic zone in the PSD and an impaired recycling of AMPA receptors261. Oligophrenin1 also plays a role in the correct functioning of the presynapse. In the axon terminus, it interacts with endophilin A1 and triggers SV retrieval262. In vivo, mice in which Ophn1 is disrupted exhibit deficits in presynaptic plasticity due to dysregulation of the cAMP/PKA signaling pathway263. Oligophrenin1 belongs to the GTPase regulator associated with focal adhesion kinase (GRAF) protein subfamily, characterized by a N-terminal BAR domain, a pleckstrin homology (PH) domain and a GAP domain, which negatively regulates RhoA264. Several case studies have provided evidence of potential loss of function mutations – either deletion or insertions – in the region coding for the BAR domain linked to intellectual 256,264,265 disability, suggestingACCEPTED an important role of this MANUSCRIPT domain in oligophrenin1 function . The BAR domain could indeed play several roles, notably in the control of the membrane curvature for SV recycling or the binding and regulation of RhoA in conjunction with the PH domain. Finally, rare missense variants in OPHN1 gene have been identified in patients with ASD206,266.

Although this review focuses on the link between actin in dendritic spines, and thus excitatory synapses, and ASD, several genes that regulate the formation of inhibitory synapses are also related to ASD. One example is Cdc42 guanine nucleotide exchange factor (GEF) 9

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(ARHGEF9), also known as collybistin or posterior end mark 2 (PEM2). ARHGEF9 was first identified in a yeast two hybrid screen as an interactor with the scaffolding protein gephyrin267,268 (Figure 6). ARHGEF9 contains a diffuse B-cell lymphoma (Dbl) homology (DH) domain that interacts with RhoGTPases, a PH domain and a SH3 domain269. ARHGEF9 is a gephyrin binding partner predominantly expressed in the brain. Its expression is induced during the second half of murine embryogenesis, in postmitotic neurons of the cortex269,270. Multiple isoforms have been identified, based on different sizes and the presence or absence of the SH3 domain269,271. The PH domain of ARHGEF9 binds to phosphatidylinositol 3-monophosphate and 4-monophosphate (PI3P, PI4P)269, whereas the SH3 domain inhibits this membrane-targeting function271. However, PH affinity to PI binding is not sufficient for the correct targeting of the protein to the plasma membrane, and the binding of ARHGEF9 to gephyrin is therefore necessary for its membrane targeting. Xiang, et al. 269 (2006) proposed a model in which the activation of PI3K by an unknown presynaptic factor results in an increased phosphatidylinositol 3,4,5-triphosphate concentration at the plasma membrane. This leads to the recruitment of ARHGEF9, which then initiates actin cytoskeleton regulation and gephyrin recruitment that in turn triggers the clustering of glycine receptors. A mutation in the PH domain linked to intellectual disability has been shown to alter the binding of ARHGEF9 to PI3K, causing the loss of gephyrin clustering affinity272. A mouse model genetically ablated for Arhgef9 displays a loss a postsynaptic gephyrin and GABA receptors, together with a disruption of synaptic plasticity and changes in anxiety and learning behavior273. The ASD-associated postsynaptic adhesion protein NL2 is specific for inhibitory synapses274. It binds to gephyrin and functions as a specific activator of ARHGEF9 by relieving the SH3-mediated inhibition of its membrane targeting function. The recruitment of gephyrin and ARHGEF9 to the synaptic membrane by activated NL2 therefore triggers the clustering of GABA and glycinergic receptors271. ARHGEF9 also binds to mTOR, a regulator of essential cellular processes such as cell growth and protein synthesis. The interaction of ARHGEF9 with mTOR promotes the activation of the mTOR complex 1 (mTORC1) signaling pathway and proteinACCEPTED synthesis. Deregulation ofMANUSCRIPT the mTORC signaling pathway results in severe neuronal disorders, deficits observed in a patient presenting an ARHGEF9 complete deletion associated with marked intellectual disability275. Furthermore, several case studies present patients with various modification of chromosomal region Xq11, the site of the gene that codes for ARHGEF9, associated with severe neurological traits and ASD276-278.

ARHGAP15 (ARHGAP15) is a Rac1 regulator that is expressed in the pyramidal cells and the GABAergic interneurons of the hippocampus in adult mice279. ARHGAP15 acts as a GAP for

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Rac1 with which it interacts through its C-terminus domain279,280. It has also been shown to interact with the Rac1 downstream effectors PAK1/2 via its N-terminus (Figure 6). This interaction leads to a mutual inhibition of the two proteins, resulting in decreased GTPase activity280. The genetic ablation of Arhgap15 in the mouse hippocampus leads to decreased complexity of the dendritic tree of neurons. Moreover, cultured hippocampal neurons from rat embryos knocked down for Arhgap15 have fewer spines, although the morphology of these spines is not changed compared to control neurons279. ARHGAP15 was linked to ASD in a case study of a young man with marked intellectual disability. Here, a severe 6 Mb de novo deletion was discovered in the 2q22 locus, covering several genes including ARHGAP15281.

ARHGAP32 (ARHGAP32), also known as RICS or p250GAP, is expressed in the cerebral cortex during early postnatal development282. Knockdown of Arhgap32 in cultured cortical neurons by miRNA has demonstrated its role in the regulation of the dendritic tree complexity282. Arhgap32 gene silencing in cultured hippocampal neurons has been shown to produce an increased dendritic spine head width without affecting the length of the spines or their density along the dendritic shaft283. In contrast, a gain-of-function experiment in hippocampal neurons overexpressing ARHGAP32 led to a decrease in the spine width but an increase in the neck length283. ARHGAP32 interacts with the NR2B subunit of the NMDA receptor and promotes the activation of the RhoGTPase RhoA in response to NMDA activation283 (Figure 6). Several genetic studies have identified de novo LGD mutations in the ARHGAP32 gene in ASD patients, with notably patients presenting a 11q deletion characteristic of Jacobsen syndrome284,285.

ARHGAP33 (ARHGAP33) (also known as TC10β/CDC42 (TC)-GAP, NOMAGAP or SOX26) is concentrated in the dendritic spines of cortical pyramidal neurons where it colocalizes with PSD-95286 (Figure 6). Although ARHGAP33 has not been genetically linked to ASD, its genetic ablation in mice results in major deficits in social behavior, a typical autistic trait286. Moreover, ARHGAP33 regulatesACCEPTED several neuronal morphogenesis MANUSCRIPT mechanisms that are closely related to ASD cellular phenotypes. Most notably, ARHGAP33 regulates dendrite morphogenesis, with Golgi impregnations of knockout mouse brains providing evidence of its involvement in the promotion of dendritic complexification287,288. This is achieved by the inactivation of Cdc42 by ARHGAP33, which triggers a molecular signaling cascade that leads to the activation of the actin depolymerization factor cofilin, and the regulation of the branching of the dendritic tree289. More recently, two studies have reported the important role of ARHGAP33 in regulating spine morphogenesis. First, Arhgap33-/- mice exhibit an impaired spine development, with a reduced

ACCEPTED MANUSCRIPT number of total spines together with a decreased proportion of mature spines. In these mice, the trafficking and surface expression of the BDNF receptor TrkB are impaired, resulting in a deficit in spine and synapse formation287. The second study shows that Arhgap33-/-mice also present morphological variations of their dendritic spines, with a longer length of the spine neck without modification of the head width and a decrease in the density of synapses containing PSD-95286. The deletion of Cdc42 does not restore this phenotype. Co-immunoprecipitations performed on cortical lysates of wildtype mice have revealed the interaction of ARHGAP33 with the scaffolding protein PSD-95. ARHGAP33 regulates the phosphorylation and localization of PSD- 95, in association with a loss of AMPA receptors at the synaptic membrane286. The recruitment of AMPA receptors at the synapse leads to the strengthening and the maturation of spines via the regulation of signaling pathways that modulate the actin cytoskeleton290.

ARHGAP24 (ARHGAP24), also known as Rac1 regulator, was identified in 2004 based on its homology to ARHGAP22291. ARHGAP24 is a binding partner of A, an F-actin cross- linking protein292. ARHGAP24 is a canonical target of the nonsense-mediated mRNA decay (NMD) factor UPF3B that regulates the degradation of mutated transcripts, and mutations in the genes coding for UPF3B have been linked to intellectual disability293. ARHGAP24 regulates neuronal morphology. Notably, overexpression of ARHGAP24 in murine hippocampal neurons leading to decreased axon outgrowth, together with a reduction in the number of dendritic termini293. This suggests that ARHGAP24 could play a role in the development of dendritic spines but this has not yet been demonstrated. A de novo deletion in the locus 4q21.23q21.3, a region that only harbors the gene which codes for ARHGAP24, has been identified in ASD patients294.

Other RhoGTPase regulating proteins have been linked to ASD. For example, two CNVs have been isolated in the gene ARHGAP11B coding for ARHGAP11B, in ASD patients71. ARHGAP11B has ACCEPTED been implicated in the evolutionary MANUSCRIPT expansion of the neocortex. Its genetic link to ASD and its presence in the brain during development suggest a potential role for ARHGAP11B in dendritic spine morphogenesis and plasticity, but this is still to be demonstrated295,296.

Finally, other actors of the small GTPases superfamily and their regulators are involved in the development of dendritic spines. Among them, the Synaptic GTPase activating protein 1 (SynGAP1) (SYNGAP1), an excitatory synapse-specific RasGAP, is enriched at excitatory

ACCEPTED MANUSCRIPT synapses where it associates with PSD-95 and regulates cofilin activity297-299 (Figure 6). A disruption in the balance between excitatory and inhibitory neurotransmission in the developing brain has been linked with autism, mental retardation and intellectual disability. Excitatory synapse strength is regulated by controlling the number of postsynaptic AMPA receptors in the CNS. SynGAP1 regulates AMPA receptor trafficking, the number of silent synapses, and excitatory synaptic transmission in cultured hippocampal and cortical neurons. In response to CaMKII phosphorylation, SynGAP1 maintains dendritic spine stability through its role in regulating Ras/Rap activity and mitogen-activated protein kinases (MAPK) signaling299-302. Overexpression of SynGAP1 in neurons decreases synaptic AMPA receptor surface expression and insertion into the plasma membrane, and decreases AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs)301,303,304. Cultured hippocampal neurons from syngap1+/− and syngap1−/− mice, as well as from neuronal cultures treated with SynGAP1 siRNA, exhibit increased actin polymerization and phospho-cofilin levels, and an increase in the synaptic strength and the in number of “mushroom” spines297,301,305, which is one of the notable classes of spine shape in addition to "thin", "stubby", and "branched". SYNGAP1 haploinsufficiency has been found in individuals with autism, intellectual disability, and a specific form of epilepsy306. In mice, SynGAP1 accelerates the maturation of dendritic spines leading to disruptions in synaptic transmission and cognitive function307-310. Several de novo mutations of SYNGAP1 have also been associated with ASD43,64,306,311-315. Together these results indicate that disruption of SynGAP1 activity in regulating F-actin structure within spines leads to spine destabilization and changes in spine morphology.

Table II. Summary of autism-associated small GTPases superfamily members regulating actin cytoskeleton. Gene Protein name/ Role Chr. Genetic Category*, References Symbol Aliases band Gene Score (GS)#

SRGAP3 SLIT-ROBOACCEPTED GAP for Rac1 and possibly MANUSCRIPT for 3p25. Rare Single Gene 118 Rho GTPase Cdc42, but not for RhoA. May 3 Mutation activating protein attenuate Rac1 signaling in neurons. GS4 3 Plays a role in spine initiation. OPHN1 Oligophrenin 1 A RhoA-GAP involved in cell Xq12 Rare Single Gene 206,266 migration and outgrowth of axons Mutation and dendrites. GS3 ARHGEF9 Cdc42 guanine Cdc42-GEF, which promotes the Xq11. Rare Single Gene 316,317 nucleotide formation of GPHN clusters. 1- Mutation exchange factor q11.2 9, collybistin

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ARHGAP1 Rho GTPase Activates Rho-like small 15q13.2 Rare Single Gene 71 1B activating protein GTPases. Function in neurons Mutation 11B unknown. ARHGAP1 Rho GTPase Rac1-GAP regulates positively 2q22. Rare Single Gene 143,318 5 activating protein complexity of the dendritic tree and 2- Mutation 15 spine density. q22.3 GS6 ARHGAP2 Rho GTPase Rac1-GAP, possibly having a role 4q21.23 Rare Single Gene 319 activating protein 4 in axon outgrowth and dendritic -q21.3 Mutation 24, FilGAP spine formation. GS5 ARHGAP3 Rho GTPase A neuron-associated RhoA-GAP 11q24.3 Rare Single Gene 43,223 activating protein 2 that regulates dendritic spine Mutation, Functional 32, RICS, p250GAP morphology. GS4 ARHGAP3 Rho GTPase Cdc42-GAP involved in the 19q13.1 Rare Single Gene 320 3 activating protein regulation of the branching of the 2 Mutation, Functional 33, TC10-beta- dendritic tree as well as in GS5 GAP, dendritic spine and synapse NOMAGAP, formation. Involved in SOX26 intracellular trafficking. SYNGAP1 Synaptic Ras An excitatory synapse-specific 6p21.32 Rare Single Gene 43,64,223,226,312- GTPase Ras-GAP regulates AMPA Mutation, Syndromic 314,321 activating protein receptor trafficking and synaptic GS1 1 transmission. *Rare single gene variants indicate disruptions/mutations, and submicroscopic deletions/duplications directly linked to ASD. Syndromic indicates genes implicated in syndromes in which a significant subpopulation develops symptoms of autism (for example in FXS). #GS= SFARI Gene Score gives an estimation of the strength of the evidence indicating that a gene is relevant for ASD. GS1, High confidence; GS2, strong candidate; GS3, suggestive evidence; GS4, minimal evidence; GS5, hypothesized but untested; GS6, evidence does not support a role. The table is modified from the SFARI gene collection (https://sfari.org/).

3. Treatment

The studies reviewed here suggest that dysregulation of actin cytoskeleton-related pathways that control dendritic structureACCEPTED may contribute significantlyMANUSCRIPT to the pathogenesis of ASD. Although ASDs show a high rate of heritability, genetic research alone has not been able to provide a complete understanding of the underlying pathophysiology in the pursuit of unique pharmacogenetic approaches. ASDs are genetically and clinically heterogeneous and effective medications to treat the core symptoms are still lacking. However, a number of drugs and alternative strategies have shown some promise. Genetic and pharmacological interventions in mouse models of ASD, including TSC322, FXS323, Rett syndrome324,325 and Angelman syndrome326, as well as in Shank2−/− mice carrying a mutation

ACCEPTED MANUSCRIPT identical to the ASD-associated microdeletion in the human SHANK2 gene327, have shown promise in improving ASD phenotypes, offering hope for the development of therapeutics for ASD. Autism-like social deficits and repetitive behaviors have been rescued by targeting actin regulators in Shank3-deficient mice178. This autism-mouse model shows significantly diminished NMDA receptor function and synaptic distribution in the prefrontal cortex, as well as a marked loss of cortical actin filaments associated with reduced Rac1/PAK activity and increased cofilin activity. The social deficits and NMDA receptor hypofunction displayed by the mice was rescued by inhibiting cofilin activity or by activating Rac1178. In agreement with these results, treatment of Shank2−/− mice with 3-cyano-N-1,3-diphenyl-1H-pyrazol-5-yl benzamide (CDPPB), which increases the responsiveness of mGluR5 to glutamate and enhances NMDA receptor function328, markedly improves autistic-like social behavior327. Targeting the deficits of actin cytoskeleton in the most common inherited form of autism and intellectual disability, FXS, has also shown promise329. Fragile X syndrome is the most common type of inherited mental retardation caused by the absence of FMRP protein, a RNA-binding protein implicated in the regulation of mRNA translation and transport, leading to protein synthesis. The Fmr1 knockout mice display hyperactivity, seizures, repetitive behaviour, and abnormal spine densities similarly to the human condition. Among many other proteins, FMRP negatively regulates expression of Rac1 and pharmacological manipulation of Rac1 partially reverses the altered long-term plasticity seen in Fmr1 KO mice20. Accordingly, by inhibiting the Rac1 effector, group I p21-activated kinase (PAK) protein, which regulates spines through modulation of actin cytoskeleton dynamics, with a drug called FRAX486, the dendritic spine phenotype is reversed in Fmr1 KO mice329. PAK inhibition also rescues seizures and behavioural abnormalities such as hyperactivity and repetitive movements, indicating that targeting spine abnormalities can also treat neurological and behavioural symptoms329. Remarkably, Dolan and colleagues329 showed in 2013 that a single administration of FRAX486 is sufficient for the phenotype rescue in adult Fmr1 knockout mice, demonstrating that a postdiagnostic therapy could be possible for FXS adults. Nutritional factors ACCEPTED affecting ASD have also MANUSCRIPT recently received attention. An increase in postsynaptic zinc (Zn) level induced by clioquinol (a Zn chelator and ionophore), and the subsequent activation of NMDA receptors through the tyrosine kinase Src, rescued social interaction in Shank2−/− mice330. Shank3 mutations and large duplications both cause a spectrum of neuropsychiatric disorders, indicating that the correct protein expression level is critical for normal brain function. Shank3 transgenic mice, modeling a human SHANK3 duplication, exhibit manic-like behavior and seizures consistent with a synaptic excitatory/inhibitory imbalance, similar to the symptoms of two hyperkinetic disorder patients carrying the smallest SHANK3-

ACCEPTED MANUSCRIPT spanning duplications reported so far179. SHANK3 was found to directly interact with Arp2/3 and to increase F-actin levels in the Shank3 transgenic mice, and treating the mice with the mood- stabilizing drug valproate, but not lithium, ameliorated the manic-like behavior179. Consistent with previous results showing that NMDA receptor membrane delivery and stability depend on the integrity of actin cytoskeleton331, these findings further highlight that the dysregulation of synaptic actin filaments and the loss of synaptic NMDA receptors contribute to the manifestation of autism-like phenotypes, and thus provide strategies for the treatment of autism178. Drug trials in human patients have also taken place. One example is the recent therapeutic study on TSC patients with autistic symptoms332. TSC disease is caused by loss-of-function mutations in TSC1 or TSC2 genes94,95, which code the proteins TSC1 and TSC2. TSC1 and TSC2 bind together to form a complex that inhibits mTOR, which controls translation, proliferation, and cell growth333. Treating children and adolescent TSC patients displaying emotional and behavioral symptoms and refractory epilepsy with everolimus, an inhibitor of mTOR, produced beneficial effects in relation to the autistic, ADHD, and depressive symptoms, as well as controlling the seizures332. Consistent with the hypothesis that excessive metabotropic glutamatergic signaling in autism may cause some of the core symptoms of the disorder, several studies using mGluR5 antagonists in animal models of autism have reported beneficial behavioral improvements, with varying side effects in mice334-336. The mGluR5 antagonists that have been evaluated for efficacy in FXS are an example of therapeutics that have been developed to treat a specific disorder, but that might have more general applicability. However, challenges to translate results from FXS animal models to humans include uncertainties regarding optimal patient selection, age of treatment onset, dosages and durations of treatment, differences in pharmacokinetics and pharmacodynamics, dose-limiting side effects, and biomarkers of CNS improvement. In recent years, there have been some preliminary results to indicate that targeted treatment with mGluR5 antagonists may be beneficial for autistic children337. However, to date, there are no reports of successful randomized,ACCEPTED double-blind, placebo MANUSCRIPT-controlled mGluR5 antagonist drug trials on humans. Defining how different ASD-associated mutations disrupt synaptic structure and function may be crucial for the development of therapeutics that target a particular molecular defect. Understanding the molecular basis of ASD will hopefully allow rational development of therapies for a spectrum of autistic disorders.

4. Final words

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Here, we reviewed the literature regarding a number of ASD-associated genes, which encode proteins that regulate actin dynamics. Although synaptic deficiency is only one possible cause underlying the symptoms of ASD, in this review we focused on the roles of ASD-associated actin regulators in the development and function of excitatory synapses, as it is becoming clear from human genetic and brain tissue studies, as well as from mouse models, that dysfunction of the actin cytoskeleton at excitatory spine synapses is likely to be a molecular pathology that is shared across different neurodevelopmental and psychiatric disorders. As the autistic symptoms can be rescued by either manipulating actin regulators or by reversing the dendritic spine density or morphology further emphasizes the importance to understand the role of synaptic actin regulation in ASD. Based on this review, we draw three main conclusions. First, many actin regulators associated with ASDs have a role in the regulation of synaptic structure and/or function, suggesting that a perturbation in the interplay between the PSD and dendritic spine actin cytoskeleton is one of the key cellular processes in ASD pathology. Actin regulators such as SHANKs, can also act as synaptic scaffolding platforms, or they can link postsynaptic scaffolding proteins, such as PSD- 95 to the actin cytoskeleton, as in the case of IRSp53/BAIAP2. Alternatively, proteins can mediate the effects of various receptors on the cytoskeleton; DISC1 mediates the effects of NMDA receptors on Rac1 signaling, whereas -actinin-4 facilitates mGluR-induced actin remodeling105. Furthermore, some proteins, such as gelsolin and -actinin-4, can be regulated by a neuronal activity-induced increase in postsynaptic Ca2+ concentration. Defective stabilization of neuronal structures may also be important in ASD pathology338 as many associated proteins link actin cytoskeleton either to plasma membrane (Irsp53, -actinin-4) or to the extracellular matrix (dystrophin and utrophin) or regulate the connection between actin filaments and extracellular matrix (SHANKs)170. Our second conclusion is that Rho signaling plays a central role in ASD pathology. Here, we evaluated nine different Rho regulators, most of which have well-established roles in dendritic spine or synapse development.ACCEPTED These GEFs andMANUSCRIPT GAPs are often linked to synaptic proteins and mediate the changes in actin dynamics that occur in response to synaptic activity. Finally, we report that the information about the function of different proteins in the brain and in particular the effects of mutations on synapses is still very limited and, therefore, it is difficult to conclude any common defects which would be consistent between different causative genes. However, it is clear that, based on the different pathways associated with ASD, the causative gene products participate in synaptic plasticity by modulating synaptic strength and/or number, and that, in ASD

ACCEPTED MANUSCRIPT animal models, mutations in the ASD-associated genes result in synaptic activity and synaptic density that are either too high or too low. To better understand the pathophysiology of ASDs, we would need comprehensive information on a) the functions of ASD-associated proteins in the brain, b) how mutations affect the expression level and function of these proteins, c) how mutations affect their function in neurons, and d) how changed neuronal function impacts the circuits and behavior. We currently have a good overview of the different pathways affected in ASD and the next step will be to characterize the proteins encoded by these genes, as well as the effects of mutations. The recent CAMK2A study121 provides a good example on how we should proceed with each protein and mutation.

Authors contributions M. Joensuu, V. Lanoue and P. Hotulainen co-wrote the manuscript. V. Lanoue prepared the schematic figures and M. Joensuu the tables.

Acknowledgements We thank Rowan Tweedale (Queensland Brain Institute, The University of Queensland) and Iryna Hlushchenko (Minerva Minerva Foundation Institute for Medical Research) for critical appraisal of the manuscript.

Funding M. Joensuu is supported by The Academy of Finland Postdoctoral Research Fellowship (298124) and P. Hotulainen by Instrumentarium Foundation senior researcher fellowship.

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Figure legends Figure 1. Regulators of actin polymerization and depolymerization. CYFIP1 and DRF3 regulate actin polymerization while GSN severs F-actin filament. CTTNB2 is involved in the branching and the stabilization of the actin . In colour are the proteins linked to ASD and in grey their cytoplasmic partners. Figure 2. Actin cross-linking proteins. ACTN4 antiparallel dimers are a link between membrane receptors and actin cytoskeleton. Villin is a Ca2+-regulated actin binding protein. In colour are the proteins linked to ASD and in grey their cytoplasmic partners. Figure 3. Actin-binding myosin motors. MYO16 is a molecular motor with an ATPase activity; MYO9B is a motorized RhoGAP that inhibits RhoA. In colour are the proteins linked to ASD and in grey their cytoplasmic partners. Figure 4. Dystrophin and Utrophin. These cohesive proteins form a link between the extracellular matrix and the cytoplasm. In colour are the proteins linked to ASD and in grey their cytoplasmic partners. Figure 5. Synaptic scaffolding proteins. Scaffolding proteins scaffold numerous proteins to complexes and link these complexes to the cytoskeleton. In colour are the proteins linked to ASD and in grey their cytoplasmic partners. Figure 6. RhoGTPases regulation. GTPase-activating proteins (GAP) and guanine-exchange factors (GEF) play a key role in modulating the activity of RhoGTPases, central regulators of intracellular actin dynamics. In colour are the proteins linked to ASD and in grey their cytoplasmic partners.

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Highlights

 The autistic symptoms can be rescued by manipulating actin regulators in Shank3 or Fmr1-deficient mice.  Many actin regulators associated with ASDs have a role in the regulation of synaptic structure and/or function  Rho signaling plays a central role in ASD pathology  Dysfunction of the actin cytoskeleton at excitatory spine synapses is likely to be a molecular pathology that is shared across different neurodevelopmental and psychiatric disorders.

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