REVIEW
published: 17 November 2016
doi: 10.3389/fncel.2016.00263
Edited by:
Hansen Wang,
University of Toronto, Canada
Reviewed by:
Elva Diaz,
University of California, Davis, USA
Annalisa Scimemi,
University at Albany, SUNY, USA
Nicoletta Landsberger,
University of Milan, Italy
*Correspondence:
Yu-Chih Lin
yclin@hussmanautism.org
Received: 19 July 2016
Accepted: 28 October 2016
Published: 17 November 2016
Citation:
Lin Y -C, Frei JA, Kilander MBC,
Shen W and Blatt GJ (2016)
A Subset of Autism-Associated
Genes Regulate the Structural
Stability of Neurons.
Front. Cell. Neurosci. 10:263.
doi: 10.3389/fncel.2016.00263
A Subset of Autism-Associated
Genes Regulate the Structural
Stability of Neurons
Yu-Chih Lin
1
*
, Jeannine A. Frei
1
, Michaela B. C. Kilander
1
, Wenjuan Shen
1
and
Gene J. Blatt
2
1
Laboratory of Neuronal Connectivity, Program in Neuroscience, Hussman Institute for Autism, Baltimore, MD, USA,
2
Laboratory of Autism Neurocircuitry, Program in Neuroscience, Hussman Institute for Autism, Baltimore, MD, USA
Autism spectrum disorder (ASD) comprises a range of neurological conditions that
affect individuals’ ability to communicate and interact with others. People with ASD
often exhibit marked qualitative difficulties in social interaction, communication, and
behavior. Alterations in neurite arborization and dendritic spine morphology, including
size, shape, and number, are hallmarks of almost all neurological conditions, including
ASD. As experimental evidence emerges in recent years, it becomes clear that
although there is broad heterogeneity of identified autism risk genes, many of them
converge into similar cellular pathways, including those regulating neurite outgrowth,
synapse formation and spine stability, and synaptic plasticity. These mechanisms
together regulate the structural stability of neurons and are vulnerable targets in ASD.
In this review, we discuss the current understanding of those autism risk genes
that affect the structural connectivity of neurons. We sub-categorize them into (1)
cytoskeletal regulators, e.g., motors and small RhoGTPase regulators; (2) adhesion
molecules, e.g., cadherins, NCAM, and neurexin superfamily; (3) cell surface receptors,
e.g., glutamatergic receptors and receptor tyrosine kinases; (4) signaling molecules,
e.g., protein kinases and phosphatases; and (5) synaptic proteins, e.g., vesicle and
scaffolding proteins. Although the roles of some of these genes in maintaining neuronal
structural stability are well studied, how mutations contribute to the autism phenotype is
still largely unknown. Investigating whether and how the neuronal structure and function
are affected when these genes are mutated will provide insights toward developing
effective interventions aimed at improving the lives of people with autism and their
families.
Keywords: autism-risk genes, neurite outgrowth, dendrite, dendritic spine, synapse formation, actin, adhesion
molecule
INTRODUCTION
Autism spectrum disorder (ASD) is a neurodevelopmental clinical condition currently diagnosed
based on the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental
Disorders, Fifth Edition (DSM-5) criteria reflecting symptoms, possibly of varying severity, in
social interaction, communication and behavior (American Psychiatric Association, 2013; Lord
and Jones, 2013). ASD occurs in 1:68 individuals in the United States (Baio, 2014) and complex
Frontiers in Cellular Neuroscience | www.frontiersin.org 1 November 2016 | Volume 10 | Article 263
Lin et al. Autism Genes Regulate Neuronal Structures
genetic interactions appear responsible for a high degree of
heterogeneity of the clinical symptoms in ASD. Individuals
with ASD often co-express other comorbidities including
epilepsy which often complicates diagnosis and treatment.
Alterations in neuronal structures in different brain regions
have been reported in ASD individuals, including increased
dendritic spine density in cortical pyramidal neurons (
Hutsler
and Zhang, 2010; Tang et al., 2014) as well as stunting
of dendritic branching in the hippocampus (Raymond
et al., 1996
; Bauman and Kemper, 2005). In addition,
subcortical band heterotopia, representing alterations in
cell migration has also been found in a child with ASD
(Beaudoin et al., 2007). These brain regions are often
characterized with neuroanatomical irregularities in ASD
(Donovan and Basson, 2016). The defective regulation for
structural stability of neurons may be one of the underlying
mechanisms that contribute to the anatomical changes
in ASD.
Autism spectrum disorder is typically diagnosed during the
first 3 years of life, a period of extensive neurite formation,
synaptogenesis and refinement (
Huttenlocher and Dabholkar,
1997; Zoghbi and Bear, 2012; St amou et al., 2013; McGee et al.,
2014). Indeed, brain imaging studies from individuals with
ASD and anatomical measurements of neuronal structure in
post-mortem tissues exhibit differences in neuronal connectivity
derived from the disruption of neurite outgrowth, synapse
formation and stabilization (
Raymond et al., 1996; Hutsler and
Zhang, 2010; Penzes et al., 2011). Studies of human induced
pluripotent stem cells (iPSCs) derived from people with ASD
also have identified defects of neuronal structure (Habela et al.,
2015; Nestor et al., 2015). Genome-wide association studies on
individuals with ASD and their f amilies revealed several risk
genes that may be the common molecular targets in autism
(
Bucan et al., 2009; Glessner et al., 2009; Hussman et al., 2011;
O’Roak et al., 2011, 2012a; Buxbaum et al., 2012, 2014; Sanders
et al., 2012; Shi et al., 2013; Stamou et al., 2013; Yu et al.,
2013; An et al., 2014; Brett et al., 2014; Cukier et al., 2014; De
Rubeis et al., 2014; Iossifov et al., 2014; McGee et al., 2014;
Pinto et al., 2014; Ronemus et al., 2014; Toma et al., 2014;
Yuen et al., 2015). Animal studies of these genes further identify
several specific cellular pathways during brain development
that are vulnerable in ASD, including the disruption of neurite
outgrowth, dendritic spine formation, and synaptic function
(Figure 1) (
Walsh et al., 2008; Bourgeron, 2009; Hussman
et al., 2011; Penzes et al., 2011; Zoghbi and Bear, 2012; Ebert
and Greenberg, 2013; Stamou et al., 2013; Bernardinelli et al.,
2014; De Rubeis et al., 2014; Pinto et al., 2014; Phillips and
Pozzo-Miller, 2015
). Differences in environment as well as the
presence of multiple gene mut ations occurring in the s ame
individual wit h autism complicate studies of the relationship
between each gene and the phenotype observed. However,
because similar cellular pathways (e.g., neurite outgrowth) are
altered in different affected individuals, we can potentially
develop therapeutic interventions to help mitigate the autism
phenotypes.
During development, neurite outgrowth and synapse
formation are dynamic processes and their maturation is
FIGURE 1 | Diagram of autism-risk genes implicated in regulating the
structural stability of neurons. Each circle represents a cellular pathway to
regulate the structural stability of neurons, including neurite outgrowth (red),
dendritic spine or synapse formation (blue), and synaptic plasticity (gold).
Experimental evidence shows that many autism-risk genes regulate at least
one cellular pathway to maintain the integrity of neuronal structures. Genes
that regulate only one pathway are labeled in light gray. Genes that regulate
two pathways are labeled in dark gray. Genes that regulate three pathways
are labeled in black. The summaries of autism-risk genes that affect each
cellular pathway can be found in Tables 1–3.
mutually dependent on proper guidance. Neurites initially
exhibit frequent branch additions and retractions. Once dendrite
arbors are established, productive synapse formation later in
life and the accompanying activation of post-synaptic signaling
machinery promotes arbor stability (
Dailey and Smith, 1996;
Wu and Cline, 1998; Rajan et al., 1999; Wong et al., 2000; Cline,
2001; Niell et al., 2004). Conversely, a loss of synaptic inputs
leads to dendritic loss (
Jones and Thomas, 1962; Matthews
and Powell, 1962; Coleman and Riesen, 1968; Sfakianos et al.,
2007). This reciprocal regulation contributes to the refinement
of dendrites and synapses as the neurons mature (Wu et al.,
1999; Trachtenberg et al., 2002; Holtmaat et al., 2005; Koleske,
2013). Thus, maintaining the structural stability of neurons
and synapses is critical for proper brain function. Alterations
in these processes likely underlie the disruption of normal
dendrite and dendritic spine structure in neurological disorders,
including neurodevelopmental conditions, psychiatric disorders,
and neurodegenerative diseases (Fiala et al., 2002; Lin and
Koleske 2010; Penzes et al., 2011; Kulkarni and Firestein, 2012;
Zoghbi and Bear, 2012; Koleske, 2013; Bernardinelli et al.,
2014
).
It is well-accepted that ASD is not a monogenetic disorder,
instead, it is often a neurologic al condition resulted from multiple
mutations of several different genes. Although knockout,
knockin, or transgenic approaches of autism-risk genes in
animal models have demonstrated some of the autistic-like
behaviors (Kazdoba et al., 2016), the limitation of the number
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Lin et al. Autism Genes Regulate Neuronal Structures
TABLE 1 | Summary of autism-associated genes that regulate neurite outgrowth.
Functional category Gene Representative references
Cytoskeletal regulator
MYO16 Patel et al., 2001; Yokoyama et al., 2011
CTTNBP2 Shih et al., 2014
ELMO1 Franke et al., 2012; Lanoue et al., 2013
Adhesion molecule
CDHs
Esch et al., 2000; Bekirov et al., 2008; Tan et al., 2010; Friedman et al., 2015b
PCDH Uemura et al., 2007; Morrow et al., 2008; Keeler et al., 2015
NRXN Gjorlund et al., 2012
NLGN Gjorlund et al., 2012
CNTNAP2 Anderson et al., 2012
CNTN Ye et al., 2008
NCAM2 Sheng et al., 2015
Surface receptor
GRIN2B Ewald et al., 2008; Espinosa et al., 2009; Sepulveda et al., 2010; Bustos et al., 2014
NTRK Joo et al., 2014
Signaling molecule
DYRK1A
Hammerle et al., 2003; Benavides-Piccione et al., 2005; Gockler et al., 2009; Lepagnol-Bestel et al.,
2009
CDKL5 Chen et al., 2010b; Amendola et al., 2014; Fuchs et al., 2014
PTEN Jaworski et al., 2005; Kwon et al., 2006; Zhou et al., 2009
Synaptic protein
STXBP5 Sakisaka et al., 2004
PRICKLE1 Liu et al., 2013a
Syndromic disorder related gene
FMR1
Galvez et al., 2003; Antar et al., 2006; Tucker et al., 2006; Berman et al., 2012; Amiri et al., 2014
MECP2 Fukuda et al., 2005; Jugloff et al., 2005; Zhou et al., 2006; Ballas et al., 2009; Belichenko et al., 2009;
Kishi and Macklis, 2010; Cohen et al., 2011; Marshak et al., 2012; Nguyen et al., 2012; Stuss et al.,
2012
; Jiang et al., 2013a; Baj et al., 2014
UBE3A Dindot et al., 2008; Miao et al., 2013; Valluy et al., 2015
TSC1/2 Floricel et al., 2007; Choi et al., 2008
of genes being manipulated in animals makes it difficult to
recapitulate the human condition experimentally. Furthermore,
ASD is a common comorbid condition in individuals with other
neurodevelopmental disorders. The similar representation of
the symptoms but different contribution of genetic mutations
often complicates the diagnosis and the treatment. The complex
profile of gene mutations makes it difficult to call a gene
“the autism gene.” However, the list of autism-risk genes
provides us a direction to understand the potentially vulnerable
pathways in neurons that may be therapeutic targets to
develop more efficient interventions for ASD. Indeed, in
addition to the structural stability of neurons, several cellular
pathways including transcriptional regulation (
De Rubeis et al.,
2014; Sanders, 2015), excitatory/inhibitory (E/I) balance (Blatt
et al., 2001; Hussman, 2001; Rubenstein and Merzenich, 2003;
Gao and Penzes, 2015; Nelson and Valakh, 2015), cerebellar
development (Wang et al., 2014; Hampson and Blatt, 2015),
and autoregulatory feedback loops (Mullins et al., 2016)
have been proposed to be vulnerable in autism. In this
review, we focus on recent identified autism-risk genes t hat
have been shown to regulate neuronal structures and circuit
formation, including aspects of neurite outgrowt h (Table 1),
synapse formation and spine stability (Table 2), and synaptic
plasticity (Table 3). We will discuss the known biological
function of those individual autism-risk genes in neurons
and how they converge into common pathways. We have
categorized these genes into cytoskeletal regulators, adhesion
molecules, cell surface receptors, signaling molecules, as well
as synaptic proteins (Figure 2). In addition, we include genes
causing syndromic disorders in the discussion to highlight the
importance of maintaining the neuronal structures for proper
brain function.
CYTOSKELETAL REGULATION IS KEY
TO THE MAINTENANCE OF PROPER
STABILITY AND PLASTICITY OF
NEURONS
The actin and microtubule cytoskeletons are the major
components of dendritic spine and neurite structure,
respectively (
Hoogenraad and Akhmanova, 2010; Hotulainen
and Hoogenraad, 2010; Dent et al., 2011; Shirao and Gonzalez-
Billault, 2013). Precise regulation of these actin and microtubule
networks is thus central to guide the proper development,
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Lin et al. Autism Genes Regulate Neuronal Structures
TABLE 2 | Summary of autism-associated genes that regulate synapse/spine formation.
Functional
category
Gene Representative references
Cytoskeletal regulator
ADNP
Oz et al., 2014
CTTNBP2 Chen and Hsueh, 2012; Chen et al., 2012; Hsueh, 2012
SYNGAP1 Chen et al., 1998; Krapivinsky et al., 2004; Oh et al., 2004; Rumbaugh et al., 2006; Clement et al., 2012; Aceti
et al., 2015
ELMO1 Kim et al., 2011a
Adhesion molecule
CDHs Benson and Tanaka, 1998; Huntley and Benson, 1999; Bozdagi et al., 2000; Togashi et al., 2002; Paradis et al.,
2007
; Suzuki et al., 2007; Bekirov et al., 2008; Williams et al., 2011; Friedman et al., 2015b
PCDH Tsai et al., 2012a
NRXN de Wit et al., 2009; Ko et al., 2009; Gokce and Sudhof, 2013
NLGN Scheiffele et al., 2000; Prange et al., 2004; Chih et al., 2005; Levinson et al., 2005; Varoqueaux et al., 2006;
Kwon et al., 2012;
CNTNAP2 Anderson et al., 2012; Gdalyahu et al., 2015; Varea et al., 2015
CNTN Li et al., 2003; Takeda et al., 2003; Sakurai et al., 2009, 2010; Toyoshima et al. 2009
Surface receptor
GRIN2B
Akashi et al., 2009; Espinosa et al., 2009; Brigman et al., 2010; Ohno et al., 2010; Kelsch et al., 2012
GRIK2/4 Huettner, 2003; Lanore et al., 2012
NTRK Menn et al., 2000
Signaling molecule
DYRK1A Benavides-Piccione et al., 2005; Park et al., 2012; Thomazeau et al., 2014
CDKL5 Zhu et al., 2013; Della Sala et al., 2016
PTEN Kwon et al., 2006; Fraser et al., 2008; Luikart et al., 2011; Pun et al., 2012; Zhang et al., 2012; Haws et al.,
2014
; Cupolillo et al., 2016
Synaptic protein
SHANK3 Sala et al., 2001; Roussignol et al., 2005; Hung et al., 2008; Peça et al., 2011; Verpelli et al., 2011; Wang et al.,
2011b
; Peixoto et al., 2016
DLGAP2 Jiang-Xie et al., 2014
Syndromic disorder related gene
FMR1 Comery et al., 1997; Weiler and Greenough, 1999; Braun and Segal, 2000; Irwin et al., 2000; Nimchinsky et al.,
2001
; Segal et al., 2003; Galvez and Greenough, 2005; Koekkoek et al., 2005; McKinney et al., 2005; Antar
et al., 2006
; Grossman et al., 2006, 2010; Dictenberg et al., 2008; Cruz-Martin et al., 2010; Pan et al., 2010;
Levenga et al., 2011a; Qin et al., 2011; Berman et al., 2012; He and Portera-Cailliau, 2013; Amiri et al., 2014;
Wijetunge et al., 2014
MECP2 Fukuda et al., 2005; Zhou et al., 2006; Chapleau et al., 2009; Nguyen et al., 2012; Stuss et al., 2012; Jiang
et al., 2013a
; Baj et al., 2014
UBE3A Dindot et al., 2008; Greer et al., 2010; Yi et al., 2015; Kim et al., 2016; Valluy et al., 2015
TSC1/2 Tavazoie et al., 2005
plasticity, and long-term stability of these structures (Matus,
2000; Matus et al., 2000; Luo, 2002; Lin and Webb, 2009;
Korobova and Svitkina, 2010; Lin and Koleske, 2010; Svitkina
et al., 2010; Dent et al., 2011; Nicholson et al., 2012; Penzes
and Cahill, 2012; Penzes and Rafalovich, 2012; Saneyoshi and
Hayashi, 2012).
Actin and Microtubule Regulators Are
Associated with Autism
Myosins are motors that utilize ATPase activity to provide
motility of actin or cargo transport on actin filaments (Pollard
and Korn, 1973; Oliver et al., 1999; Tyska and Warshaw,
2002). Several myosin isoforms play central roles in regulating
neurite outgrowth, as well as dendritic spine structural plasticity
(Wylie et al., 1998; Wylie and Chantler, 2003; Ryu et al.,
2006; Hammer and Wagner, 2013; Kneussel and Wagner,
2013; Yoshii et al., 2013; Koskinen et al., 2014; Ultanir et al.,
2014). Among all isoforms, MYO16 (Myr8 or NYAP3) was
recently implicated in ASD (Wang et al., 2009; Connolly
et al., 2013; Kenny et al., 2014; Roberts et al., 2014; Liu
et al., 2015b). MYO16 is expressed predominantly in the
cortex and cerebellum. Le vels and phosphorylation of MYO16
protein peak during early developmental stages, consistent
with a role in regulating neuronal migration and neurite
extension (
Patel et al., 2001; Yokoyama et al., 2011). In addition
to binding directly to filamentous- (F-)actin, MYO16 also
physically interacts with PI3K and WAVE complex to regulate
stress fiber remodeling in fibroblasts as well as the adhesion-
dependent neurite outgrowth in neurons (
Yokoyama et al.,
2011
).
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Lin et al. Autism Genes Regulate Neuronal Structures
TABLE 3 | Summary of autism-associated genes that regulate synaptic plasticity.
Functional category Gene Representative references
Cytoskeletal regulator
SYNGAP1 Vazquez et al., 2004; Rumbaugh et al., 2006; Carlisle et al., 2008; Clement et al., 2012, 2013; Ozkan et al.,
2014
Adhesion molecule
CDHs Bozdagi et al., 2000; Manabe et al., 2000; Togashi et al., 2002; Bozdagi et al., 2010; Mendez et al., 2010
NRXN Levinson et al., 2005; Etherton et al., 2009
NLGN Chih et al., 2005; Varoqueaux et al., 2006; Chubykin et al., 2007; Tabuchi et al., 2007
Surface receptor
GRIN2B
Brigman et al., 2010; Ohno et al., 2010; Wang et al., 2011a; Yang et al., 2012a; Ryan et al., 2013; Dupuis
et al., 2014
GRIK2/4 Contractor et al., 2001; Huettner, 2003; Youn and Randic, 2004; Lanore et al., 2012; Aller et al., 2015
Signaling molecule
CDKL5 Della Sala et al., 2016
PTEN Fraser et al., 2008; Luikart et al., 2011
Synaptic protein
SHANK3 Bangash et al., 2011; Peça et al., 2011; Wang et al., 2011b; Peixoto et al., 2016
DLGAP2 Jiang-Xie et al., 2014
STXBP5 Barak et al., 2013; Ben-Simon et al., 2015
Syndromic disorder related gene
FMR1 Segal et al., 2003; Koekkoek et al., 2005; Bureau et al., 2008; Auerbach and Bear, 2010
MECP2 Collins et al., 2004; Dani et al., 2005; Asaka et al., 2006; Moretti et al., 2006; Chao et al., 2007; Zhang
et al., 2008
; Cohen et al., 2011; Li et al., 2011; Noutel et al., 2011; Blackman et al., 2012; Na et al., 2012;
Qiu et al., 2012; Zhong et al., 2012; Na et al., 2013; Della Sala and Pizzorusso, 2014; Deng et al., 2014; De
Filippis et al., 2015
UBE3A Yashiro et al., 2009; Sato and Stryker, 2010; Smith et al., 2011; Hayrapetyan et al., 2014
CTTNBP2 encodes cortactin-binding protein 2 t hat interacts
with cortactin, a nucleation-promoting factor of actin (Ohoka
and Takai, 1998
). CTTNBP2 is highly expressed in dendritic
spines where it locally interacts with cortactin, striatin, a
calcium binding protein, and PP2A, a serine/threonine protein
phosphatase 2A, to control the formation and the maintenance
of dendritic spines (
Chen et al., 2012; Chen and Hsueh, 2012;
Hsueh, 2012). In addition, oligomerization of CTTNBP2 induces
microtubule bundling to promote dendrite arborization (
Shih
et al., 2014). Several mutations of CTTNBP2 have been reported
in ASD cases, further indicating the importance of neurite
outgrowth and dendritic spine formation for proper brain
function (Cheung et al., 2001; Iossifov et al., 2012; De Rubeis et al.,
2014).
Activity-dependent neuroprotective protein (ADNP) is a
homeobox-containing protein secreted from glia and neurons
(Bassan et al., 1999; Zamostiano et al., 2001; Furman et al.,
2004
; Mandel et al., 2008; Nakamachi et al., 2008). Through its
interaction with the chromatin remodeling complex SWI/SNF,
ADNP regulates hundreds of genes to modulate brain function
(
Pinhasov et al., 2003; Mandel and Gozes, 2007; Mandel
et al., 2007). In addition to its traditional role in regulating
transcription, ADNP has also been suggested to have function
in regulating dendritic spines through interactions with
microtubule end binding proteins (
Oz et al., 2014). Mutations
in or the alteration of the protein expression of ADNP have
been associated wit h several neurological disorders, including
schizophrenia and Alzheimer’s Disease (Vulih-Shultzman et al.,
2007; Fernandez-Montesinos et al., 2010; Dresner et al., 2011;
Yang et al., 2012b). Intriguingly, the association of mutations
in ADNP and ASD is further emphasizing that the cytoskeletal
integrity of neurons is vulnerable in ASD (O’Roak et al.,
2012a,b; Ben-David and Shifman, 2013; De Rubeis et al., 2014;
Helsmoortel et al., 2014; Vandeweyer et al., 2014; D’Gama et al.,
2015).
Small RhoGTPase Regulation Is a Key
Mechanism in Controlling Neurite and
Spine Stability
Small RhoGTPases including Rho, Rac, and Cdc42 are central
cytoskeletal re gulators that control cell motility and morphology
(
Govek et al., 2005; Newey et al., 2005; Lin and Koleske,
2010; Tolias et al., 2011). Genetic mutations or dysregulation
of the small RhoGTPase regulators, including guanine-exchange
factors (GEFs) and GTPase-activating proteins (GAPs), have
been implicated in several neurological conditions, including
ASD (Newey et al., 2005; Lin and Koleske, 2010; Antoine-
Bertrand et al., 2011; Stankiewicz and Linseman, 2014). Here, we
will hig hlight those that regulate the morphological stability of
neurons.
Engulfment and cell motility 1 (ELMO1) was first identified in
a complex with a RacGEF, DOCK180, to activate Rac1 activity,
which is essential for cell migration and phagocytosis (
Gumienny
et al., 2001; Brugnera et al., 2002; Grimsley et al., 2004). In
hippocampal neurons, ELMO1 and DOCK180 colocalize at
synaptic sites and together are required for spine formation
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