the tiny protrusions emerging from dendrites known as dendritic spines are the primary subcellular locations of excitatory synapses in the mammalian central nervous system .
dendritic spines are typically ~1 - 2  m in length and 0.51  m in width of the spine head , with diverse morphologies , such as mushroomlike , stubby , and thin spines . these structures are mainly supported by the f - actin cytoskeleton .
thus , f - actin cytoskeletal proteins and regulators are important factors for generating dendritic spines .
many membrane proteins and adaptor and signaling molecules are also involved in controlling dendritic spine formation and maintenance .
the most popular mechanism is that dendritic filopodia serve as precursors for dendritic spine formation .
interestingly , filopodia are ubiquitously found on various cell types . in contrast , dendritic spines are neuron - specific structures .
thus , the transition from filopodia to spines should be controlled by neuron - specific factors .
these proteins or responses directly or indirectly regulate f - actin rearrangement and dynamics to promote dendritic spine formation .
studies of cytoskeleton - associated cortactin - binding protein 2 ( cttnbp2 ) and heparan sulfate transmembrane proteoglycan ( hspg ) syndecan-2 serve as examples for these two categories , respectively .
cttnbp2 is a neuron - specific cytoskeleton - associated protein and that is enriched at dendritic spines of mature neurons .
although syndecan-2 is widely expressed in many cell types , it is highly concentrated at synapses in neurons .
syndecan-2 cooperates with other proteins to trigger neurotransmission through a neuron - specific signal to induce dendritic spine formation .
genomic analyses of patients with autism spectrum disorders ( asds ) indicated that both cttnbp2 and syndecan-2 were associated with asds [ 4 , 5 ] .
additionally , neurofibromin , cask , and vcp coordinate with syndecan-2 to control dendritic spinogenesis and were also associated with neurological disorders .
these findings suggest that these genes are critical for neuronal function , likely through their regulation of dendritic spine formation . in this review
, we will summarize the functions of these proteins in dendritic spinogenesis and use these proteins as examples to discuss how neuron - specific molecules coordinate with ubiquitously expressed proteins to generate neuron - specific signals for dendritic spine formation .
syndecan-2 is a type i membrane protein with a heparan sulfate modification at its ectodomain ( figure 1 ) . in mammals ,
the syndecan protein family contains four members , syndecan-1 , syndecan-2 , syndecan-3 , and syndecan-4 . in rodent brains ,
syndecan-2 and syndecan-3 are the two major syndecans expressed in neurons with differential distribution ; syndecan-2 is highly concentrated at synapses , while syndecan-3 is distributed along the axonal shaft .
syndecan-2 is involved in cell - cell and cell - matrix interactions through its heparan sulfate modification .
it can also bind growth factors , such as fibroblast growth factor ( fgf ) and epidermal cell growth factor , and it acts as a coreceptor for these growth factors .
syndecan-2 is broadly and dynamically expressed in several tissues and cell types [ 7 , 8 ] . during neural development ,
its expression gradually increases concurrent with synapse formation [ 8 , 9 ] . in mature neurons , such as cultured rat hippocampal neurons at 18 days after plating in vitro ( div ) or later
more importantly , overexpression of syndecan-2 in immature rat hippocampal cultured neurons , such as 1 - 2 div , when endogenous syndecan-2 is not yet expressed , dendritic filopodia are massively induced at 4 - 5 div and dendritic filopodia are then transformed to dendritic spines at 8 - 9 div [ 9 , 11 ] . those dendritic spines are expected to be functional , as they are adjacent to the presynaptic marker synaptophysin based on confocal microscopy [ 11 , 12 ] . syndecan-2-induced dendritic spinogenesis serves as a model to explore the mechanisms underlying the initiation of dendritic spinogenesis ( namely , dendritic filopodia formation ) , the transition from filopodia to spines , and dendritic spine maturation and maintenance .
the ectodomain of syndecan-2 heparan sulfate modification is involved in cell - cell and cell - matrix interactions .
its transmembrane domain is required for homodimerization or oligomerization , which is critical for the protein - protein interactions of syndecan-2 .
the cytoplasmic domain of syndecan-2 contains only 32 amino acid residues ( figure 1 ) .
although it is short , it is divided into three motifs , conserved domain 1 ( c1 ) , the variable region ( v ) , and conserved domain 2 ( c2 ) .
the c1 and c2 motifs are conserved among different syndecans , while the sequences of the v regions vary ( figure 1 ) .
the c1 motif is essential for syndecan-2-induced dendritic filopodia formation of rat hippocampal cultured neurons , as the syndecan-2c1 mutant completely loses the ability to promote filopodia formation and spine formation at 5 as well as 9 div [ 11 , 15 ] .
the c2 is required for the dendritic filopodia - spines transition and dendritic spine maintenance [ 15 , 16 ] .
expression of the c2 deletion mutant syndecan-2c2 at 2 div promotes dendritic filopodia formation at 5 div .
however , those filopodia are unable to transform into dendritic spine at 9 div [ 11 , 15 , 16 ] .
these analyses indicate that the function of syndecan-2 in dendritic spinogenesis can be separated into two sequential steps , namely , filopodia and spine formation , which are controlled by two distinct motifs in syndecan-2 . because both c1 and c2 motifs are short and lack recognizable enzymatic domains , syndecan-2 binding partners have been identified to determine its molecular mechanism underlying dendritic spine formation .
several direct binding partners ( summarized in table 1 ) have been identified for the c1 domains of syndecan-2 , including neurofibromin   and ezrin .
, the interactions between syndecan-2 and neurofibromin and cask have been shown to be relevant in dendritic spine formation .
because the cytoplasmic tail of syndecan-2 is very short , it is unlikely that a single syndecan-2 molecule can simultaneously interact with all of its binding partners .
because the c1 and c2 motifs are involved in two sequential processes , it is likely that neurofibromin and cask sequentially interact with syndecan-2 .
alternatively , it is possible that because syndecan-2 forms at least a dimer through its transmembrane domain , different syndecan-2 molecules in dimers or oligomers separately interact with neurofibromin and cask
. this would suggest that syndecan-2 , neurofibromin , and cask form a single large complex .
neurofibromin encoded by the neurofibromatosis type i ( nf1 ) gene is characterized by its rasgap- ( ras gtpase activating protein- ) related domain ( grd ) ( figure 2(a ) ) [ 2124 ] .
similar to syndecan-2 , neurofibromin is widely expressed in different cell types , though its expression level is much higher in the nervous system .
nf1 is one of the most common human inherited disorders featured by changes in skin pigmentation , benign tumor growth , and learning difficulty [ 26 , 27 ] .
in addition to its ras activity , neurofibromin can increase camp concentration by activating adenylate cyclase .
although the molecular mechanisms are less clear , the grd and c - terminal region of neurofibromin are required for camp pathway activation ( figure 2(a ) ) .
both gs - dependent and gs - independent pathways are involved in neurofibromin - regulated adenylate cyclase activation .
the camp pathway has been shown to be involved in learning and memory in drosophila   and dendritic spine formation in the mammalian nervous system . in a yeast two - hybrid screen using different fragments of neurofibromin as baits , syndecan-2 was identified as a neurofibromin binding partner .
one is the jn fragment corresponding to amino acid residues 13571473 in the grd of human neurofibromin ; the other is the pn fragment containing amino acid residues 26192719 ( figure 2(a ) ) .
in addition to biochemical studies demonstrating the direct interaction between syndecan-2 and neurofibromin , fluorescence immunostaining further demonstrated the colocalization of syndecan-2 and neurofibromin at synapses in cultured hippocampal neurons .
moreover , both nf1 knockdown and haploinsufficiency reduce the density of dendritic spines in both rat hippocampal and mouse cortical cultured neurons and in brains [ 11 , 32 ] , consistent with a function of neurofibromin in regulating dendritic spine formation .
one study examined syndecan-2 downstream signaling for triggering filopodia formation . using a panel of inhibitors to suppress various kinase activities , protein kinase a ( pka ) was identified to be required for syndecan-2-induced filopodia formation .
combined with the analysis using different motif deletion mutants of syndecan-2 , we found that the c1 motif of syndecan-2 is essential for pka - dependent filopodia formation . because neurofibromin interacts with the c1 motif and also activates the camp pathway , cultured hippocampal neurons
were then used to investigate whether neurofibromin mediates syndecan-2-induced filopodia formation . both nf1 knockdown and jn fragment expression , which acts as a dominant - negative to disrupt the interaction between endogenous neurofibromin and syndecan-2 ,
thus , neurofibromin mediates the signal from syndecan-2 to the camp pathway to initiate dendritic spinogenesis .
because filopodia are supported by f - actin bundles , the syndecan-2-neurofibromin - camp pathway has to induce f - actin polymerization and bundle formation to promote dendritic filopodia formation .
the ena ( enabled)/vasp ( vasodilator - stimulated phosphoprotein ) protein family is a group of f - actin regulators that initiate actin polymerization and bundling .
pka phosphorylation promotes ena / vasp protein activity to regulate the f - actin cytoskeleton . upon syndecan-2 overexpression , ena / vasp phosphorylation increases , consistent with camp pathway activation
moreover , disruption of ena / vasp activity impairs syndecan-2-induced dendritic filopodia formation . in summary ,
these studies indicate that syndecan-2 overexpression enhances the ability of neurofibromin to activate the pka pathway , which then induces the ena / vasp activity to promote f - actin bundling and filopodia formation .
although the pka pathway is required for dendritic filopodia formation , increased intracellular camp concentrations alone can not induce dendritic filopodia formation , suggesting that other factor(s ) are involved . from an immunoprecipitation - mass spectrometry study , valosin - containing protein ( vcp , also known as p97 ) was identified as a neurofibromin - binding protein .
the entire d1 and d2 atpase domains of vcp are required for the interaction with the leucine - rich domain ( lrd ) of neurofibromin .
vcp is a causative gene of inclusion body myopathy associated with paget 's disease of bone and frontotemporal dementia ( ibmpfd ) .
in addition , vcp mutations are associated with asds and amyotrophic lateral sclerosis [ 36 , 37 ] .
a combination of human genetic studies , mouse genetic models , and cultured hippocampal and cortical neurons have indicated that neurofibromin interacts with vcp and guides vcp to promote dendritic spinogenesis .
the roles of vcp and neurofibromin in dendritic spine formation may account for the neural phenotypes in patients with mutations in the nf1 and vcp genes .
however , it is still unclear how vcp regulates dendritic spine formation . to fully address the molecular regulation of neurofibromin and vcp in dendritic spinogenesis ,
the function of the syndecan-2-neurofibromin interaction in dendritic spine formation is summarized in figure 2(b ) .
mutations in the human cask gene result in x - linked mental retardation and microcephaly with pontine and cerebellar hypoplasia [ 3943 ] .
cask belongs to the membrane - associated guanylate kinase ( maguk ) family and functions as a scaffold protein to interact with more than two dozen cellular proteins .
it is widely distributed in neurons , including synapses , dendrites , axons , and soma . at synapses ,
pontine explants and rat hippocampal cultured neurons , cask knockdown impairs synapse formation at the pre- and postsynapse , respectively [ 16 , 45 ] .
at presynaptic sites , it binds the membrane protein neurexin and other scaffold proteins , such as mint1 , mlin7 , and liprin , to control presynaptic button formation [ 4548 ] .
cask uses its pdz domain at the postsynaptic site to interact with the c2 motif of syndecan-2 . in cultured hippocampal neurons , expression of the pdz alone of cask or the c - terminal tail of syndecan-2 that disrupts
the interaction between endogenous cask and syndecan-2 reduces dendritic spine density , narrows spine heads , and shortens spine length at 18 div , suggesting that the cask - syndecan-2 interaction is critical for dendritic spine formation . to investigate whether cask is involved in dendritic spinogenesis initiation or dendritic spine stabilization , a time course study using a knockdown approach in cultured hippocampal neurons
the time window of 1518 div covering the initiation toward maturation of dendritic spinogenesis was used for analysis . at 15 div ,
wild - type dendritic spines are immature , long , and thin , and they are present at a low density . as they mature at 18 div , dendritic spine density increases , spine length decreases , and spine width increases . compared to control neurons ,
cask knockdown does not affect spine density , length , or size at 15 div , suggesting that cask is not critical for dendritic spinogenesis initiation . at 18 div ,
the data indicate that cask is important for dendritic spine maturation , likely by linking the membrane protein syndecan-2 to the f - actin cytoskeleton via protein 4.1 to stabilize dendritic spines ( figure 3 ) . in human embryonic kidney hek293 cells , syndecan-2
however , these filopodia can not mature into spines . because neurofibromin and cask are also expressed in hek293 cells
, the aforementioned studies can not explain why syndecan-2-induced dendritic spines are only present in neurons .
a neuron - specific factor must be present to control dendritic spine formation . because neurotransmission is a neuron - specific event and
because dendritic filopodia are able to receive neurotransmission signals from presynaptic buttons , neurotransmission seems a likely factor that triggers the filopodia - spines transformation in a neuron - specific manner . indeed , egta treatment to chelate extracellular calcium or ap5 treatment to block nmdar activity , a major neurotransmitter gated calcium channel , impairs the endogenous filopodia - spines transition at 1517 div and syndecan-2-induced filopodia - spines transition at 59 div .
in syndecan-2-overexpressing neurons , intracellular calcium concentration is increased compared to control neurons at 5 div .
this increase is due to nmdar - regulated calcium influx because ap5 treatment effectively reduced the intracellular calcium concentration induced by syndecan-2 .
the c2 motif of syndecan-2 is required for syndecan-2 overexpression - induced calcium influx , suggesting that the interaction with cask is involved in calcium influx .
previous studies have shown that cask interacts with mlin7 via the l27 domains in both proteins [ 5052 ] and that mlin7 interacts with the c - terminal tail of nmdar subunit 2b ( nmdar2b ) through its pdz domain .
the interaction between syndecan-2 , cask , mlin7 , and nmdar2b facilitates nmdar localization to the tips of dendritic filopodia , where nmdar may be activated by presynaptic stimulation , namely , glutamate , and induce calcium influx .
disruption of the syndecan-2 , cask , mlin7 , and nmdar complex by overexpressing the interacting domains impairs nmdar filopodial distribution , calcium influx , and the filopodia - spines transition , suggesting that syndecan-2 triggers calcium influx via the cask - mlin7-nmdar complex and induces the filopodia - spines transition ( figure 3 ) .
the morphological feature of the filopodia - spines transition is dendritic spine head enlargement and spine length shortening .
calcium is known to regulate f - actin dynamics in dendritic spines [ 5456 ] , and gelsolin is a calcium - activated f - actin regulator .
gelsolin deficiency impairs filopodial retraction of developing neurons   and inhibits activity - dependent f - actin remodeling in mature dendritic spines .
it is also critical for the filopodia - spines transition induced by the syndecan-2-cask - mlin7-nmdar complex , as gelsolin knockdown maintains syndecan-2-induced protrusions at the filopodial stage .
it is possible that other calcium regulated f - actin regulators also act downstream of syndecan-2 to control the filopodia - spines transition .
more investigations are required to further elucidate the regulation . through its interactions with intracellular binding partners ,
the ubiquitously expressing protein syndecan-2 modulates the f - actin cytoskeleton , triggers neurotransmission , and promotes neuron - specific synapse formation . from dendritic filopodia formation ,
filopodia - spines transition to dendritic spine maturation , syndecan-2 interacts with different binding partners to control f - actin behaviors .
syndecan-2 first activates the pka pathway via neurofibromin to promote f - actin polymerization and bundling for dendritic filopodia formation .
it recruits nmdar to filopodial tips through its interaction with the cask - mlin7 complex and increases the postsynaptic responsiveness to presynaptic stimulation .
calcium influx induces f - actin cytoskeleton rearrangement to allow for the morphological change from filopodia to spines . to further promote dendritic spine maturation and maintenance , syndecan-2 binds to the protein 4.1 through interactions with cask . throughout the entire process
, neuron specificity falls within nmdar - mediated calcium influx , which induces f - actin cytoskeleton remodeling to result in morphological changes to the dendritic spine .
these studies provide a comprehensive example of how a neuron - specific ion channel coordinates with other adhesion molecules and synaptic proteins to control dendritic spine formation .
to identify a neuron - specific f - actin regulator involved in dendritic spinogenesis , we searched the database and literature and focused on cortactin - binding protein 2 ( cttnbp2 ) .
cttnbp2 gene encodes a brain - specific protein that interacts with the sh3 domain of cortactin through its proline - rich domain .
cortactin promotes and stabilizes f - actin branching [ 63 , 64 ] and thus plays a critical role for dendritic spine morphological maintenance . because cortactin is a ubiquitously expressed protein , its function in controlling dendritic spinogenesis must be regulated by a neuron - specific factor .
the specific expression of cttnbp2 in the brain makes it a good candidate to control cortactin in dendritic spinogenesis .
furthermore , de novo mutations in the cttnbp2 gene have been repeatedly identified in asd patients [ 5 , 37 , 66 ] . in a genomic analysis covering 3871 asd patients
, results indicated that cttnbp2 is a high - confidence risk factor for asds with a false discovery rate less than 0.05% .
these genetic data support a critical role for cttnbp2 in brain development and function . in the expression tag sequence ( est )
database ( http://www.ncbi.nlm.nih.gov/ ) , three variants have been identified as cttnbp2 transcripts , namely , cttnbp2-short ( cttnbp2-s ) , cttnbp2-intron ( cttnbp2-i ) , and cttnbp2-long ( cttnbp2-l ) .
based on the nucleotide sequence , the first 625 predicted amino acid residues are shared among all variants .
using an antibody against the common region of the cttnbp variants , immunoblotting revealed that the short form of cttnbp2 is the predominant protein product in brains .
it is still unclear whether the cttnbp2-i and cttnbp2-l variants play any role in neurons .
therefore , mutation analysis of asd patients is meaningful when the mutation was located within the cttnbp2-s variant sequences .
seven de novo asd mutations in the cttnbp2 gene have been identified in the exons encoding cttnbp2-s . to further explore the association of cttnbp2 with asd
, these mutations should be investigated in detail to determine their effects on cttnbp2 molecular function and neuronal morphogenesis .
analysis of the amino acid sequence of cttnbp2-s predicts a coiled - coil domain at the n - terminal region and proline - rich domain at the c - terminus .
the n - terminal coiled - coil domain mediates cttnbp2-s homooligomerization and heterooligomerization of cttnbp2-s and the striatin family [ 68 , 69 ] .
both endogenous cttnbp2-s and overexpressed myc - tagged cttnbp2-s were found to be highly concentrated at dendritic spines in mature cultured hippocampal neurons .
immunofluorescence analysis of adult brains also indicated that cttnbp2-s colocalized with f - actin puncta in vivo , presumably to dendritic spines .
cttnbp2-s is critical for dendritic spine formation , as cttnbp2 knockdown right before dendritic spinogenesis at 12 div reduces spine density and spine head width measured at 18 div .
consistent with the morphological changes , the frequency of mepsc ( miniature excitatory postsynaptic synaptic current ) is lower in cttnbp2 knockdown neurons at 18 div .
in addition to dendritic spine formation , cttnbp2-s is involved in dendritic spine maintenance , as cttnbp2-s knockdown in mature neurons , such as 20 div , still reduces dendritic spine density at 26 div .
cortactin is required for cttnbp2-s 's regulation of dendritic spinogenesis , as a cttnbp2-s mutant that can not interact with cortactin can not rescue cttnbp2 knockdown - induced spine deficiency .
moreover , fluorescence recovery after photobleaching ( frap ) analysis indicates that cttnbp2-s regulates cortactin mobility in mature dendritic spines . in the presence of cttnbp2-s ,
the data suggest that cttnbp2-s retains cortactin in dendritic spines and controls dendritic spine formation and maintenance .
the striatin protein family contains three mammalian members , namely , striatin , zinedin , and sg2na .
they function as b - type regulatory subunits of protein phosphatase 2a ( pp2a ) to control pp2a subcellular location and substrate specificity [ 70 , 71 ] .
striatin protein distribution to synapses is mediated by its interactions with cttnbp2-s through the n - terminal coiled - coil domains of both cttnbp2-s and striatin family members .
, cttnbp2-s regulates f - actin dynamics and pp2a signaling at dendritic spines . in cos cells ,
exogenous cttnbp2-s was unexpectedly associated with the microtubule cytoskeleton in addition to the cortactin - f - actin cytoskeleton .
cell - matrix interactions influence the cytoskeleton association of cttnbp2-s . in cos cells , cttnbp2-s
cttnbp2-s gradually shifts its preference to the microtubule cytoskeleton when establishing cell - matrix interactions .
cttnbp2-s is highly concentrated at dendritic spines in mature neurons where cttnbp2-s interacts with f - actin cytoskeletons . in the premature stages
when dendritic spines have not yet formed , cttnbp2-s is already expressed and forms puncta attached on microtubule bundles along the dendritic shaft .
the mid domain is required for the association of microtubule , and the n - terminal coiled - coil domain is involved in cttnbp2-s oligomerization .
oligomerization allows the cttnbp2-s oligomer to contain multiple microtubule binding sites to induce microtubule bundling . during the dendritic extension stage , cttnbp2-s knockdown or disruption of microtubule bundling by overexpression of the n - terminal coiled - coil domain or mid domain impairs dendritic arborization .
the studies suggest that , in addition to controlling the f - actin cytoskeleton , cttnbp2-s regulates microtubule stability to influence dendrite morphology .
the dual roles of cttnbp2-s in controlling f - actin and the microtubule cytoskeletons require further investigation . as a neuron - specific morphology regulator and a high - confidence risk factor for asds
for instance , what is the molecular mechanism regulating the association between cttnbp2-s and f - actin and microtubules ?
are the associations of cttnbp2-s with f - actin and microtubules mutually exclusive ? alternatively , can cttnbp2-s act as a bridge to link f - actin and microtubules ?
only cultured hippocampal neurons have been examined in functional studies of cttnbp2-s . in the future , in vivo studies should be considered . particularly , to address the association of cttnbp2 with asds , a mouse genetic model is required .
the impact of cttnbp2 asd mutations on the molecular function of cttnbp2-s , brain development , and cognition must be studied to further understand the biological significance of cttnbp2 .
although hundreds of genes are involved in dendritic spine formation , they should be either neuron - specific or directly or indirectly controlled by or linked to neuron - specific signaling or proteins to specifically regulate dendritic spine formation in neurons . in this review ,
syndecan-2-induced dendritic spine formation and the role of cttnbp2-s in controlling neuronal morphology provide two distinct examples of how neuronal morphology can be regulated in a neuron - specific manner .
understanding these regulations is crucial for basic research and for understanding neurological disorder etiology , which could contribute to potential therapeutic treatments of the diseases .
 dendritic spines are the location of excitatory synapses in the mammalian nervous system and are neuron - specific subcellular structures essential for neural circuitry and function . 
 dendritic spine morphology is determined by the f - actin cytoskeleton . 
 f - actin remodeling must coordinate with different stages of dendritic spinogenesis , starting from dendritic filopodia formation to the filopodia - spines transition and dendritic spine maturation and maintenance . 
 hundreds of genes , including f - actin cytoskeleton regulators , membrane proteins , adaptor proteins , and signaling molecules , are known to be involved in regulating synapse formation . 
 many of these genes are not neuron - specific , but how they specifically control dendritic spine formation in neurons is an intriguing question . here , we summarize how ubiquitously expressed genes , including syndecan-2 , nf1 ( encoding neurofibromin protein ) , vcp , and cask , and the neuron - specific gene cttnbp2 coordinate with neurotransmission , transsynaptic signaling , and cytoskeleton rearrangement to control dendritic filopodia formation , filopodia - spines transition , and dendritic spine maturation and maintenance . 
 the aforementioned genes have been associated with neurological disorders , such as autism spectrum disorders ( asds ) , mental retardation , learning difficulty , and frontotemporal dementia . 
 we also summarize the corresponding disorders in this report . 
1. Introduction
2. The HSPG Syndecan-2 Triggers Dendritic Spine Formation
3. The Neuron-Specific Cytoskeleton Regulator CTTNBP2 Is Highly Associated with Autism Spectrum Disorders
4. Conclusions