Dendritic spines are actin-rich structures, and regulation of the actin cytoskeletal reorganization is critical for dendritic spine formation and plasticity (Fischer et al., 1998
). The Rho-family GTPase Rac is well known as a molecular switch for signal transduction regulating the actin cytoskeleton in diverse cellular functions (Hall, 1998
) and also as a key player in dendritic spine morphogenesis in excitatory neurons (Nakayama et al., 2000
; Tashiro et al., 2000
). In the present study, we show that the Rac activator Dock4 is localized in dendritic spines in hippocampal neurons and positively regulates spine formation. Our results suggest that both the Rac GFF domain and the C-terminal, proline-rich region of Dock4 play important roles in spine formation. We also find that the F-actin–binding protein cortactin interacts with the C-terminal, proline-rich region of Dock4 and is required for Dock4-mediated spine formation. Collectively our findings reveal a novel function of Dock4 in dendritic spines in hippocampal neurons.
Recent studies showed that several Rac GEFs are localized in dendritic spines and involved in spine morphogenesis (Tolias et al., 2011
). Among them, Kalirin-7 and Tiam1 form complexes with N
-aspartate (NMDA) receptors, and their GEF activities are enhanced by calcium/calmodulin-dependent kinase II (CaMKII)–mediated phosphorylation after glutamate stimulation of the NMDA receptor (Xie et al., 2007
; Tolias et al., 2005
). Kalirin-7 and Tiam1 are also translocated into the postsynaptic region by interacting with the EphB receptor after ephrinB ligand stimulation and enhance Rac activity in the spines (Penzes et al., 2003
; Tolias et al., 2007
). βPIX is recruited to spines by G-protein–coupled receptor kinase–interacting protein 1, and its GEF activity is also enhanced by CaMKI-mediated phosphorylation (Zhang et al., 2003
; Saneyoshi et al., 2008
). On the other hand, Dock4 forms a complex with the actin-binding protein cortactin in dendritic spines and regulates spine formation via activation of Rac. Therefore Dock4 and other Rac GEFs are targeted to dendritic spines by different systems and may generate spatiotemporally diverse regulation of Rac activity.
Cortactin binds to F-actin via the central tandem repeat region and to Arp2/3 complex via the N-terminal acidic region and promotes polymerization, branching, and stabilization of the actin cytoskeleton (Weed et al., 2000
; Uruno et al., 2001
; Weaver et al., 2001
). Cortactin is highly enriched in dendritic spines, where it colocalizes with F-actin and positively regulates spine formation (Hering and Sheng, 2003
; Chen and Hsueh, 2012
). Dock4 also colocalizes with F-actin and cortactin in spines, and Dock4-mediated spine formation requires cortactin. Cortactin interacts with the C-terminal, proline-rich region of Dock4, and deletion of this region causes a dramatic change in the localization of Dock4. Therefore the interaction with cortactin may play an important role in spine localization of Dock4. However, we could not provide direct evidence of the involvement of cortactin in spine targeting of Dock4 because knockdown of cortactin results in the loss of dendritic spines. On the other hand, previous studies reported that cortactin interacts with various signaling molecules, such as N-WASP, WIP, Fgd1, ZO-1, and dynamin, through the C-terminal SH3 domain and contributes to diverse cellular processes, including cell migration, adhesion, and endocytosis (Daly, 2004
; Ammer and Weed, 2008
; Kirkbride et al., 2011
). Thus it is possible that cortactin functions as a scaffold protein to recruit these signaling molecules to the actin cytoskeleton. Of interest, the intracellular localization of cortactin is regulated by the activation of Rac1 in fibroblasts (Weed et al., 1998
). On the basis of these findings, we hypothesize that Dock4 may be recruited to the F-actin–rich region in dendritic spines via interaction with cortactin and promote actin polymerization via activation of Rac, which in turn recruits additional cortactin with Dock4 to the actin cytoskeleton, providing a positive feedback mechanism regulating the actin cytoskeleton by cortactin and Dock4 in dendritic spines.
Thin and long dendritic protrusions found in immature neurons are called dendritic filopodia. They are believed to be precursors of dendritic spines and important for initiation of synaptogenic contacts (Ziv and Smith, 1996
). In this study, expression of a mutant of Dock4 lacking the cortactin-binding region in Dock4-knockdown neurons or overexpression of wild-type Dock4 in the absence of cortactin results in a decrease in the number of mushroom-shaped mature spines and an increase in the number of dendritic filopodia. These observations suggest that interaction between Dock4 and cortactin is necessary for spine maturation and/or mature spine maintenance. This is supported by a previous report that mutants of cortactin lacking the C-terminal region, which contains the Dock4-binding domain, significantly reduced the spine head width, even though these mutants can localize to dendritic spines (Hering and Sheng, 2003
). On the other hand, it remains unclear whether Dock4 also regulates spine initiation. From the result that expression of Dock4 without cortactin interaction increases filopodia density, it is possible that Dock4 also regulates spine initiation by a cortactin-independent manner. Further investigations are required to determine at what stages (e.g., initiation, maturation, stabilization, maintenance, and plasticity) Dock4 is involved in spine formation and to understand how Dock4 regulates spine morphology in hippocampal neurons during development.
has been reported to be a susceptibility gene for several neuropsychiatric disorders, such as ASDs, dyslexia, and schizophrenia, by family-based, genome-wide association studies (Maestrini et al., 2010
; Pagnamenta et al., 2010
; Alkelai et al., 2012
). These disorders are believed to be associated with the disturbance of neuronal connectivity (Penzes et al., 2011
). ASDs make up a behaviorally defined syndrome characterized by deficits in social interaction, disruption of verbal and nonverbal communication, and the presence of repetitive, stereotyped behaviors (Lord et al., 2000
); they become established usually around 2–3 yr of age (Cox et al., 1999
). On the other hand, schizophrenia is a mental disorder characterized by auditory and visual hallucinations, paranoia, delusions, blunted affect, avolition, and social withdrawal; it typically emerges in late adolescence or early adulthood (Lewis and Lieberman, 2000
). Anatomical studies of hippocampal neurons have reported reduction in dendritic complexity and decrease in spine volume and number in individuals with autism and schizophrenia, respectively (Raymond et al., 1996
; Kolomeets et al., 2005
). Because Dock4 is involved in regulation of spine density in addition to its role in dendritic growth and branching in hippocampal neurons (Ueda et al., 2008
), dysfunction of Dock4 in dendrites during development might contribute to the pathogenesis of those psychiatric disorders. Further studies might allow us to better understand the biological mechanisms underlying these disorders and provide insights for new therapies.