N-WASP and the Arp2/3 Complex Are Critical Regulators of Actin in the Development of Dendritic Spines and Synapses

Abstract

Changes in the number, size, and shape of dendritic spines are associated with synaptic plasticity, which underlies cognitive functions such as learning and memory. This plasticity is attributed to reorganization of actin, but the molecular signals that regulate this process are poorly understood. In this study, we show neural Wiskott-Aldrich syndrome protein (N-WASP) regulates the formation of dendritic spines and synapses in hippocampal neurons. N-WASP localized to spines and active, functional synapses as shown by loading with FM4 -64 dye. Knock down of endogenous N-WASP expression by RNA interference or inhibition of its activity by treatment with a specific inhibitor, wiskostatin, caused a significant decrease in the number of spines and excitatory synapses. Deletion of the C-terminal VCA region of N-WASP, which binds and activates the actinrelated protein 2/3 (Arp2/3) complex, dramatically decreased the number of spines and synapses, suggesting activation of the Arp2/3 complex is critical for spine and synapse formation. Consistent with this, Arp3, like N-WASP, was enriched in spines and excitatory synapses and knock down of Arp3 expression impaired spine and synapse formation. A similar defect in spine and synapse formation was observed when expression of an N-WASP activator, Cdc42, was knocked down. Thus, activation of N-WASP and, subsequently, the Arp2/3 complex appears to be an important molecular signal for regulating spines and synapses. Arp2/3-mediated branching of actin could be a mechanism by which dendritic spine heads enlarge and subsequently mature. Collectively, our results point to a critical role for N-WASP and the Arp2/3 complex in spine and synapse formation.

F-BAR Proteins of the Syndapin Family Shape the Plasma Membrane and Are Crucial for Neuromorphogenesis

“…Surprisingly, the C terminus of N-WASP, which strongly activates the Arp2/3 complex but is unable to mediate interactions with further N-WASP-binding proteins, causes neuronal phenotypes similar to Arp2/3 complex loss of function (Strasser et al, 2004;Pinyol et al, 2007;Wegner et al, 2008). These observations suggest that a recruitment of N-WASP to the cell cortex is of utmost importance for N-WASP/Arp2/3 complex-dependent processes during neuromorphogenesis.…”

“…This shows that N-WASP integrates several molecular stimuli leading to Arp2/3 complex-mediated actin nucleation. The cooperative action of several components upstream of N-WASP may provide a molecular solution to the need for spatial and temporal fine control of N-WASP function during endocytosis, neuronal development, and neuronal network formation-cellular functions impaired by N-WASP loss of function (Kessels and Qualmann, 2002;Benesch et al, 2005;Kakimoto et al, 2006;Pinyol et al, 2007;Wegner et al, 2008).…”

“…To control and fine-tune actin nucleation, the neural Wiskott-Aldrich syndrome protein (N-WASP) is kept in an autoinhibited state (Pollard, 2007). N-WASP reduction results in defects in proper axon development (Kakimoto et al, 2006;Pinyol et al, 2007) and in reduction of postsynaptic spines and synapses (Wegner et al, 2008). N-WASP overexpression in neurons leads to excessive formation of neurites and neuritic branches .…”

The Rho family GEF Asef2 regulates cell migration in three dimensional (3D) collagen matrices through myosin II

“…Asef2 shRNAs were prepared by ligating 64-mer oligonucleotides into pSUPER vector as previously described. 36,37 The target sequences for Asef2 were previously described in Bristow et al 12 The non-target sequence, 5 0 -CAGTCGCGTTTGCGACTGG-3 0 , was used as a control.…”

Neuritic complexity of hippocampal neurons depends on WIP‐mediated mTORC1 and Abl family kinases activities

“…2006; Wegner et al. 2008; Padrick and Rosen 2010). There is, however, much less information about the role of the cortactin‐ and N‐WASP‐binding protein WIP (WASP‐interacting protein) in neurons.…”