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 actin-related 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.
Highlights
Rich dendritic spines [1] that are small extensions from the dendrite that form connections with axonal terminals
To begin to test our hypothesis, we examined the subcellular localization of neural Wiskott-Aldrich syndrome protein (WASP) (N-WASP) in hippocampal neurons by immunostaining low density cultures with N-WASP antibody along with a presynaptic marker, SV2, or a postsynaptic marker, PSD-95
As shown in the higher magnification images, N-WASP puncta were in close apposition to the presynaptic marker SV2, suggesting it was in postsynaptic terminals (Fig. 1A)
Summary
Reagents—SV2 (1:250) and GAD-6 (1:250) monoclonal antibodies were from the Developmental Studies Hybridoma Bank (The University of Iowa, Iowa City, IA). N-WASP (1:50) polyclonal antibody was a generous gift from Marc Kirschner (Harvard Medical School, Boston, MA). Arp antibody (1:100) was described previously [14]. Immunohistochemistry and Image Analysis—For most antibodies, neurons were fixed with 4% paraformaldehyde/4% glucose in phosphate-buffered saline (PBS) for 15 min. Coverslips were permeabilized with 0.2% Triton X-100 for 5 min and washed three times with PBS. Antibodies were diluted in 5% goat serum in PBS and incubated at the indicated dilutions for 1 h followed by three washes with. Rhodamine and Alexa Fluor 555 were imaged with a TRITC/Cy3 cube (excitation HQ545/30, emission HQ610/75, Q570LP dichroic mirror). Neurons were washed three times with calcium-free solution, fixed with 4% paraformaldehyde/glucose for 15 min, and visualized in fluorescence
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