Abstract
Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (Vil2kd/kd) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (Vil2+/+) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the Vil2kd/kd mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the Vil2kd/kd neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the Vil2kd/kd neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.
Highlights
Establishment of neural circuits in the central nerve system requires generation and development of multiple dendrites and single axon
ERM proteins that link plasma membrane proteins and the actin cytoskeleton are expressed in various cultured neurons; hippocampal, cortical and dorsal root ganglion neurons [11,13,28]
C-E, The number (C) and length (D) of neurites, and length of axon (E) were quantified in the Vil2+/+ and Vil2kd/kd neurons treated with DMSO or 50 mM blebbistatin
Summary
Establishment of neural circuits in the central nerve system requires generation and development of multiple dendrites and single axon. Neuritogenesis that is the first step in neuronal morphogenesis is driven by exocytic and cytoskeletal machinery [3]. Several neurites extended from a symmetrical cell body become an axon or dendrites, and subsequently, neurons establish synaptic connections and networks. Small GTPases, RhoA, Rac and Cdc modulate the neuronal morphogenesis through regulating cytoskeletal dynamics in different pathways [4]. Rac and Cdc promote neurite outgrowth through phosphorylation of p21-activated kinase (PAK) family of serine/threonine kinases [5]. RhoA and its downstream effector Rho kinase mediate neurite retractions [6]. Myosin II activity is determined by phosphorylation of myosin light chains (MLCs) and mediated by RhoA/Rho kinase pathway. Activated myosin II generates formation of cortical actin filaments and leads to inhibition of neuritogenesis [7]
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