Gas7 Functions with N-WASP to Regulate the Neurite Outgrowth of Hippocampal Neurons

Jhong-Jhe You,Sue Lin-Chao

doi:10.1074/jbc.m109.051094

Abstract

Neuritogenesis, or neurite outgrowth, is a critical process for neuronal differentiation and maturation in which growth cones are formed from highly dynamic actin structures. Gas7 (growth arrest-specific gene 7), a new member of the PCH (Pombe Cdc15 homology) protein family, is predominantly expressed in neurons and is required for the maturation of primary cultured Purkinje neurons as well as the neuron-like differentiation of PC12 cells upon nerve growth factor stimulation. We report that Gas7 co-localizes and physically interacts with N-WASP, a key regulator of Arp2/3 complex-mediated actin polymerization, in the cortical region of Gas7-transfected Neuro-2a cells and growth cones of hippocampal neurons. The interaction between Gas7 and N-WASP is mediated by WW-Pro domains, which is unique in the PCH protein family, where most interactions are of the SH3-Pro kind. The interaction contributes to the formation of membrane protrusions and processes by recruiting the Arp2/3 complex in a Cdc42-independent manner. Importantly, specific interaction between Gas7 and N-WASP is required for regular neurite outgrowth of hippocampal neurons. The data demonstrate an essential role of Gas7 through its interaction with N-WASP during neuronal maturation/differentiation.

Highlights

Neurite outgrowth, which begins with the extension of membrane protrusions of neurons, is an important characteristic of neuronal differentiation that largely depends on actin dynamics and/or cortical cytoskeleton rearrangement
Neural-Wiskott Aldrich syndrome protein (N-WASP) overexpression alone did not affect or We examined the correlation of Gas7 and N-WASP in induce any membrane protrusions without an activator; in contrast, Gas7-transfected cells had ing dissociated neurons at 5 days in vitro (DIV) with specific membrane protrusions, as previously reported [9]
Cultured hippocampal neurons were transfected with GFP, shGas7/GFP, or shN-WASP/GFP at DIV2 and immunostained at DIV4

Summary

Introduction

Neurite outgrowth, which begins with the extension of membrane protrusions of neurons, is an important characteristic of neuronal differentiation that largely depends on actin dynamics and/or cortical cytoskeleton rearrangement. To determine whether the interaction between Gas7 and N-WASP is involved in the formation of these structures, Gas7 truncates lacking different functional domains (Fig. 4A) were transiently expressed in Neuro-2a cells. Formation of membrane protrusions (Fig. barely detectable after an antigen competition assay (Fig. 2C, 4B) was reduced in Gas7⌬WW-transfected Neuro-2a cells bottom panel), from which it can be concluded that N-WASP (44.7% had processes) after transfection for 12–16 h, compared was present in the nucleus, cytoplasm, and neurites.

Results

Conclusion