The Mental Retardation Associated Protein, srGAP3 Negatively Regulates VPA-Induced Neuronal Differentiation of Neuro2A Cells

Abstract

The Slit-Robo GTPase-activating proteins (srGAPs) are important multifunctional adaptor proteins involved in various aspects of neuronal development, including axon guidance, neuronal migration, neurite outgrowth, dendritic morphology and synaptic plasticity. Among them, srGAP3, also named MEGAP (Mental disorder-associated GTPase-activating protein), plays a putative role in severe mental retardation. SrGAP3 expression in ventricular zones of neurogenesis indicates its involvement in early stage of neuronal development and differentiation. Here, we show that overexpression of srGAP3 inhibits VPA (valproic acid)-induced neurite initiation and neuronal differentiation in Neuro2A neuroblastoma cells, whereas knockdown of srGAP3 facilitates the neuronal differentiation in this cell line. In contrast to the wild type, overexpression of srGAP3 harboring an artificially mutation R542A within the functionally important RhoGAP domain does not exert a visible inhibitory effect on neuronal differentiation. The endogenous srGAP3 selectively binds to activated form of Rac1 in a RhoGAP pull-down assay. We also show that constitutively active (CA) Rac1 can rescue the effect of srGAP3 on attenuating neuronal differentiation. Furthermore, change in expression and localization of endogenous srGAP3 is observed in neuronal differentiated Neuro2A cells. Together, our data suggest that srGAP3 could regulate neuronal differentiation in a Rac1-dependent manner.

The Mental Retardation-Associated Protein srGAP3 Regulates Survival, Proliferation, and Differentiation of Rat Embryonic Neural Stem/Progenitor Cells

“…This result further confirms that srGAP3 is essential in regulating NSCs/NPCs survival and proliferation. The Slit-Robo signaling pathway and Rac1 may be involved [2,10,28]. In addition, we noticed that approximately two thirds of cells in the srGAP3 knockdown group were nestinpositive progenitors after 7 days culturing in the differentiation medium.…”

“…The lentivirus encoding shRNA for srGAP3, LV3-srGAP3 was prepared by GenePharma. The shRNA sequence is as follows: 5¢-GAATATGAAGCCCAAATAA-3¢, which was demonstrated to knockdown srGAP3 expression dramatically [10]. The sequence for control shRNA (LV3-NC) was 5¢-TTCTCCGAAC GTGTCACGT-3¢.…”

“…LV3-NC and LV3-srGAP3 were added to the culture system at an multiplicity of infection of 20. The total protein was subjected for immunoblotting analysis to confirm the knockdown efficiency of LV3-srGAP3, which followed Chen's protocol [10]. The immunolabeled protein was detected by BM Chemiluminescence Western Blotting kit (Roche).…”

“…Immunochemistry staining was performed following the standard protocol and optimized in the authors' laboratory [25]. Primary antibodies, including polyclonal rabbit antisrGAP3-3A1 antibody [10], polyclonal mouse anti-ki-67 (Chemicon, 1:300), and mouse anti-nestin, mouse anti-btubulin III, mouse anti-GFAP, and mouse anti-O4 (described as above) were diluted in PBS with 2% normal goat serum (NGS). The blocking solution contained 5% NGS and 0.25% Triton X-100 in PBS.…”

“…In addition, srGAP3 is critical to neural development acting as a downstream effector of neurogenin 2, participating in the specification of neural fates [9]. The nuclear-localized srGAP3 has also been shown to play a crucial role in the neuronal differentiation of Neuro2A [10]. During neuronal development, srGAP3 has been implicated in axon guidance, neuronal migration, neurite outgrowth, and dendritic morphogenesis [7,[11][12][13][14][15].…”