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

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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.

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Abbreviations

srGAP:

Slit-Robo GTPase-activating protein

VPA:

Valproic acid

CA:

Constitutively active

MEGAP:

Mental disorder-associated GAP

GAP-43:

Growth associated protein-43

FCH:

Fer/Fes CIP4 homology

SH3:

Src homology 3

F-BAR:

FCH-bin/amphiphysin/Rvs

CC:

Coiled-coil

WRP:

WAVE-associated Rac GAP

GST:

Glutathione S-transferase

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

Rac1:

Ras-related C3 botulinum toxin substrate 1

RhoA:

Ras homolog gene family, member A

Cdc42:

Cell division control protein 42 homolog

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Acknowledgments

We thank Dr. Yuan XB (Institute of Neuroscience, SIBS, CAS, Shanghai, China) for the gift of GFP-tagged CA-Rac Q61L, CA-Rho Q63L, CA-Cdc42 Q61L, and DN-Rac T17N constructs and Dr. Burridge K (UNC, Chapel Hill, NC) for the gift of GST-Rho GTPases (CA-Rac Q61L, CA-Rho Q63L, and CA-Cdc42 Q61L) constructs. We are grateful for the generous donation of the human KIAA0411 and KIAA 0456 clones from the Kazusa DNA Research Institute (Chiba, Japan). This work was supported by National Natural Science Foundation of China (No. 30770671 and No. 309700936) and Shanghai Leading Academic Discipline Project (No. B205).

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Authors and Affiliations

  1. Institute of Neurosciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China

    Keng Chen, Ya-Jing Mi, Yue Ma & Wei-Lin Jin

  2. Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Centre for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, China

    Hua-Lin Fu

  3. Institute of Neurosciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Rm. 509, Wen Xuan Medical Building, 800 Dong Chuan Road, Minhang, Shanghai, 200240, China

    Wei-Lin Jin

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  1. Keng Chen
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Correspondence to Wei-Lin Jin.

Electronic supplementary material

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10571_2011_9664_MOESM1_ESM.tif

Supplementary Fig. 1. Expression of srGAPs and their upstream regulators in Neuro2A cells. RT–PCR is performed on total RNA from Neuro2A cells. The specific primers used to detect expression of Slit2, Robo1, Robo2, Robo3, srGAP1, srGAP2, srGAP3, GAPDH and β-actin in Neuro2A cells, the corresponding product size are listed in Table 1. The PCR products are subjected to a 1.5% agarose gel and visualized by ethidium bromide staining. (TIFF 1050 kb)

10571_2011_9664_MOESM2_ESM.tif

Supplementary Fig. 2. Quantification of the differentiation rates of control or srGAP3-knockdown Neuro2A cells cultured in DMEM containing 2% FBS and 20 μM Retinoic Acid. Values are means ± SEM, * p < 0.05. (TIFF 1088 kb)

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Chen, K., Mi, YJ., Ma, Y. et al. The Mental Retardation Associated Protein, srGAP3 Negatively Regulates VPA-Induced Neuronal Differentiation of Neuro2A Cells. Cell Mol Neurobiol 31, 675–686 (2011). https://doi.org/10.1007/s10571-011-9664-7

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