Molecular And Cellular Mechanismunderlyingdendritic Development And Neuronal Differentiation Of The Srgap Family Members

The Slit-Robo GTPase-activating proteins (srGAPs) were originally identified as adownstream mediator of neuronal repellent factor Slit and Robo receptor. In mammals,the srGAP family consists of three members, srGAP1, srGAP2and srGAP3.They areimplicating in neuronal development and may be linked to mental retardation,schizophrenia and seizure in humans.The molecular mechanisms of srGAPscontrolling normal and abnormal higher brain functions have not been adequatelycarried out. SrGAP1, srGAP2and srGAP3are dynamically expressed in cerebralcortex developmentally and are highly distributed in subventricular/subgranular zonesof neurogenesis in adult brain. The present study will focus on srGAPsregulatingdendritic development andneuronal differentiation.ThesrGAP family members share considerable homology structurally and functionally,and there may exist partial compensation among the familymembers at the geneexpression level.To investigate the function of srGAP familiy in regulating dendriticdevelopment, we generate a miR-shRNA lentivirus expression systems by chainingthree shRNA targeting different srGAP. And the dendritic arborization is morecomplex when endogenous srGAPs were down-regulated simultaneously.We found previously that srGAP2and srGAP3, not srGAP1were expressed in mouseneuroblastoma Neuro2A cells and srGAP3can negatively regulate VPA (valporicacid)–induced neuronal differentiation in a Rac1-dependent manner. We demonstratethe three family members can form both homo-and hetero-dimers throughtheirF-BAR domains. RhoGAP pull-down assaysprove that srGAP1and srGAP3havepreferred substrate towards GTP-bound Rac1in vivo, while srGAP2does not. InNeuro2A cells, exogenously expressed srGAP1and srGAP2are sufficient to inhibitvalporic acid (VPA)-induced neurite initiation and growth. Different from the datafrom srGAP3R542A, ectopic-or over-expression of RhoGAP-defective mutants,srGAP1R542Aand srGAP2R527Aalso exert a visible inhibitory effect on neuronal differentiation. Unexpectedly, knockdown of endogenous srGAP2fails to facilitatethe neuronal differentiation induced by VPA, and down-regulation of both srGAP2and srGAP3can promote neuronal differentiation. Furthermore, when knock down ofsrGAP3and meanwhile overexpressingthere full-lengthsrGAPs respectively inNeuro2A cells, overexpressed srGAP1and srGAP3both exhibitinhibitory effect onneuronal differentiation, whereas srGAP2does not.The results mean that srGAP3isrequiredfor overexpressed srGAP2in inhibiting neuronal differentiation.Moreover, we applied two antagonists to interupt the interaction between Slit andRobo. Overexpression of Robo1-Fc and Robo1-ecto-TM can phenocopy srGAP3inhibition, indicating that srGAP3are dedicated to the Slit–Robo pathway.Our results show srGAP family members can regulate dendritic development and theinterplay between srGAP1, srGAP2and srGAP3regulates neuronal differentiationand neurite outgrowth.