Rhoa Relevant Gtp Enzyme Activation Protein Regulation Made The Cortex Neuron Dendritic Development Research

Neuronal network development of an organism is an extremely complicated process. Generally, neurons have a main axon that typically relays information to other neurons, and several dendrites that typically receive inputs from other neurons. Therefore, proper morphogenesis of axons and dendrites has a big impact on neuronal information processing. Neuronal dendrites are usually highly branched and the extent of the arborization of the dendritic tree correlates with the number and distribution of inputs that the neuron can receive and process. The development of dendrites involves several discrete morphological steps, which depends on the regulation of the cytoskeleton inside and outside the neurons. Defect in regulation of dendritic cytoskeleton will affect both dendritic structure and function, and may lead to cognitive impairment.Rho GTPases, including RhoA, Rac1and Cdc42, play a key role in dendritic development. Racl/Cdc42promotes dendrites branching and growth, while RhoA does the opposite. Over-activation of RhoA leads to developmental disorder of dendrites. It has been suggested that the activity of RhoA must be kept low to allow dendritic growth. However, how neurons restrict the activation of RhoA for proper dendritic development is not clear. It has been proved in many in vitro studies that RhoA GTPase-activating proteins (RhoA GAPs) are one of the negative regulators of RhoA. But it’s still not clear whether these GAPs are involved in the regulation of RhoA activity in neuronal dendrite development. In the present study, we undertook a comprehensive loss-of-function analysis of putative RhoA GAPs in cultured cortical neurons in vitro and developing cortical neurons in vivo, exploring the role of RhoA GAPs in the regulation of dendritic development and its mechanism.Our results showed that:(1) RhoA/ROCK signaling pathway was involved in the regulation of dendritic development in both cultured cortical neurons in vitro and developing cortical neurons in vivo. Over-activation of RhoA/ROCK caused an inhibition of dendrite growth, while inhibition of ROCK activity led to longer and more branches.(2) We tested the expression of18putative RhoA GAPs in rat developing cerebral cortex by real-time PCR and16of them had been detected.(3) We employed miRNAs to interfere with the expression of RhoA GAPs and observed the morphology of the cultured cortical neurons on DIV4. Down-regulation of Myo9a, Myo9b or RICS in cultured neurons with miRNAs came out a decrease in dendrite length and number.(4) Down-regulation of Myo9b or RICS through in utero electroporation inhibited the growth of developing cortical neurons, while inhibition of Myo9a didn’t show the similar phenomenon.(5) Interference of both Myo9b and RICS cooperated to inhibit dendrite branching and growth. Overexpression of Myo9b or RICS significantly increased the length and number of dendritic processes, whereas overexpression of GAP activity-deficient mutants of Myo9b or RICS had no such effects.(6) Inhibition of RhoA/ROCK signaling pathway rescued the growth impairment of dendrites caused by down-regulation of Myo9b or RICS.In conclusion, our data proved for the first time that restricted RhoA/ROCK signaling is essential for normal dendritic development in vivo. Besides, our study first systematically analyzed the expression of RhoA GAPs in nerve system and screened out that Myo9b and RICS played a key role in dendritic development in both cultured cortical neurons in vitro and developing cortical neurons in vivo, which conducted by RhoA/ROCK signaling pathway. Our study provided a novel insight into the physiological functions of RhoA GAPs in the dendritic development.