Mechanisms of mechanical force evoked Ca2+ influx in developing neurons

描述(申请人提供)：神经系统的发展需要神经元的适当分化、迁移和形态发生。单个神经元的形态发生和定义人类神经系统的数万亿个神经元回路的组装，是通过轴突和树突的引导延伸发生的。这项研究的目的是更好地了解负责大脑正确连接的钙通道和下游效应器机制。为此，我们必须了解神经生长锥如何检测、整合和响应环境中的可溶性、细胞和基质相关的指导分子。参与检测轴突引导信息并将其转化为定向轴突生长的基因突变与许多认知功能缺陷有关，包括自闭症、阅读障碍、心理障碍和智力障碍。引导轴突的环境因素通常会刺激生长锥体内的细胞内钙变化。有趣的是，促进生长和抑制轴突生长的引导信号都需要细胞内钙波动。目前尚不清楚这种简单的离子如何对生长锥行为产生不同甚至相反的影响，但许多研究表明，生长锥内局部钙信号的频率、幅度和分布决定了下游效应机制的激活。最近的证据表明，钙离子内流或释放所涉及的特定通道类型决定了对生长锥运动的影响。这项提议将测试不同的瞬时受体潜力(Trp)通道在生长锥生理和运动中的作用。色氨酸通道是由四个亚基组成的质膜阳离子通道，可被不同的化学和机械刺激激活。Aim 1通过测试机械门控通道如何控制轴突生长和引导，使用增益法和功能损失法来确定哪些亚单位形成机械门控通道。我们的初步数据显示，钙蛋白酶的活性受到机械诱导的钙内流的严格调控。在目标2中，我们将研究钙蛋白酶蛋白分解的分子底物，它对黏附、周转和轴突引导很重要。更好地了解钙对生长锥运动产生不同影响的分子机制，将为发育障碍和神经元损伤的治疗策略提供支持。

DESCRIPTION (provided by applicant): The development of the nervous system requires the proper differentiation, migration and morphogenesis of neurons. The morphogenesis of individual neurons and the assembly of the trillions of neuronal circuits that define the human nervous system occur through guided extension of axons and dendrites. The objective of this research is to better understand the calcium channels and downstream effector mechanisms that are responsible for the proper wiring of the brain. For this we must understand how nerve growth cones detect, integrate and respond to soluble, as well as cell- and substratum- associated guidance molecules in their environment. Mutations in genes involved in the detection and transduction of axon guidance information into directed neurite outgrowth are responsible for many deficits in cognitive function, including autisms, dyslexias, psychological disorders and mental retardations. Environmental factors that guide axons often stimulate intracellular calcium changes within growth cones. Interestingly, both growth promoting and inhibiting axon guidance cues have been shown to require intracellular calcium fluctuations. It is unclear how this simple ion can mediate distinct and even opposite effects on growth cone behavior, but many studies suggest that the frequency, amplitude and distribution of local calcium signals within growth cones determine the downstream effector mechanisms activated. Recent evidence suggests that the specific channel types involved in calcium influx or release from stores determines the effect on growth cone motility. This proposal will test the role of distinct transient receptor potential (TRP) channels on growth cone physiology and motility. TRP channels are plasma membrane cation channels composed of four subunits that are activated by diverse chemical and mechanical stimuli. Aim 1 uses gain- and loss-of-function approaches to determine which subunits form mechanically gated channels by testing how these channels control axon outgrowth and guidance. As our preliminary data shows calpain activity is tightly regulated by mechanically induced calcium influx. In Aim 2 we will investigate the molecular substrates of calpain proteolysis important for adhesion turnover and axon guidance. A better understanding of the molecular mechanisms through which calcium exerts such varied effects on growth cone motility will support treatment strategies for developmental disorders and neuronal injuries.