Molecular mechanisms of axon guidance and neural connectivity

描述(申请人提供)：正常运作的人类神经系统需要数十亿个神经元的相互连接。这些连接的不正确形成或维持会导致神经异常，从而导致许多精神疾病和障碍。这些电路是如何组装和集成的？信号素是参与轴突连接形成和维持的最大蛋白质家族之一。信号素是在无脊椎动物和脊椎动物中发现的系统发育保守的分泌型和跨膜型蛋白质。信号素利用神经丛蛋白(轴突表面发现的一种大的跨膜蛋白家族)作为受体来指导其作用。丛状蛋白实际上是如何转导信号素信号的，人们对此知之甚少，但对于了解信号素如何塑造和维持神经系统很重要。那么，什么策略将进一步定义这些重要的机制，信号素和丛状蛋白通过这些机制来指导神经连接？过去二十年的研究表明，在简单和复杂的动物之间，轴突引导和连接的分子机制非常保守。像苍蝇这样的简单动物使用许多与哺乳动物相同的轴突引导信号。鉴于这种保守，我的研究计划的目标是专注于苍蝇胚胎简单神经系统内的一小群轴突，并描述引导它们到达目标的分子和机制。使用这一策略，我最近发现了一个新的细胞内蛋白质家族，MICALs，它们对引导信号素/神经丛非依赖性神经连接至关重要。在像苍蝇这样的简单生物中只有一个Mical基因，而在包括人类在内的哺乳动物中发现了三个独立的Mical基因，它们也对调节信号素和丛蛋白的影响起着重要作用。有趣的是，Mical蛋白包含几个已知与细胞骨架结构相互作用的区域，这些结构是轴突生长、导航和形成连接所必需的。MICAL还含有氧化还原酶结构域，该结构域的完整性是Semaphorin轴突连接所必需的。这个氧化还原酶结构域的存在首次暗示了信号素介导的连接中的氧化还原信号机制。这项提议的焦点之一是确定Mical引导轴突通过的分子。对这个问题的初步认识来自我们最近发现的Mical与神经元中含有SH3结构域的蛋白Cas相互作用。Cas是非神经细胞中肌动蛋白细胞骨架动力学的关键调节因子，我们发现Cas和Mical将丛状蛋白和整合素联系在一起，介导轴突引导。我们的初步结果显示，Cas与G蛋白信号的特定介体相互作用，这表明Mical和Cas可能在调节GTP酶在轴突导航中发挥作用。我们将使用体内遗传和生化方法以及模型苍蝇轴突系统来检验这一假说，即特定的GTP酶及其调节因子是轴突导航的媒介，并在信号素在轴突引导过程中利用的细胞内信号机制中发挥重要作用。与公共健康相关：我们的神经系统控制着这样一种非凡的能力，比如把我们的想法写在纸上，因为我们的神经元在高度组织的网络中进行交流。这项提议的目标是更好地描述使神经元能够找到并相互连接的分子和机制。了解这些网络是如何组装、整合和维护的，将提出减轻精神疾病负担的解决方案，揭示潜在的思想、情感和行为的基本机制，确定一些精神障碍的治疗策略，并有助于神经创伤后的健康恢复。

DESCRIPTION (provided by applicant): A normal functioning human nervous system requires the interconnection of billions of neurons. Improper formation or maintenance of these connections leads to neurological abnormalities that result in a number of mental diseases and disorders. How are these circuits assembled and integrated? The semaphorins are one of the largest protein families involved in the formation and maintenance of axonal connections. Semaphorins are phylogenetically conserved secreted and transmembrane proteins found in invertebrates and in vertebrates. Semaphorins utilize plexins, a family of large transmembrane proteins found on the axonal surface, as receptors to direct their effects. How plexins actually transduce semaphorin signals is poorly understood but is of importance for learning how semaphorins sculpt and maintain the nervous system. So what strategies will further define these important mechanisms by which semaphorins and plexins direct neural connectivity? Work over the past twenty years has revealed that the molecular mechanisms of axon guidance and connectivity are remarkably well-conserved between simple and complex animals. Simple animals like flies use many of the same axon guidance signals as mammals. In light of this conservation, the goal of my research program is to focus on a small group of axons within the simple nervous system of the fly embryo and characterize the molecules and mechanisms that guide them to their targets. Using this strategy, I recently identified a new family of intracellular proteins, the MICALs, that are critical for directing semaphorin/plexindependent neural connectivity. There is one MICAL gene in simple organisms like flies, while three separate MICAL genes are found in mammals including humans that are also important for mediating the effects of semaphorins and plexins. Interestingly, MICAL proteins contain several regions known to interact with the cytoskeletal machinery necessary for allowing axons to grow, navigate, and form their connections. MICALs also contain an oxidoreductase domain, the integrity of which is required for Semaphorin axonal connectivity. The presence of this oxidoreductase domain implicates for the first time oxidation-reduction signaling mechanisms in semaphorin-mediated connectivity. One important question that is the focus of this proposal is to identify the molecules through which MICAL steers an axon. Initial insight into this question has come with our recent identification that MICAL interacts with the SH3-domain containing protein Cas in neurons. Cas is a critical regulator of actin cytoskeletal dynamics in non-neuronal cells and we find that Cas and MICAL link Plexins and integrins to mediate axon guidance. Our preliminary results now reveal that Cas interacts with a specific mediator of G protein signaling suggesting the possibility that MICAL and Cas play a role in regulating GTPases in navigating axons. We will use in vivo genetic and biochemical approaches and the model fly axon system to test the hypothesis that specific GTPases and their regulators are mediators of axon navigation and play an important role in the intracellular signaling mechanisms utilized by semaphorins during axon guidance. PUBLIC HEALTH RELEVANCE: Our nervous systems control such remarkable abilities as putting our thoughts to paper only because our neurons communicate in highly organized networks. The goal of this proposal is to better characterize the molecules and mechanisms that enable neurons to find and connect with one another. Understanding how these networks are assembled, integrated, and maintained will suggest solutions to diminish the burden of mental illness, reveal fundamental mechanisms underlying thought, emotion, and behavior, identify therapeutic strategies for a number of mental disorders, and contribute to healthy recovery following neural trauma.