MICALs and semaphorin-mediated 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。许多信号素利用丛蛋白(一种大型跨膜蛋白家族)作为受体。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。我最近发现了果蝇Mical，一种大的、多结构域的胞浆蛋白。Mical在轴突中表达，与神经元丛状蛋白A受体相互作用，是果蝇信号蛋白1a(Sema-1a)/丛状A介导的轴突引导和连接所必需的。Mical蛋白包含几个已知与轴突延伸所需的细胞骨架机制相互作用的结构域。此外，Mical含有黄素蛋白单加氧酶结构域，其完整性是形成Sema-1a-PlexA轴突回路所必需的。这个黄素蛋白单加氧酶结构域的存在首次暗示了信号素介导的轴突引导中的氧化还原信号机制。这项建议侧重于表征这些不寻常的蛋白质在轴突连接中所起的作用。哺乳动物基因组中编码的三种Mical蛋白与果蝇Mical高度相似。我的初步分析表明，它们也是神经性表达的，是丛状蛋白相互作用的蛋白质。结合体内遗传方法和体外基因转移方法，我将确定这些脊椎动物Mical蛋白是否在脊椎动物神经系统的发育中发挥作用。我的结果还表明，MICAL是协调与信号素介导的轴突连接相关的细胞骨架改变的极佳候选者。为了进一步确定Mical在信号素/丛蛋白信号转导中所起的作用，我将使用生化方法来确定Mical是否与神经元细胞骨架和几种调节其组装的蛋白质相互作用。拟议实验的结果，以及我将获得的概念和技术培训，将补充我的实验背景，并促进我向作为学术实验室首席研究员的独立、富有成效的研究项目过渡。

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. Many semaphorins utilize plexins, a family of large transmembrane proteins as receptors. How plexins actually transduce semaphorin signals is poorly understood but is of importance for learning how semaphorins sculpt and maintain the nervous system. I recently identified Drosophila MICAL, a large, multidomain, cytosolic protein. MICAL is expressed in axons, interacts with the neuronal plexin A (PlexA) receptor, and is required for Drosophila semaphorin la (Sema-la)/PlexA mediated axon guidance and connectivity. The MICAL protein contains several domains known to interact with the cytoskeletal machinery necessary for axonal extension. Furthermore, MICAL contains a flavoprotein monooxygenase domain, the integrity of which is required for Sema-la-PlexA axonal circuit formation. The presence of this flavoprotein monooxygenase domain implicates for the first time oxidation-reduction signaling mechanisms in semaphorin-mediated axon guidance. This proposal focuses on characterizing the role these unusual proteins play in axonal connectivity. There are three MICAL proteins encoded in the mammalian genome that are highly similar to Drosophila MICAL. My preliminary analyses show that they are also neuronally expressed and are plexin-interacting proteins. Combining in vivo genetic approaches with in vitro gene transfer approaches, I will determine if these vertebrate MICAL proteins play a role in the development of the vertebrate nervous system. My results also suggest that MICALs are excellent candidates for orchestrating the cytoskeletal alterations associated with semaphorin-mediated axonal connectivity. To further define the role played by MICAL in semaphorin/plexin signaling, I will use biochemical approaches to determine whether MICAL interacts with the neuronal cytoskeleton and several proteins that regulate its assembly. The results from the proposed experiments, as well as the conceptual and technical training I will acquire, will supplement my experimental background and promote my transition to an independent, productive research program as the principal investigator of an academic laboratory.