Interrogating Local Microtubule Regulation Required for Presynapse Formation and Maintenance

在人类大脑发育过程中，在高度复杂但刻意建立神经元连接的过程中形成了数万亿个突触。当这个复杂的过程出错时，许多发育和神经退行性疾病状态可能会出现，包括癫痫，智力迟钝，自闭症，阿尔茨海默氏症，帕金森氏症和亨廷顿氏病。尽管突触连接的数量和功能的重要性令人惊讶，但令人惊讶的是，人们对中枢神经系统突触的建立位置或指导突触形成或持续功能的细胞机制知之甚少。最近对轴突微管马达调节的认识提供了与局部突触前囊泡递送所需的细胞骨架基础的联系，这是适当突触功能的必要条件。KIF 1A是一种高度进行性的神经元特异性驱动蛋白-3马达蛋白，已显示优先从存在于突触前位点的富含GTP的微管末端分离，从而促进通过KIF 1A递送突触货物。微管网络是如何局部调节以促进突触前KIF 1A卸载的问题仍然未被探索。在这里，我建议测试的假设，多聚谷氨酰化和痉挛活性的串联作用，功能区分微管沿着轴突建立和维持突触。先前的工作已经揭示，痉挛的行为，切断微管扩大其质量，多聚谷氨酰化水平决定痉挛的活动，痉挛与突触前囊泡共定位，使这些因素突触前微管调节剂的优秀候选人。在目标1中，我将使用重建的体外单分子测定法直接检测多聚谷氨酰化和痉挛素活性对KIF 1A行为的影响。通过最小系统的策略性使用，这一目标将解开微管网络调控的复杂性，并建立两个重要因素，多聚谷氨酰化和痉挛，优先调节KIF 1A活性。目的2将探讨多聚谷氨酰化和痉挛蛋白在体内的作用，形成和维持突触前位点沿着轴突在人多能干细胞来源的神经元。这一目标将表征多聚谷氨酰化和痉挛蛋白在富含GTP的微管和高空间分辨率的突触前囊泡中的位置，并通过遗传破坏酶来功能性地评估它们在突触生成和维持中的作用。

During human brain development, trillions of synapses are formed during the highly complex yet deliberate establishment of neuronal connectivity. When this intricate process goes awry, numerous developmental and neurodegenerative disease states can manifest, including epilepsy, mental retardation, autism, Alzheimer’s, Parkinson’s, and Huntington’s disease. Despite the astonishing number and functional importance of synaptic connections, surprisingly little is known about where central nervous system synapses are established or the cellular mechanisms that guide synapse formation or continued function. Recent insights into the regulation of axonal microtubule motors provide a link to the cytoskeletal foundation required for local presynaptic vesicle delivery, a requisite for appropriate synapse function. KIF1A, a highly-processive, neuron-specific kinesin-3 motor protein, has been shown to preferentially detach from GTP-rich microtubule ends, which are present at presynaptic sites and thus promote the delivery of synaptic cargos by KIF1A. The question of how the microtubule network is locally regulated to facilitate presynaptic KIF1A off-loading remains unexplored. Here, I propose to test the hypothesis that polyglutamylation and spastin activity act in tandem to functionally distinguish microtubules along the axon to establish and maintain synapses. Previous work has revealed that spastin acts to sever microtubules to expand their mass, that polyglutamylation level dictates spastin activity, and that spastin co-localizes with presynaptic vesicles, making these factors excellent candidates for presynaptic microtubule regulators. In Aim 1, I will directly test the consequences of polyglutamylation and spastin activity on KIF1A behavior using reconstituted in vitro single molecule assays. Through the strategic use of minimal systems, this aim will untangle the complexity of microtubule network regulation and establish how two important factors, polyglutamylation and spastin, preferentially modulate KIF1A activity. Aim 2 will explore the in vivo role of polyglutamylation and spastin on the formation and maintenance of presynaptic sites along the axon in neurons derived from human pluripotent stem cells. This aim will characterize the location of polyglutamylation and spastin in relation to GTP-rich microtubules and presynaptic vesicles with high spatial resolution as well as functionally assess their role in synapse generation and maintenance by genetically disrupting the enzymes.