Interrogating Local Microtubule Regulation Required for Presynapse Formation and Maintenance

探究突触前形成和维持所需的局部微管调节

• 批准号: 10474396
• 负责人: Jayne E Aiken
• 金额: $ 2.48万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-11 至 2023-01-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10474396
• 关键词: Address; Alzheimer' s Disease; Axon; Behavior; Biological Assay; Brain; Code; Cognition; Complex; DNA; Degenerative Disorder; Dendrites; Deposition; Development; Disease; Environment; Enzymes; Epilepsy; Exhibits; Family suidae; Foundations; Generations; Genes; Guanosine Triphosphate; Human; Human Engineering; Huntington Disease; Hydrolysis; Image; In Vitro; Intelligence; Interneurons; Kinesin; Label; Lead; Link; Location; Longevity; Maintenance; Mediating; Mental Retardation; Microscopy; Microtubule-Associated Proteins; Microtubules; Modeling; Molecular; Motor; Motor Activity; Neuraxis; Neurodegenerative Disorders; Neurons; Parkinson Disease; Perception; Phase; Population; Post-Translational Protein Processing; Presynaptic Terminals; Process; Proteins; Regulation; Research; Resolution; Role; Site; Synapses; Synaptic Vesicles; System; Techniques; Testing; Tubulin; Vesicle; Work; analog; autism spectrum disorder; cell motility; developmental disease; experimental study; human pluripotent stem cell; immunocytochemistry; in vivo; insight; interest; knock-down; motor behavior; mutant; neuron development; neuronal circuitry; overexpression; presynaptic; reconstitution; response; single molecule; spastin; synaptic function; synaptogenesis; trafficking

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.

在人脑发育过程中，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。当这一复杂的过程出错时，可能会出现许多发育和神经退行性疾病状态，包括癫痫、智力低下、自闭症、阿尔茨海默氏症、帕金森氏症和亨廷顿病。尽管突触连接的数量和功能重要性令人震惊，但令人惊讶的是，人们对中枢神经系统突触在哪里建立，或者指导突触形成或继续发挥功能的细胞机制知之甚少。最近对轴突微管马达的调控提供了与局部突触前囊泡传递所需的细胞骨架基础的联系，这是适当的突触功能所必需的。KIF1A是一种高度进行性的神经元特异性Kinesin-3马达蛋白，已被证明优先从突触前部位的富含GTP的微管末端分离，从而促进KIF1A递送突触货物。微管网络如何在局部调节以促进突触前KIF1A的卸载的问题仍未被探索。在这里，我建议检验一种假设，即多聚谷氨酰化和痉挛蛋白活性协同作用，在功能上区分沿着轴突的微管，以建立和维持突触。以前的工作揭示了spastin作用于切断微管以扩大其质量，多谷氨酰化水平决定了spastin的活性，以及spastin与突触前小泡共定位，使这些因素成为突触前微管调节的极佳候选者。在目标1中，我将使用重组的体外单分子分析直接测试多谷氨酰化和spastin活性对KIF1A行为的影响。通过最小系统的战略性应用，这一目标将解开微管网络调控的复杂性，并确定两个重要因素--多聚谷氨酰化和痉挛蛋白--如何优先调节KIF1A的活性。目的2探讨多聚谷氨酰化和痉挛蛋白在人多能干细胞来源神经元突触前轴突形成和维持中的作用。这一目标将以高空间分辨率表征多谷氨酰化和痉挛蛋白与富含GTP的微管和突触前小泡的位置，并从功能上评估它们通过基因干扰这些酶在突触生成和维持中的作用。