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: 10041536
• 负责人: Jayne E Aiken
• 金额: $ 6.46万
• 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/10041536
• 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.

在人类大脑发育过程中，数万亿个突触是在高度复杂但经过深思熟虑的神经元连接建立过程中形成的。当这个复杂的过程出错时，许多发育和神经退行性疾病就会出现，包括癫痫、智力迟钝、自闭症、阿尔茨海默病、帕金森病和亨廷顿病。尽管突触连接有着惊人的数量和功能重要性，但令人惊讶的是，关于中枢神经系统突触在哪里建立，以及指导突触形成或持续功能的细胞机制，人们知之甚少。最近对轴突微管运动调节的见解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。KIF1A是一种高进程性的神经元特异性运动蛋白，已被证明可以优先从富含gtp的微管末端分离，这些微管末端存在于突触前位点，从而促进KIF1A传递突触货物。微管网络如何局部调控以促进突触前KIF1A卸载的问题仍未被探索。在这里，我提出测试假设，即聚谷氨酰化和痉挛蛋白活性协同作用，在功能上区分沿轴突的微管，以建立和维持突触。先前的研究表明，spastin可以切断微管以扩大其质量，多谷氨酰化水平决定了spastin的活性，并且spastin与突触前囊泡共定位，使这些因子成为突触前微管调节剂的优秀候选因子。在目的1中，我将使用体外重组单分子测定法直接测试多谷氨酰化和spastin活性对KIF1A行为的影响。通过战略性地使用最小系统，该目标将理清微管网络调节的复杂性，并确定多谷氨酰化和痉挛蛋白这两个重要因子如何优先调节KIF1A活性。目的2将探讨多谷氨酰化和spastin在人类多能干细胞衍生的神经元轴突突触前位点形成和维持中的体内作用。这一目标将以高空间分辨率表征与富含gtp的微管和突触前囊泡相关的多谷氨酰化和痉挛蛋白的位置，并从功能上评估它们通过基因破坏酶在突触产生和维持中的作用。