S-acylation dependent regulation of fast axonal transport in neurons

神经元快速轴突运输的 S-酰化依赖性调节

• 批准号: RGPIN-2021-02547
• 负责人: Sanders, Shaun
• 金额: $ 2.7万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761163
• 关键词: acylation; dependent; regulation; fast; axonal

Neurons are large, morphologically complex cells that receive input at synapses or neuron-neuron connections located on short projections called dendrites. Information is then transmitted to downstream neurons via long output projections known as axons. The efficient trafficking and delivery of neuronal proteins and organelles to specific subcellular locations is critical for neuronal function but can be challenging over long axonal distances, up to a metre in humans. Despite the importance of trafficking in neurons, the governing mechanisms are poorly understood. Fast axonal transport (FAT) is the continuous, fast movement of vesicles carrying neurotransmitters and neurotrophins, endosomes and lysosomes, mRNA granules, autophagosomes, and pro-degenerative signaling complexes along microtubules by dynein and kinesin motors. To achieve FAT, motors must move cargo continuously over long distances, which requires a constant source of energy. Interestingly, glycolytic enzymes were recently found on FAT vesicles where they provide motors with an `on-board' constant supply of energy. However, how molecular motors function during FAT and how the cytosolic glycolytic enzymes are tethered to vesicles is not known. One mechanism to direct proteins to specific membranes is the covalent addition of fatty acids to protein cysteines residues, a process known as S-acylation. Recent curation of S-acyl proteomic studies reveals that glycolytic enzymes as well as a number of kinesin and dynein motor subunits and their activators are S-acylated. I therefore hypothesize that S-acylation tethers multiple motor proteins, their activators, and glycolytic enzymes to vesicles to provide `on-board' energy and continuous movement required for FAT. This proposal seeks to establish a research program focused on understanding the molecular basis of protein and organelle transport in axons. My short-term goals for this research project will focus on elucidating the role of S-acylation in FAT through the following specific objectives: (1) define the enzymatic and molecular regulation of S-acylation of FAT transport machinery, (2) determine if S-acylation of transport machinery is required for FAT, and (3) characterize the dependence of FAT on S-acylation in vivo. This work will be carried out using a range of advanced biochemical and cell biological techniques including conventional and microfluidic neuronal cell culture, viral mediated knockdown/rescue, live cell confocal imaging in neurons and optic nerve explants, and specialized S-acylation assays. Overall, this multifaceted project will position my group at the forefront of research into the molecular mechanisms that govern FAT with implications for neurodevelopment, synaptic function, and axon degeneration.

神经元是大的、形态复杂的细胞，通过突触或位于称为树突的短投射上的神经元-神经元连接接受输入。然后，信息通过被称为轴突的长输出投射传递到下游神经元。神经元蛋白质和细胞器的有效运输和传递到特定的亚细胞位置对神经元功能至关重要，但在人类长达一米的轴突距离上可能是具有挑战性的。尽管贩运神经元很重要，但人们对其管理机制知之甚少。快速轴突运输是携带神经递质和神经营养素、内小体和溶酶体、信使核糖核酸颗粒、自噬小体和变性信号复合体的囊泡在动力蛋白和动蛋白马达的作用下沿微管持续、快速运动的过程。为了实现肥胖，发动机必须在长距离持续运送货物，这需要持续不断的能源。有趣的是，最近在脂肪小泡上发现了糖酵解酶，它们为马达提供持续的能量供应。然而，分子马达在脂肪过程中如何发挥作用，以及胞浆糖酵解酶是如何与囊泡捆绑在一起的，目前尚不清楚。将蛋白质定向到特定膜上的一种机制是脂肪酸与蛋白质半胱氨酸残基的共价加成，这一过程被称为S酰化。最近对S-酰基蛋白质组的研究表明，糖酵解酶以及一些动蛋白和动力蛋白马达亚基及其激活剂都是S-酰化的。因此，我假设S酰化将多个马达蛋白、它们的激活剂和糖酵解酶连接到囊泡上，以提供脂肪所需的能量和持续的运动。这项提议旨在建立一个研究计划，专注于了解蛋白质和细胞器在轴突中运输的分子基础。我这个研究项目的短期目标将集中于通过以下具体目标来阐明S酰化在脂肪中的作用：(1)确定脂肪运输机制的S-酰化的酶和分子调节，(2)确定脂肪是否需要运输机械的S-酰化，以及(3)在体内表征脂肪对S-酰化的依赖。这项工作将使用一系列先进的生化和细胞生物学技术，包括常规和微流控神经细胞培养、病毒介导的击倒/挽救、神经元和视神经外植体的活细胞共聚焦成像，以及专门的S酰化分析。总体而言，这个多方面的项目将使我的团队处于研究脂肪与神经发育、突触功能和轴突退化相关的分子机制的前沿。