Regulation of biosynthetic cargo transport in neurons

神经元中生物合成货物运输的调节

• 批准号: 10602433
• 负责人: Iryna Pustova
• 金额: $ 2.04万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10602433
• 关键词: Affect; Affinity; Amyotrophic Lateral Sclerosis; Apoptosis; Architecture; Axon; Binding; Biochemistry; Biosynthetic Proteins; Brain; Bypass; CRISPR/Cas technology; Capsid Proteins; Cell membrane; Cells; Central Nervous System; Chemicals; Communication; Complex; Confocal Microscopy; Corticospinal Tracts; Data; Data Analyses; Defect; Dendrites; Destinations; Development; Diffuse; Disease; Distal; Electron Microscopy; Endoplasmic Reticulum; Endosomes; Ensure; Environment; Epithelial Cells; Exhibits; Experimental Designs; Fellowship; Fibroblasts; Fostering; Foundations; Future; Gene Mutation; Genetic; Glutamates; Goals; Golgi Apparatus; Growth; Health; Heart; Hereditary Motor and Sensory Neuropathies; Hereditary Spastic Paraplegia; Human; Impairment; Infrastructure; Integral Membrane Protein; Kinetics; Label; Leadership; Liquid substance; Liver; Maintenance; Mammalian Cell; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Mentors; Molecular; Motor Neurons; Movement; Mutation; Neurites; Neurodegenerative Disorders; Neurons; Nuclear; Pathologic; Pathway interactions; Patients; Phase; Physiological; Play; Point Mutation; Predisposition; Process; Productivity; Protein Export Pathway; Protein Sortings; Recycling; Regulation; Research; Research Personnel; Resolution; Role; Science; Secretory Component; Signal Transduction; Site; Structure; Surface; System; Technical Expertise; Testing; Therapeutic; Tissues; Training; Tubular formation; Variant; axonopathy; cell type; comparative; data acquisition; early onset; endoplasmic reticulum stress; fusion gene; gene function; genome editing; high resolution imaging; human stem cells; human tissue; imaging study; induced pluripotent stem cell; insight; live cell imaging; mRNA Translation; melanocyte; membrane synthesis; neurotransmitter release; protein complex; protein transport; secretory protein; skills; stem cell biology; stem cells; success; therapeutic development; tool; trafficking; transmission process

Project Summary The goal of this proposal is to understand the mechanisms by which newly synthesized secretory proteins emerge from the endoplasmic reticulum (ER) in neurons, move through the endomembrane system, and ultimately reach the surface of axons and dendrites. Previous studies in numerous mammalian cell types have clearly demonstrated that the coat protein complex II (COPII) machinery plays an integral role in directing most secretory proteins from the ER to the ER-Golgi intermediate compartment (ERGIC), a vesicular-tubular cluster of membranes directly adjacent to budding sites on the ER. Cargoes are then typically transported to the perinuclear Golgi apparatus and subsequently delivered to their final destinations. However, comparatively little is known about the architecture of the early secretory pathway in neurons, and even less is understood about local protein export from the ER within axons and dendrites. Using differentiated human induced pluripotent stem cells (iPSCs), I have developed a physiologically relevant system to study biosynthetic secretory protein transport in glutamatergic cortical neurons. Additionally, I will leverage genome edited human iPSCs to define the impact of a pathological variant in Trk-fused gene (TFG), which underlies an early onset form of hereditary spastic paraplegia (HSP) that is characterized by progressive axonopathy within the corticospinal tract. My studies will provide new insights into the importance of localized protein transport from the ER in distal portions of neurites, while simultaneously providing me with outstanding training in stem cell biology, biochemistry, high resolution imaging, and genetics. In preliminary studies, I have already generated a large number of tools to study biosynthetic protein trafficking in neurons, including CRISPR/Cas9-modified iPSCs that natively express tagged subunits of the COPII machinery and other components of the secretory pathway (using HaloTag), as well as fluorescently-labeled cargoes that can be released upon demand from the ER. Thus, I am exceptionally well- prepared to tackle the studies outlined in my proposal, which should help to uncover new pathomechanisms that contribute to corticospinal axonopathy observed in patients with HSP, laying the foundation for the future development of therapeutic approaches to ameliorate disease. Importantly, the fellowship training plan created specifically for me will facilitate growth of my operational and technical skills in experimental design, data acquisition, and data analysis, while simultaneously fostering development of my professional skills in science communication, mentoring, and leadership. Overall, the intellectual environment at UW-Madison and its infrastructure are ideal to ensure my long-term success as a productive biomedical researcher.

项目概要 该提案的目的是了解新合成的分泌蛋白的机制 从神经元的内质网（ER）中出现，穿过内膜系统，并且 最终到达轴突和树突的表面。先前对多种哺乳动物细胞类型的研究表明 清楚地表明外壳蛋白复合物 II (COPII) 机制在指导大多数 从 ER 到 ER-高尔基中间室 (ERGIC) 的分泌蛋白，一个囊泡管状簇 与内质网出芽位点直接相邻的膜。然后货物通常被运送到 核周高尔基体，随后被运送到最终目的地。但相对较少 人们对神经元早期分泌途径的结构了解甚少，但对 轴突和树突内内质网的局部蛋白质输出。使用分化的人类诱导多能干细胞 细胞（iPSC），我开发了一个生理相关系统来研究生物合成分泌蛋白运输 在谷氨酸能皮质神经元中。此外，我将利用基因组编辑的人类 iPSC 来定义影响 Trk 融合基因 (TFG) 的病理变异，它是遗传性痉挛的早期发作形式的基础 截瘫（HSP），其特征是皮质脊髓束内进行性轴突病变。我的学业将 提供了关于神经突远端部分内质网局部蛋白质转运重要性的新见解， 同时为我提供干细胞生物学、生物化学、高分辨率方面的出色培训 影像学、遗传学。在前期的研究中，我已经生成了大量的学习工具 神经元中的生物合成蛋白质运输，包括原生表达标记的 CRISPR/Cas9 修饰的 iPSC COPII 机制的亚基和分泌途径的其他组成部分（使用 HaloTag），以及 带有荧光标记的货物，可根据急诊室的要求放行。因此，我特别好—— 准备好处理我的提案中概述的研究，这应该有助于发现新的病理机制 有助于在 HSP 患者中观察到的皮质脊髓轴突病变，为未来奠定基础 开发改善疾病的治疗方法。重要的是，奖学金培训计划制定 特别适合我，将促进我在实验设计、数据方面的操作和技术技能的增长 采集和数据分析，同时促进我在科学方面的专业技能的发展 沟通、指导和领导。总体而言，威斯康星大学麦迪逊分校的智力环境及其 基础设施是确保我作为一名高效的生物医学研究人员取得长期成功的理想选择。