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: 10402264
• 负责人: Iryna Pustova
• 金额: $ 3.52万
• 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/10402264
• 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; 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; Genes; Genetic Translation; Glutamates; Goals; Golgi Apparatus; Growth; Growth and Development function; 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; Neuraxis; Neurites; Neurodegenerative Disorders; Neurons; Nuclear; Pathologic; Pathway interactions; Patients; Phase; Physiological; Play; Point Mutation; Process; 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; base; cell type; comparative; data acquisition; early onset; endoplasmic reticulum stress; gene function; genome editing; high resolution imaging; human stem cells; human tissue; imaging genetics; imaging study; induced pluripotent stem cell; insight; live cell imaging; melanocyte; neurotransmitter release; protein complex; protein transport; secretory protein; skills; stem cell biology; stem cells; success; therapeutic development; tool; trafficking

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.

项目总结