Regulation of biosynthetic cargo transport in neurons

项目摘要 这项提议的目标是了解新合成的分泌蛋白的机制 从神经元的内质网(ER)出来，穿过内膜系统，以及 最终到达轴突和树突的表面。之前对多种哺乳动物细胞类型的研究已经 清楚地表明，外壳蛋白复合体II(COPII)机制在指导MOST 从内质网到内质网高尔基体中间隔(ERGIC)的分泌蛋白是一个囊状管状簇 与内质网上的萌发部位直接相邻的膜。然后，货物通常被运送到 核周高尔基体，并随后运往其最终目的地。然而，相对较少的 人们知道神经元早期分泌途径的结构，而对此了解更少 轴突和树突内的内质网输出局部蛋白质。利用分化的人诱导多能干细胞 细胞(IPSCs)，我已经开发了一种生理相关系统来研究生物合成分泌蛋白的运输 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ipscs来定义其影响 Trk融合基因(TFG)的一种病理变异，这是一种早发性遗传性痉挛的基础 以皮质脊髓束内进行性轴索病变为特征的截瘫(HSP)。我的学习将会 对内质网在神经突起远端的局部蛋白质运输的重要性提供了新的见解， 同时为我提供了出色的干细胞生物学、生物化学、高分辨率 成像学和遗传学。在初步研究中，我已经生成了大量的研究工具 神经元中的生物合成蛋白运输，包括固有表达标签的CRISPR/Cas9修饰的IPSCs COPII机制的亚单位和分泌途径的其他组件(使用HaloTag)，以及 可根据急诊室要求放行的带有荧光标记的货物。因此，我非常好- 准备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， 有助于观察HSP患者的皮质脊髓轴索病变，为以后的研究奠定基础 开发治疗方法以改善疾病。重要的是，团契培训计划创建了 特别是对我来说，将促进我在实验设计、数据和数据方面的操作和技术技能的增长 获取和数据分析，同时培养我的科学专业技能发展 沟通、指导和领导力。总体而言，威斯康星大学麦迪逊分校及其 基础设施是确保我作为一名多产的生物医学研究人员取得长期成功的理想选择。

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.