Regulation of biosynthetic cargo transport in neurons

项目摘要 这项提议的目的是了解新合成的分泌蛋白质 从神经元的内质网（ER）中出现，穿过内膜系统， 最终到达轴突和树突的表面。先前在许多哺乳动物细胞类型中的研究 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-高尔基体中间室（ERGIC）的分泌蛋白，ERGIC是一个囊泡-管状簇 与内质网上的出芽位点直接相邻的膜。然后货物通常被运输到 高尔基体，随后被运送到它们的最终目的地。然而，相对较少 关于神经元中早期分泌途径的结构， 轴突和树突内ER的局部蛋白输出。使用分化的人诱导多能干细胞 细胞（iPSCs），我已经开发了一个生理相关的系统来研究生物合成分泌蛋白的运输 大脑皮层神经元中。此外，我将利用基因组编辑的人类iPSC来定义 Trk融合基因（TFG）的病理变异，这是遗传性痉挛性痴呆的早期发病形式的基础。 截瘫（HSP），其特征在于皮质脊髓束内的进行性轴突病。我的研究会 提供了新的见解的重要性，局部蛋白质运输从ER在远端部分的神经突， 同时在干细胞生物学，生物化学，高分辨率， 成像和遗传学。在初步研究中，我已经产生了大量的工具来研究 神经元中的生物合成蛋白运输，包括天然表达标记的CRISPR/Cas9修饰的iPSC， COPII机制的亚基和分泌途径的其他组分（使用HaloTag），以及 荧光标记的货物，可以根据需要从ER释放。因此，我非常好- 我准备处理我的建议中概述的研究，这应该有助于揭示新的病理机制， 有助于HSP患者皮质脊髓轴突病变的观察，为今后的研究奠定基础 开发治疗方法以改善疾病。重要的是， 特别是对我来说，将促进我在实验设计，数据， 采集和数据分析，同时培养我在科学方面的专业技能 沟通、指导和领导力。总的来说，在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富有成效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.