Subcellular Proteomic Investigation of Projection Neuron Growth Cones in Developing Mouse Cortex

项目总结/摘要 在大脑皮质发育过程中，皮质投射神经元亚型延伸轴突以支配 不同的目标位于很大的距离（103-105细胞体直径），从他们的含核胞体。 这种精确的导航和电路发展是由生长锥（GC）调节的：亚细胞区室 在生长的轴突的尖端，快速整合细胞外信号以控制神经回路的发育， 发育成突触由于GC对化学信号的反应迅速，而运输需要数小时/天， 由于GCs与其亲本胞体之间的分子之间的相互作用，GCs极有可能半自主地发挥功能。以前的工作有 还揭示了GC中的局部蛋白质合成和降解途径是对一些 方向提示然而，大多数GC蛋白在轴突导向和回路发育中的功能尚未被阐明。 鉴定，甚至更少的是已知的GC蛋白的亚型特异性作用。该提案结合了最近 开发了具有超低输入蛋白质组学的亚型特异性GC纯化，以1）研究亚型特异性GC 蛋白质组在不同的发展阶段（前和后中线交叉），以确定和功能 研究具有阶段特异性作用的蛋白质，以及2）研究功能障碍GC的亚型特异性蛋白质组 识别和功能研究在精确电路布线中具有关键作用的失调蛋白。 这些严格的体内研究将加深对亚型和阶段特异性机制的理解 调节GC中的亚细胞蛋白质稳态，以及这些过程如何控制轴突寻路和 皮质投射神经元亚型中的突触回路。电路形成与亚细胞的交叉 蛋白质稳态具有实质性的

Project Summary/Abstract During development of the cerebral cortex, cortical projection neuron subtypes extend axons to innervate distinct targets located at great distances (103-105 cell body diameters) from their nucleus-containing somata. This precise navigation and circuit development is regulated by growth cones (GCs): subcellular compartments at tips of growing axons that rapidly integrate extracellular signals to control development of neural circuits, then mature into synapses. Since GCs respond rapidly to chemical cues, while hours/days are required to transport molecules between GCs and their parent somata, GCs highly likely function semi-autonomously. Prior work has also revealed that local protein synthesis and degradation pathways in GCs are required for responses to some directional cues. However, function of most GC proteins in axon guidance and circuit development have not been identified, and even less is known about subtype-specific roles of GC proteins. This proposal combines recently developed subtype-specific GC purification with ultra-low-input proteomics to 1) investigate subtype-specific GC proteomes at distinct developmental stages (pre- and post-midline crossing) to identify and functionally investigate proteins with stage-specific roles, and 2) investigate subtype-specific proteomes of dysfunctional GCs to identify and functionally investigate dysregulated proteins with critical roles in precise circuit wiring. These rigorous in vivo investigations will deepen understanding of subtype-, and stage-specific mechanisms regulating subcellular proteostasis in GCs, and how these processes control axon pathfinding and formation of synaptic circuitry in cortical projection neuron subtypes. The intersection of circuit formation and subcellular proteostasis has substantial