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

项目摘要/摘要 在大脑皮层发育过程中，皮质投射神经元亚型延伸轴突以支配 明显的靶标位于距离其含核的体细胞很远的地方(103-105细胞体直径)。 这种精确的导航和回路发育由生长锥体(GC)调节：亚细胞室 在生长轴突的尖端，迅速整合细胞外信号来控制神经回路的发展，然后 成熟为突触。由于GC对化学信号反应迅速，而运输需要数小时/数天 在GC和其亲本体细胞之间的分子，GC很可能以半自主的方式发挥作用。之前的工作已经完成 还揭示了GC中的局部蛋白质合成和降解途径需要对一些 方向性提示。然而，大多数GC蛋白在轴突引导和回路发育中的功能尚未得到 对于GC蛋白亚型特有的作用，人们所知更少。这项建议最近结合了 用超低输入蛋白质组学建立亚型特异性GC纯化方法1)研究亚型特异性GC 不同发育阶段的蛋白质组(中线前后交叉)以识别和功能 研究具有阶段特定作用的蛋白质，以及2)研究功能障碍的GC的亚型特定蛋白质组 鉴定和功能研究在精确电路连接中起关键作用的失调蛋白。 这些严谨的体内研究将加深对亚型和阶段特定机制的理解 调节GC中的亚细胞蛋白平衡，以及这些过程如何控制轴突的发现和形成 皮质投射神经元亚型中的突触回路。电路形成和亚细胞的交集 蛋白质平衡有实质性的

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