Subcellular RNA-Proteome Mapping in Subtype- and Circuit-Specific Growth Cones: Development, Cell Biology, Disease, and Regeneration

在所有神经科学中，一个至关重要的中心问题是巨大神经元的特异性、修饰和功能。 特定功能电路的多样性-这是一个在多个核心方面仍然无法解决的问题。这就是为什么 大脑神经系统感知、整合、移动身体、思考、精确运作， 疾病特异性、具有回路特异性的退化、可以再生和/或可以在培养物中建模。什么 实际上实现和维持电路特异性是一个关键的核心问题，从电路的发育特异性， 发育异常，适当的功能（或功能障碍）和电路类型特异性分子调节剂， 亚型特异性变性（例如ALS、亨廷顿病、帕金森病），再生（或典型缺乏再生） 在CNS中用于脊髓损伤或在PNS中具有最佳准确性，以建立疾病的机制和治疗模型 使用iPS/ES衍生的神经元。生长锥（GC）“构建”电路并成熟为突触，其中人类基因组风险 神经精神疾病，如精神分裂症，自闭症，双相情感障碍，发育性 智力残疾。我建议在这些问题上开展独特的、具有开创性的工作--现在，我们的创新 接近。我们现在能够直接研究不同GC亚型的分子机制，从而研究不同的电路。 尽管它们很重要，但我们对电路特异性GC的多样性和专业化知之甚少-亚细胞GC 实现特定电路布线的分子机器，精确地成熟为突触前的两半， 多样的突触，并控制长期存在的“排序问题”。GC在许多天内执行这些功能， 每种途径的发育，通常距离细胞核和转录控制103-105个细胞体直径。 值得注意的是，但很少考虑，一个核，一个转录调控机制，可以控制2个或更多 发散性毒气来连接多目标电路我建议在发展方面进行全新的、高度创新的、开拓性的工作， 细胞生物学、疾病和再生（也与建模相关），以独特地解决知识中的这一关键空白。 我们开发了新的方法来研究直接来自大脑的亚型和阶段特异性GC， 定量蛋白质组学和RNA分析，并已经完成了概念验证实验，使一系列 开拓新的工作。我们根据神经元亚型、投射轨迹和发育阶段选择性地纯化GC 使用分子、解剖学和遗传标记策略的组合;亚细胞生物化学;新开发的 小颗粒分选;肽质谱;和下一代测序。同时分离蛋白质和RNA 从亲本胞体和它们的GC中鉴定出数百种在GC中富集数量级的蛋白质和转录物， 在亲本胞体中基本上没有检测到。这表明，调查GC实际上可能需要了解 子类型特定电路。GC似乎是“编程”早期，然后“准备”施加相当自主的地方控制。 我提出了雄心勃勃的和冒险的调查亚型，阶段，和目标特异性GC蛋白和RNA， 多个特定的设置来研究发育机制，细胞生物学，疾病，再生和iPS/ES模型。这些 方向范围从近、到~5年、到~10年的地平线。结果将产生新的假设和调查。

An overarching, central question in all of neuroscience is the specificity, modification, and function of the immense diversity of function-specific circuitry– a question still inaccessible in multiple core aspects. This is what underlies how the brain-nervous system senses, integrates, moves the body, thinks, functions with precision, malfunctions with specificity in disease, degenerates with circuit specificity, might be regenerated, and/or might be modeled in culture. What actually implements and maintains circuit specificity is a key, core issue from developmental specificity of circuits, to developmental abnormalities, to proper function (or dysfunction) and circuit type-specific molecular regulators, to subtype-specific degeneration (e.g. in ALS, Huntington's, Parkinson's diseases), to regeneration (or typical lack thereof) in the CNS for spinal cord injury or with optimal accuracy in the PNS, to mechanistic and therapeutic modeling of disease using iPS/ES-derived neurons. Growth cones (GCs) “build” circuits and mature into synapses, where human genomic risk associations are showing up in neuropsychiatric diseases such as schizophrenia, autism, bipolar disorder, developmental intellectual disabilities. I propose uniquely enabling, pioneering work on these issues– now possible by our innovative approaches. We are now able to directly investigate molecular machinery of distinct GC subtypes, thus distinct circuits. Despite their importance, we know little about the diversity and specialization of circuit-specific GCs– the subcellular molecular machines that implement specific circuit wiring, mature with precision into presynaptic halves of immensely diverse synapses, and control the long-standing “sorting problem”. GCs perform these functions over many days of development for each pathway, often 103-105 cell body diameters away from the nucleus and transcriptional control. Remarkably, but rarely considered, one nucleus, with one transcriptional regulatory machinery, can control 2 or more divergent GCs to wire multi-target circuitry. I propose entirely new, highly innovative, pioneering work in development, cell biology, disease, and regeneration (also relevant to modeling) to uniquely address this critical gap in knowledge. We developed new approaches to investigate subtype- and stage-specific GCs directly from brains, with high-depth, quantitative proteomic and RNA analysis, and have already completed proof-of-concept experiments enabling a range of pioneering new work. We selectively purify GCs based on neuron subtype, projection trajectory, and developmental stage using a combination of molecular, anatomic, and genetic labeling strategies; subcellular biochemistry; newly developed small-particle sorting; peptide mass spectrometry; and Next Gen sequencing. Simultaneous isolation of protein and RNA from parent somas and their GCs identifies hundreds of proteins and transcripts enriched orders of magnitude in GCs, essentially not detected in parent somas. This indicates that investigation of GCs might actually be required to understand subtype-specific circuitry. GCs appear to be “programmed” early, then “poised” to exert quite autonomous local control. I propose ambitious and venturesome investigations of subtype-, stage-, and target-specific GC proteins and RNAs in multiple specific settings to study mechanisms of development, cell biology, disease, regeneration, & iPS/ES models. These directions range from immediate, to ~5 yrs, to an ~10 yr horizon. Results will generate new hypotheses and investigations.