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，一个首要的中心问题是巨细胞的特异性、修饰和功能 功能特定的电路的多样性--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、 疾病中的特异性，与电路特异性一起退化，可以再生，和/或可以在培养中建模。什么 实际实现和维护电路的专用性是一个关键、核心的问题，从电路的发展专用性，到 发育异常，到正常的功能(或功能障碍)和电路类型特定的分子调节器，到 亚型特异性变性(如在肌萎缩侧索硬化症、亨廷顿病、帕金森病中)，到再生(或典型的缺乏) 对于脊髓损伤的CNS或在PNS中的最佳准确性，到疾病的机械和治疗模型 使用iPS/ES来源的神经元。生长锥体(GC)“构建”环路并成熟为突触，人类基因组在突触中存在风险 在精神分裂症、自闭症、双相情感障碍、发育性疾病等神经精神疾病中都出现了关联。 智力障碍。我建议在这些问题上开展独特的、开创性的工作--现在可以通过我们的创新 接近了。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--亚细胞--的多样性和专门化知之甚少 实现特定电路连接的分子机器，以精确的方式成熟为极大的突触前半部分 多样化的突触，并控制了长期存在的“分类问题”。GCS在许多天的时间里执行这些功能 发育的每个途径，往往103-105细胞体直径远离细胞核和转录控制。 值得注意的是，但很少有人考虑到，一个核，带有一个转录调控机制，可以控制两个或更多 分散的GC连接多目标电路。我提出了全新的、高度创新的、开创性的发展工作， 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差距。 我们开发了新的方法来直接从大脑中研究特定亚型和特定阶段的GC，具有高深度， 定量蛋白质组和RNA分析，并已经完成了概念验证实验，使一系列 开拓性的新工作。我们根据神经元亚型、投射轨迹和发育阶段选择性地提纯GC 使用分子、解剖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；多肽质谱学；下一代测序。蛋白质和RNA的同时分离 从母公司SoMAS及其GC中鉴定出数百种蛋白质和转录产物在GC中富含数量级， 基本上没有在父SoMAS中检测到。这表明，实际上可能需要对GC进行调查，以了解 子类型特定的电路。GCS似乎很早就被“编程”，然后“准备”施加相当自主的本地控制。 我建议对亚型、阶段和靶标特定的GC蛋白和RNA进行雄心勃勃和大胆的研究 多个特定环境，研究发育机制、细胞生物学、疾病、再生和iPS/ES模型。这些 方向从即刻到约5年，到约10年的水平。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.