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

亚型和电路特异性生长锥中的亚细胞 RNA 蛋白质组图谱：发育、细胞生物学、疾病和再生

• 批准号: 10223443
• 负责人: JEFFREY D MACKLIS
• 金额: $ 118.3万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10223443
• 关键词: Address; Anatomy; Biochemistry; Bipolar Disorder; Brain; Caliber; Cell Nucleus; Cells; Cellular biology; Development; Disease; Disease model; Functional disorder; Genetic; Genetic Transcription; Genomics; Growth Cones; Human; Huntington Disease; Intellectual functioning disability; Investigation; Knowledge; Label; Mass Spectrum Analysis; Modeling; Modification; Molecular; Molecular Machines; Natural regeneration; Neurons; Neurosciences; Parents; Parkinson Disease; Pathway interactions; Peptides; Peripheral Nerves; Pharmaceutical Preparations; Protein Analysis; Proteins; Proteome; Proteomics; RNA; RNA analysis; Risk; Schizophrenia; Sorting - Cell Movement; Specificity; Spinal cord injury; Subcellular structure; Synapses; Therapeutic; Transcript; Transcriptional Regulation; Work; autism spectrum disorder; base; experimental study; induced pluripotent stem cell; innovation; neuronal cell body; neuropsychiatric disorder; next generation sequencing; novel strategies; particle; presynaptic

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.

所有神经科学中一个首要的、核心的问题是巨大神经元的特异性、修饰和功能。 功能特定电路的多样性——这个问题在多个核心方面仍然无法解决。这就是背后的原理 大脑神经系统感知、整合、移动身体、思考、精确运作、发生故障 疾病中的特异性，随着电路特异性而退化，可能会再生，和/或可能在培养中建模。什么 实际上实现和维持电路特异性是从电路的发展特异性到 发育异常，正常功能（或功能障碍）和电路类型特异性分子调节器， 亚型特异性退化（例如 ALS、亨廷顿病、帕金森病）到再生（或典型缺乏再生） 在 CNS 中治疗脊髓损伤或在 PNS 中以最佳精度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，人类基因组风险 这种关联出现在神经精神疾病中，如精神分裂症、自闭症、双相情感障碍、发育障碍 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来实现 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（亚细胞）的多样性和专业化知之甚少。 实现特定电路布线的分子机器，精确地成熟为突触前的一半 多样化的突触，并控制长期存在的“排序问题”。 GC 在许多天内执行这些功能 每条途径的发育，通常远离细胞核和转录控制103-105个细胞体直径。 值得注意的是，但很少有人考虑到，一个具有一种转录调控机制的细胞核可以控制 2 个或更多转录调控机制。 不同的 GC 连接多目标电路。我提出全新的、高度创新的、开拓性的发展工作， 细胞生物学、疾病和再生（也与建模相关）独特地解决了这一知识方面的关键差距。 我们开发了新方法来直接从大脑研究亚型和阶段特异性 GC，具有高深度、 定量蛋白质组学和 RNA 分析，并且已经完成了概念验证实验，使一系列 开拓新工作。我们根据神经元亚型、投射轨迹和发育阶段选择性纯化 GC 结合使用分子、解剖和基因标记策略；亚细胞生物化学；新开发的 小颗粒分选；肽质谱分析；和下一代测序。同时分离蛋白质和 RNA 从亲代体细胞及其 GC 中鉴定出数百种蛋白质和转录本，这些蛋白质和转录物在 GC 中富集了几个数量级， 在亲本体细胞中基本上未检测到。这表明实际上可能需要对 GC 进行调查才能了解 子类型特定的电路。 GC 似乎很早就被“编程”，然后“准备好”发挥相当自主的本地控制。 我建议对亚型、阶段和目标特异性 GC 蛋白和 RNA 进行雄心勃勃且大胆的研究 多种特定设置来研究发育、细胞生物学、疾病、再生和 iPS/ES 模型的机制。这些 方向范围从立即到〜5年，到〜10年。结果将产生新的假设和调查。