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: 9354029
• 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 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9354029
• 关键词: Address; Anatomy; Autistic Disorder; 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; 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.

在所有神经科学中，一个总体的中心问题是巨大的特异性，修改和功能 功能特异性电路的多样性 - 这个问题在多个核心方面仍然无法访问。这就是如何基础 大脑障碍系统感官感官，整合，移动身体，思考，精确，功能不当，与 疾病的特异性，与电路特异性退化，可以再生和/或可能在培养中建模。什么 实际实施和维护电路特异性是关键，核心问题，从电路的发展特异性到 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，在ALS中，亨廷顿，帕金森氏病），再生（或典型缺乏） 在中枢神经系统中，用于脊髓损伤或PN的最佳精度，用于疾病的机械和治疗模型 使用IPS/ES衍生的神经元。生长锥（GCS）“构建”电路和成熟的人类基因组风险 社会出现在精神分裂症，自闭症，躁郁症，发育中的神经精神疾病中 智力残疾。我建议在这些问题上进行独特的实现，开创性的工作 - 现在我们的创新性可能 方法。现在，我们能够直接研究不同GC亚型的分子机械，从而不同的电路。 尽管它们的重要性，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 实现特定电路接线的分子机，精确成熟为突触前的一半 潜水员突触并控制长期存在的“分类问题”。 GC在许多天内执行这些功能 每种途径的发育通常远离细胞核和转录对照的103-105细胞体直径。 值得注意但很少考虑的是，一个核具有一种转录调节机制，可以控制2个或更多 与电线多目标电路的发散GC。我提出了全新的，高度创新的，开创性的开发工作， 细胞生物学，疾病和再生（也与建模有关），以唯一解决知识的关键差距。 我们开发了新的方法来直接从大脑中研究亚型和阶段特异性GC，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，使一系列能够 开拓新作品。我们根据神经元亚型，投影轨迹和发育阶段有选择地纯化GCS 结合分子，解剖和遗传标记策略；亚细胞生物化学；新开发 天粒子分类；胡椒质谱；和下一个一代测序。同时分离蛋白质和RNA 从父som及其GC识别出数百种蛋白质和转录本，富含GC中的数量级， 基本上未在父somas中检测到。这表明实际上可能需要对GC进行调查才能了解 亚型特异性电路。 GC似乎很早就被“编程”，然后“准备”以发挥相当自主的本地控制。 我提出了对亚型，阶段和特异性GC蛋白和RNA的雄心勃勃和冒险研究 多种特定的环境，用于研究发展，细胞生物学，疾病，再生和IPS/ES模型的机制。 方向范围从立即到约5年，到约10年的视野。结果将产生新的假设和研究。