Defining motor neuron diversity from embryo to adulthood and generating tools for in vivo and in vitro access

定义从胚胎到成年的运动神经元多样性并生成体内和体外访问工具

• 批准号: 10369830
• 负责人: Tulsi Patel
• 金额: $ 13.62万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10369830
• 关键词: ATAC-seq; Adult; Affect; Age; Amyotrophic Lateral Sclerosis; Autopsy; Award; Behavior; Bioinformatics; Biological Assay; Biological Models; Brain region; Cell Line; Cell Nucleus; Cells; Cessation of life; Chromatin; Data; Data Analyses; Development Plans; Dimensions; Disease; Disease model; Electrophysiology (science); Embryo; Embryonic Development; Gene Delivery; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Human; In Vitro; Laboratories; Lead; Life; Maps; Mentorship; Methods; Mitotic; Modeling; Molecular; Molecular Profiling; Motor; Motor Neuron Disease; Motor Neurons; Movement; Mus; Muscle; Muscular Atrophy; Nervous system structure; Neurons; Neurophysiology - biologic function; Nucleic Acid Regulatory Sequences; Paralysed; Patients; Phase; Predisposition; Property; Research; Research Personnel; Signal Transduction; Sorting - Cell Movement; Specific qualifier value; Spinal; Spinal Cord; Spinal cord injury; System; Testing; Therapeutic; Time; TimeLine; Training; Universities; Viral; Virus; Work; base; career; career development; cell type; disease phenotype; in vitro Model; in vivo; induced pluripotent stem cell; infancy; method development; motor neuron function; mutant; nervous system disorder; neural circuit; post-doctoral training; programs; sarcopenia; skills; stem cells; therapy development; time use; tool; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY/ABSTRACT In order to understand neurological diseases, it is essential to identify the affected neuronal cell types, create model systems that accurately recapitulate normal function and disease phenotypes, and develop tools that allow cellular manipulations. Motor neurons in the spinal cord control body movement by communicating central motor commands with muscle targets. All spinal motor neurons are born and specified during embryonic development, but their molecular identities and electrophysiological properties evolve for weeks in mice and months in humans, until motor circuits and behavior become fully mature in post-natal life. In diseases like Amyotrophic Lateral Sclerosis (ALS), sarcopenia, or spinal cord injury, the degeneration of specific subsets of mature, adult motor neurons can lead to loss of muscle control, paralysis, and death. Although several studies have mapped the mechanisms of motor neuron diversification in the embryonic spinal cord, our understanding of motor neuron diversity in the adult spinal cord is in its infancy. This hinders the study of adult motor neuron diseases as the affected motor neuron subtypes are not thoroughly defined. Furthermore, in vitro models that faithfully recapitulate adult motor neuron identity do not exist, and adequate tools to access specific motor neuron subtypes in vivo are lacking. This research plan aims to map the trajectory of post-mitotic motor neurons from embryo to adulthood and use this data to both create viral tools that provide genetic access to specific subtypes of motor neurons in vivo, and develop methods for generating adult-like motor neurons in vitro. This will be done by first performing single cell transcriptome and chromatin profiling in mouse spinal motor neurons at various embryonic to adult ages. The temporal chromatin profiles will be used to develop AAV tools that provide genetic access to specific motor neuron subtypes at all ages, and to computationally identify candidate regulators of subtype- and adult-specific identity. The identified regulators will then be used to mature the age of mouse stem cell derived motor neurons in vitro. Finally, single cell transcriptomic and chromatin accessibility profiles of adult human motor neurons will be generated and a combination of mouse and human-specific regulators will be used to program the age of iPSC-derived motor neurons. This thorough approach will define the molecular features of motor neuron subtypes that contribute to their differential susceptibility in disease, and establish tools necessary for dissecting circuits, disease modeling, and the delivery of potential therapeutics. The training phase of the award will be conducted in the laboratory of Dr. Hynek Wichterle at Columbia University, and under the co- mentorship of Dr. David Gifford and Dr. Paola Arlotta. My career development plan describes a detailed timeline for acquiring all the technical and professional skills necessary for the successful transition into a career as an independent researcher. The completion of the proposed research plan will facilitate future research in my lab aimed at understanding the temporal dynamics of neuronal identity, circuits, and disease in motor neurons and other nervous system cells.

项目摘要/摘要 为了了解神经疾病，必须识别受影响的神经元细胞类型， 创建模型系统，以准确概括正常功能和疾病表型，并开发工具 这允许细胞操作。通过通信，脊髓控制体运动中的运动神经元 带有肌肉目标的中央电动机命令。所有脊柱运动神经元均在胚胎期间出生和指定 开发，但是它们的分子身份和电生理特性在小鼠和 在人类中的几个月，直到马达电路和行为在产后生活中变得完全成熟。在类似的疾病中 肌萎缩性侧面硬化症（ALS），肌肉减少症或脊髓损伤，特定子集的变性 成熟的成年运动神经元会导致肌肉控制，麻痹和死亡的丧失。虽然有几项研究 已经绘制了胚胎脊髓中运动神经元多样化的机制，我们的理解 成人脊髓中运动神经元的多样性仍处于起步阶段。这阻碍了成人运动神经元的研究 疾病作为受影响的运动神经元亚型的疾病未彻底定义。此外，体外模型 忠实地概括成人电机神经元的身份，并且有足够的工具访问特定的电机神经元 缺乏体内亚型。该研究计划旨在绘制有丝分裂后运动神经元的轨迹 成年的胚胎并使用这些数据来创建病毒工具，可提供对特定亚型的遗传访问 体内运动神经元的体积，并开发用于体外产生成年运动神经元的方法。这将完成 首先在各种的小鼠脊柱运动神经元中执行单细胞转录组和染色质分析 成人年龄的胚胎。时间染色质曲线将用于开发提供遗传的AAV工具 在所有年龄段的特定运动神经元亚型中访问 亚型和成人特异性身份。然后，已确定的调节器将用于成熟小鼠茎的年龄 细胞衍生的运动神经元体外。最后，成人的单细胞转录组和染色质可及性概况 将产生人类运动神经元，并将使用小鼠和人类特异性调节剂的组合 编程IPSC衍生运动神经元的年龄。这种详尽的方法将定义分子特征 运动神经元亚型的疾病敏感性差异并建立工具的运动神经元亚型 解剖电路，疾病建模和潜在治疗剂的交付所必需的。 该奖项的培训阶段将在哥伦比亚的Hynek Wichterle博士实验室进行 大学，并在戴维·吉福德（David Gifford）博士和帕拉·阿洛塔（Paola Arlotta）博士的合作下。我的职业发展计划 描述了获取成功所需的所有技术和专业技能的详细时间表 过渡到独立研究人员的职业。拟议的研究计划的完成将有助于 我的实验室中的未来研究旨在了解神经元身份，电路和电路的时间动态 运动神经元和其他神经系统细胞中的疾病。