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 为了了解神经系统疾病，识别受影响的神经细胞类型是至关重要的， 创建准确概括正常功能和疾病表型的模型系统，并开发工具 使细胞操控成为可能。脊髓中的运动神经元通过沟通来控制身体的运动 肌肉靶点的中央运动指令。所有的脊髓运动神经元都是在胚胎时期出生和特化的 但它们的分子特性和电生理特性在小鼠体内进化了数周 在人类中持续数月，直到出生后生活中的运动回路和行为完全成熟。在疾病中，比如 肌萎缩侧索硬化症(ALS)、肌萎缩侧索硬化症或脊髓损伤，是指 成熟的成年运动神经元可能导致肌肉失去控制、瘫痪和死亡。尽管有几项研究 绘制了胚胎脊髓运动神经元多样化的机制，我们的理解是 成人脊髓运动神经元多样性的研究还处于初级阶段。这阻碍了对成年运动神经元的研究。 疾病作为受影响的运动神经元亚型还没有得到彻底的定义。此外，体外模型 真实地概括成人运动神经元的身份不存在，并有足够的工具来访问特定的运动神经元 体内缺乏亚型。这项研究计划旨在绘制有丝分裂后运动神经元的轨迹 从胚胎到成年，并使用这些数据创建病毒工具，提供对特定亚型的基因访问 体内运动神经元的研究，并开发在体外产生成人型运动神经元的方法。这件事会做到的 首先在小鼠脊髓运动神经元中进行单细胞转录组和染色质图谱分析 从胚胎到成年。时间染色质图谱将用于开发AAV工具，提供基因 在所有年龄段获得特定的运动神经元亚型，并通过计算确定候选的调节器 特定于亚型和成人的身份。然后，确定的调节剂将被用于成熟小鼠干细胞 细胞来源的运动神经元在体外。最后，成人的单细胞转录和染色质可及性谱 将产生人类运动神经元，并将使用老鼠和人类特有的调节剂的组合 对IPSC来源的运动神经元的年龄进行编程。这种彻底的方法将定义分子特征 运动神经元亚型在疾病中的不同易感性，并建立工具 对于解剖电路、疾病建模和提供潜在的治疗方法来说是必要的。 该奖项的培训阶段将在哥伦比亚大学的Hynek Wichterle博士的实验室进行 由大卫·吉福德博士和保拉·阿洛塔博士共同指导。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生涯。拟议研究计划的完成将有助于 我实验室的未来研究旨在了解神经元身份、电路和 运动神经元和其他神经系统细胞的疾病。