Manipulation of neuron identity towards in-vivo circuit reprogramming in the cerebral cortex

操纵神经元身份以实现大脑皮层体内电路重编程

• 批准号: 10755203
• 负责人: Lee O Vaasjo
• 金额: $ 4.87万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10755203
• 关键词: Address; Aging; Area; Axon; Biology; Brain; Brain Stem; Cells; Cerebral cortex; Characteristics; Complement; Cre driver; Development; Developmental Gene; Effectiveness; Embryo; Equilibrium; Exhibits; Future; Genes; Genetic; Genetic Transcription; Goals; Histology; Image; Induced Mutation; Injury; Institution; Investments; Knock-out; Knowledge; Laboratories; Learning; Lesion; Life; Light; Maintenance; Maps; Mediating; Mentors; Methods; Microscopy; Modeling; Molecular; Molecular Biology; Molecular Target; Morphology; Motor; Motor Cortex; Movement; Mus; Mutation; Neurodegenerative Disorders; Neurons; Outcome; Paralysed; Pathway interactions; Pattern; Phase; Population; Positioning Attribute; Prefrontal Cortex; Process; Property; RNA; Regenerative Medicine; Reporter; Research; Research Personnel; Resolution; Scientist; Sensory; Signal Transduction; Specific qualifier value; Spinal cord damage; Spinal cord injury; System; Technical Expertise; Techniques; Testing; Thalamic structure; Therapeutic Uses; Tracer; Training; Training Support; Transgenic Mice; Up-Regulation; Visualization; Vulnerable Populations; Work; brain repair; career; cell type; cellular targeting; central nervous system injury; clinically relevant; efficacy evaluation; experience; experimental study; frontotemporal lobar dementia amyotrophic lateral sclerosis; in vitro Assay; in vivo; injured; injury and repair; insight; interest; light microscopy; mature animal; molecular marker; mouse model; multi-photon; neural; neuron development; neuroregulation; novel; novel strategies; post-doctoral training; postnatal; prevent; programs; repaired; skills; success; tenure track; tool; transcriptome; transcriptome sequencing; transcriptomics

Project Summary Sub-Cerebral Projection Neurons (SCPNs) are a clinically relevant neuron class that controls voluntary movement and whose loss in Amyotrophic Lateral Sclerosis and Fronto-temporal dementia or injury (e.g., damaged by spinal cord injury) leads to paralysis. Currently, there are no methods to replenish injured or lesioned SCPNs. A milestone in regenerative medicine is to utilize cellular reprogramming for brain and circuit repair. This approach uses developmental genes and identity maintenance pathways to switch one cell type to another to replenish vulnerable neuron types. However, there is a significant gap in knowledge in understanding the mechanisms that control identity maintenance in neurons as the brain matures. Our current knowledge is limited to reprogramming projection neurons in-vivo during embryonic stages, limiting potential therapies and the advancement of in-vitro assays. This proposal directly addresses these needs by targeting a identity maintenance mechanism in Layer 6-Cortico Thalamic Neurons (CTns) to shift the balance of cell identity toward Layer 5 (L5) SCPNs. CTns share a developmental origin, differentiation pathways, and morphological characteristics with SCPNs, similarities that are known to facilitate the conversion between cell types. Our group and others have found that CTns can be switched into SCPNs upon loss of the transcriptional regulator Bce1. I hypothesize that knock-out of Bce1 is an effective means of generating SCPNs until post-natal day 14 using re- wiring the CTn axon to the brainstem and the upregulation of L5 markers as readouts of reprogramming success. To target CTns in the motor cortex for reprogramming across developmental stages, I first sought to characterize a novel and broadly necessary Cre- transgenic mouse line (Syt6-Cre) (Aim 1 Part 1). This approach allows access to previously inaccessible CTn populations in the motor areas. Next (Aim 1 Part 2), I will determine the potential and effectiveness of Bce1 mutation in CTns at different maturation stages in generating SCPNs in the motor cortex. In Aim 1 Experiment 1, I investigate the extent that Bce1 mutation induces in-vivo reprogramming of CTns into SCPNs at different cortical maturation stages. I use circuit mapping with retrograde tracers, testing for a new brainstem-projecting axon, and histology for SCPN and CTns molecular markers to determine the efficacy of the approach. Then, in Aim 1 Experiment 2, I use RNA-seq to identify downstream effectors of Bce1 required for reprogrammed CTns to acquire SCPN characteristics. For my (K00 phase), I will transition into the field of CNS repair and regenerative medicine. Aim 2 focuses on visualizing cellular repair within single neurons at the sub-cellular level. This will involve 1) learning a mouse model for CNS injury and repair. Then 2) apply Spatial Transcriptomics to the system. Then 3) test functional hypotheses with high-resolution multi-photon or light sheet microscopy to visualize the process in-vivo. Overall, these techniques will combine my refined skills in light microscopy and developing skills in molecular biology with my interest in CNS repair and are universally applicable thought my career on my path towards independence and a tenure track position.

项目概要 大脑下投射神经元 (SCPN) 是一种临床相关的神经元类别，控制自主意识 运动能力以及因肌萎缩侧索硬化症和额颞叶痴呆或损伤而丧失的能力（例如， 脊髓损伤）导致瘫痪。目前还没有办法补充受伤或病变的物质。 SCPN。再生医学的一个里程碑是利用细胞重编程来修复大脑和电路。这 方法使用发育基因和身份维持途径将一种细胞类型转换为另一种细胞类型 补充脆弱的神经元类型。然而，人们在认识上存在着巨大的差距。 随着大脑成熟，控制神经元身份维持的机制。我们目前的知识有限 在胚胎阶段体内重新编程投射神经元，限制了潜在的治疗方法和 体外测定的进展。该提案通过针对身份直接解决这些需求 第 6 层皮质丘脑神经元 (CTns) 的维持机制将细胞身份的平衡转向 第 5 层 (L5) SCPN。 CTns 具有共同的发育起源、分化途径和形态学特征 与 SCPN 的特征相似，已知可促进细胞类型之间的转换。我们组 等人发现，在转录调节因子 Bce1 丢失后，CTns 可以转变为 SCPN。我 假设敲除 Bce1 是在产后第 14 天使用重新生成 SCPN 的有效方法 将 CTn 轴突连接到脑干和 L5 标记物的上调作为重编程成功的读数。 为了针对运动皮层中的 CTns 进行跨发育阶段的重新编程，我首先试图表征 一种新颖且广泛必需的 Cre 转基因小鼠品系 (Syt6-Cre)（目标 1 第 1 部分）。这种方法允许 接触到以前无法接触到的运动区 CTn 人群。接下来（目标 1 第 2 部分），我将确定 不同成熟阶段 CTns 中 Bce1 突变在生成 SCPN 中的潜力和有效性 运动皮层。在目标 1 实验 1 中，我研究了 Bce1 突变诱导体内重编程的程度 不同皮质成熟阶段的 CTns 转化为 SCPN。我使用带有逆行示踪剂的电路测绘，测试 用于新的脑干投射轴突，以及 SCPN 和 CTns 分子标记的组织学以确定 该方法的有效性。然后，在目标 1 实验 2 中，我使用 RNA-seq 来识别 Bce1 的下游效应子 重新编程的 CTns 需要获得 SCPN 特性。对于我的（K00 阶段），我将过渡到 中枢神经系统修复和再生医学领域。目标 2 侧重于可视化单个神经元内的细胞修复 在亚细胞水平。这将涉及 1) 学习用于中枢神经系统损伤和修复的小鼠模型。然后2）申请 系统的空间转录组学。然后 3）用高分辨率多光子或测试功能假设 光片显微镜观察体内过程。总的来说，这些技术将结合我精炼的技能 我对光学显微镜和分子生物学技能的发展感兴趣，并且对中枢神经系统修复感兴趣，并且普遍 适用的想法是我的职业生涯在我走向独立和终身教职的道路上。