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.

项目摘要 脑外投射神经元（SCPN）是控制自愿的临床相关神经元类别 运动和肌萎缩性侧索硬化症和额叶痴呆或损伤的损失（例如， 脊髓损伤受损）导致瘫痪。目前，没有补充受伤或病变的方法 SCPN。再生医学中的一个里程碑是利用细胞重编程进行大脑和电路修复。这 方法使用发展基因和身份维护途径将一种单元格切换到另一种单元格 补充脆弱的神经元类型。但是，了解知识的差距很大 随着大脑的成熟，控制神经元的身份维持的机制。我们目前的知识有限 在胚胎阶段重新编程在体内的投影神经元，限制潜在疗法和 体外测定的进步。该建议通过针对身份直接解决这些需求 第6层丘脑神经元（CTN）中的维护机制将细胞身份的平衡转移到 第5层（L5）SCPN。 CTN具有发展起源，分化途径和形态学 具有SCPN的特征，已知的相似性促进细胞类型之间的转化。我们的小组 其他人发现，转录调节器BCE1丢失后，CTN可以切换到SCPN。我 假设BCE1的敲除是生成SCPN的有效手段 将CTN轴突接线到脑干，并将L5标记的上调作为重新编程成功的读数。 为了靶向运动皮层中的CTN，以跨发育阶段进行重新编程，我首先试图表征 一种新颖且广泛必要的转基因小鼠系（SYT6-CRE）（AIM 1第1部分）。这种方法允许 在运动区域中访问以前无法访问的CTN种群。接下来（目标1第2部分），我将确定 BCE1突变在不同成熟阶段的CTN中的潜在和有效性在产生SCPN的情况下 运动皮层。在AIM 1实验1中，我研究了BCE1突变诱导体内重新编程的程度 在不同的皮质成熟阶段中的CTN中的CTN。我使用逆行示踪剂的电路映射，测试 为了新的脑干项目轴突，以及用于SCPN和CTN的分子标记的组织学来确定 方法的功效。然后，在AIM 1实验2中，我使用RNA-Seq识别BCE1的下游效应子 重新编程的CTN需要获得SCPN特征所需的。对于（k00阶段），我将过渡到 中枢神经系统维修和再生医学领域。 AIM 2专注于可视化单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）具有高分辨率多光子或 轻度显微镜可视化体内过程。总体而言，这些技术将结合我的精致技能 在光学显微镜和分子生物学的发展技巧中，我对中枢神经系统的维修感兴趣，并且是普遍的 适用的是我的职业生涯在我走向独立的道路上和任期轨道位置。