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 亚脑投射神经元(SCPN)是一类临床上相关的神经元，它控制着 肌萎缩侧索硬化症和额颞痴呆或损伤中的运动及其丢失(例如， 脊髓损伤)会导致瘫痪。目前，还没有方法来补充受伤或受损的人 SCPN。再生医学的一个里程碑是利用细胞重新编程来修复大脑和电路。这 该方法使用发育基因和身份维持途径将一种细胞类型切换为另一种细胞类型，以 补充易受伤害的神经元类型。然而，在认识上存在着巨大的差距 随着大脑的成熟，控制神经元身份维持的机制。我们目前的知识是有限的 在胚胎阶段对体内的投射神经元重新编程，限制潜在的治疗和 体外检测技术的进展。该建议通过以身份为目标直接满足这些需求 6层皮质丘脑神经元维持细胞特性平衡的机制 第5层(L5)SCPN。CTN具有共同的发育起源、分化途径和形态 与SCPN的特征，已知有助于细胞类型之间转换的相似性。我们的团队 其他人发现，失去转录调控因子bce1后，CTN可以转换为SCPN。我 假设Bce1基因敲除是在出生后14天之前使用Re-Re-1产生SCPN的有效手段 将CTN轴突连接到脑干，并上调L5标记，以此作为重新编程成功的读数。 为了针对运动皮质中的CTN进行跨发育阶段的重新编程，我首先试图描述 一个新的和广泛需要的Cre转基因小鼠系(Syt6-Cre)(目标1，第1部分)。这种方法允许 在汽车区域接触到以前无法接触到的CTN人群。下一步(目标1第2部分)，我将确定 不同成熟阶段CTN中bce1基因突变在小鼠单细胞趋化因子生成中的作用 运动皮质。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。 CTN在不同的皮质成熟阶段转化为SCPN。我使用电路映射和逆行示踪器，测试 用于新的脑干投射轴突，并用组织学上的SCPN和CTNS分子标记来确定 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CTN以获取SCPN特征所必需的。对于我的(K00阶段)，我将过渡到 中枢神经系统修复与再生医学领域。目标2专注于可视化单个神经元内的细胞修复 在亚细胞水平上。这将涉及1)学习中枢神经系统损伤和修复的小鼠模型。然后2)申请 对系统进行空间转译。然后3)用高分辨率多光子或 光片显微镜在活体内可视化这一过程。总体而言，这些技巧将结合我精湛的技能 在光学显微镜和分子生物学方面的发展技能，以及我对中枢神经系统修复的兴趣 应聘者认为我的职业生涯正在走向独立和终身教职的轨道上。