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

项目摘要 大脑下投射神经元（Sub-Cerebral Projection Neurons，SCPNs）是一种临床相关的神经元， 运动和其在肌萎缩性侧索硬化症和额颞痴呆或损伤中的丧失（例如， 因脊髓损伤而受损）导致瘫痪。目前，还没有方法来补充受伤或病变 SCPNs。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 这种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，在理解这一点方面存在着巨大的知识差距。 大脑成熟时控制神经元身份维持的机制。我们目前的知识有限 在胚胎阶段对投射神经元进行体内重编程，限制了潜在的治疗方法， 体外分析的进展。该提案通过针对一个身份直接解决了这些需求 第6层-皮质丘脑神经元（CTns）中的维持机制将细胞身份的平衡转向 第5层（L5）SCPNs。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，已知促进细胞类型之间的转换的相似性。我们集团 其他人发现CTns可以在失去转录调节因子Bce 1时转换为SCPNs。我 假设敲除Bce 1是使用re-SCPNs产生SCPNs直到出生后第14天有效手段， 将CTn轴突连接到脑干和上调L5标记作为重编程成功的读数。 为了靶向运动皮层中的CTns，以便在发育阶段进行重新编程，我首先试图描述 一种新的和广泛需要的Cre转基因小鼠系（Syt 6-Cre）（目的1第1部分）。这种方法允许 在运动区接触到以前无法接触到的CTn人群。接下来（目标1第2部分），我将确定 在不同成熟阶段的CTns中Bce 1突变在产生SCPNs中的潜力和有效性 运动皮层在目标1实验1中，我研究了Bce 1突变诱导体内重编程的程度 CTns向SCPNs转化的可能性。我用逆行追踪器做电路图， 一个新的脑干投射轴突，和SCPN和CTns分子标记物的组织学，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来鉴定Bce 1的下游效应子 重新编程CTns以获取SCPN特性所需的。对于我的（K 00阶段），我将过渡到 CNS修复和再生医学领域。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CNS损伤和修复的小鼠模型。（2）应用 空间转录组系统。然后3）用高分辨率多光子或 光片显微镜观察体内过程。总的来说，这些技术将联合收割机结合我的精炼技能 在光学显微镜和发展技能的分子生物学与我的兴趣在中枢神经系统修复，并普遍 适用的思想我的职业生涯在我的道路上走向独立和终身职位。

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.