Genetic labeling and visualization of CSMN in models of motor neuron disorders

我们的大脑皮层中有成千上万种不同的神经元，但在神经退行性疾病中 只有选定的神经元群体表现出最初的脆弱性，并经历进行性退化。皮质脊髓 运动神经元(CSMN)是位于运动皮质V层的大锥体神经元。他们的手机 结构是非常独特的，它们的功能是大脑皮层的“轮辐人”，用于启动和 运动的调节。自愿行动是聪明和见多识广的头脑的行为。因此，CSMN 接收来自众多神经元的信息，包括长距离投射神经元和局部电路神经元。 CSMN将这些信息整合并翻译成一个指向脊髓目标的信号的独特能力设置了它 除了其他大脑皮层神经元。因此，它的退化具有严重的后果，导致各种 运动障碍。了解CSMN变性在疾病中的基础是一种发展中的需要。 然而，CSMN的识别和可视化并不容易，因为它们嵌入在数以千计的 大脑皮层内的神经元群。我们最近产生了一个新的报告线，UCHL1-EGFP 在小鼠中，CSMN在运动皮质中被基因标记。UCHL1基因调控下的绿色荧光蛋白表达 启动子是稳定的，在体内持续到P800，并受限于运动皮质中的CSMN。的CSMN身份 通过解剖、逆行标记、分子标志物表达鉴定运动皮质中的EGFP+神经元 侧写和电生理分析。这条记者专线提供了许多独特的优势；a)我们可以第一 时间可视化CSMN不需要任何逆行标记手术；b)CSMN可以通过FACS纯化- 生命不同阶段的中介途径；c)细胞和分子机制 可以详细和精确地研究CSMN的脆弱性和退化；d)最重要的是这一新的 报告线可以跨越各种运动障碍的小鼠模型来研究CSMN的生物学 关于疾病。在这项提议中，我们的目标是在不同的小鼠模型中为CSMN带来视觉清晰度 运动神经元病。我们将把UCHL1-EGFP小鼠与最近发现的小鼠模型进行杂交 提示上运动神经元变性可能参与疾病的病理过程。由于时间限制， R21授予，我们将在有限数量的小鼠中表征CSMN退化的时间和程度 模型，如Tdp43A315T和Alsin KO小鼠。我们生成的工具和我们开发的方法将 帮助生成和刻画其他记者鼠标模型，并将提高我们的努力 了解CSMN脆弱性和退化背后的细胞和分子机制。

There are thousands of different neuron populations in our cerebral cortex, but in neurodegenerative diseases only a select neuron population show primary vulnerability and undergo progressive degeneration. Corticospinal motor neurons (CSMN) are large pyramidal neurons that are located in layer V of the motor cortex. Their cellular structure is very unique and they function as the "spokes person"of the cerebral cortex for the initiation and modulation of movement. Voluntary movement is the act of a clever and well-informed mind. Therefore CSMN receive information from numerous neurons, including long-distance projection and local circuitry neurons. CSMN's unique ability to integrate and translate this information into one signal towards spinal cord targets sets it apart from other cortical neurons. Therefore its degeneration has severe consequences that lead to various movement disorders. There is a developing need to understand the basis of CSMN degeneration in diseases. However, identification and visualization of CSMN is not easy as they are embedded among thousands of other neuron populations within the cerebral cortex. We recently generated a novel reporter line, the UCHL1-eGFP mice, in which CSMN are genetically labeled in the motor cortex. eGFP expression under the control of UCHL1 promoter is stable, persistent up to P800 in vivo, and is restricted to CSMN in the motor cortex. CSMN identity of eGFP+ neurons in the motor cortex are identified by anatomy, retrograde labeling, molecular marker expression profile and electrophysiological analysis. This reporter line offers many unique advantages; a) we can for the first time visualize CSMN without any need for a retrograde labeling surgery; b) CSMN can be purified by FACS- mediated approaches at different stages in life; c) the cellular and molecular mechanisms that are responsible for CSMN vulnerability and degeneration can be studied in detail and with precision; d) most importantly this novel reporter line can be crossed to various mouse models of movement disorders to investigate the biology of CSMN with respect to disease. In this proposal, our goal is to bring visual clarity to CSMN in various mouse models of motor neuron diseases. We will be crossing UCHL1-eGFP mice with the recently identified mouse models that show potential involvement of upper motor neuron degeneration in disease pathology. Due to time limitations of R21 grant, we will characterize the timing and extent of CSMN degeneration in a limited number of mouse models, such as the Tdp43A315T and Alsin KO mice. The tools we generate and the approach we develop will help generation and characterization of other reporter mouse models, and will improve our efforts of understanding the cellular and molecular mechanisms behind CSMN vulnerability and degeneration.