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

描述（由申请人提供）：在我们的大脑皮层中有数千种不同的神经元群体，但在神经退行性疾病中，只有选定的神经元群体显示出原发性脆弱性并经历进行性变性。皮质脊髓运动神经元（Corticospinal motor neurons，CSMN）是位于运动皮质V层的大型锥体神经元。它们的细胞结构非常独特，它们作为大脑皮层的“辐条人”，用于启动和调节运动。自愿的运动是一个聪明和见多识广的头脑的行为。因此，CSMN接收来自许多神经元的信息，包括长距离投射和局部回路神经元。CSMN的独特能力，整合和翻译这些信息成一个信号对脊髓的目标设置它除了其他皮层神经元。因此，它的变性会产生严重的后果，导致各种运动障碍。有一个发展的需要，以了解疾病的CSMN变性的基础。然而，CSMN的识别和可视化并不容易，因为它们嵌入在大脑皮层内的数千个其他神经元群体中。我们最近产生了一种新的报告细胞系，UCHL 1-eGFP小鼠，其中CSMN在运动皮层中被遗传标记。在UCHL 1启动子控制下的eGFP表达是稳定的，在体内持续到P800，并且仅限于运动皮层中的CSMN。通过解剖学、逆行标记、分子标记物表达谱和电生理学分析来鉴定运动皮层中eGFP+神经元的CSMN身份。该报道细胞系提供了许多独特的优点; a）我们可以第一次可视化CSMN而不需要任何逆行标记手术; B）CSMN可以在生命的不同阶段通过FACS介导的方法纯化; c）可以详细和精确地研究导致CSMN脆弱性和变性的细胞和分子机制; d）最重要的是，这种新的报道细胞系可以与各种运动障碍的小鼠模型杂交，以研究CSMN与疾病有关的生物学。在这项提案中，我们的目标是在各种运动神经元疾病的小鼠模型中为CSMN带来视觉清晰度。我们将UCHL 1-eGFP小鼠与最近鉴定的小鼠模型杂交，这些小鼠模型显示上运动神经元变性可能参与疾病病理学。由于R21授权的时间限制，我们将在有限数量的小鼠模型中表征CSMN变性的时间和程度，例如Tdp 43 A315 T和Alsin KO小鼠。我们生成的工具和我们开发的方法将有助于其他报告小鼠模型的生成和表征，并将提高我们理解CSMN脆弱性和变性背后的细胞和分子机制的努力。

DESCRIPTION (provided by applicant): 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.