Dysfunctional Astroglial Exosome to (motor) Neuron Axon Signaling in ALS

摘要 运动神经元（MN）轴突形成下行运动束和周围神经的功能障碍 在肌萎缩侧索硬化症（ALS）中广泛观察到束，其通常先于MN索马（和轴突） 退化并显著促进疾病病理学。尽管星状胶质细胞粘附信号至关重要 为了正确的轴突生长和指导，胶质细胞粘附分子是否以及如何在轴突中发挥作用， ALS中的功能障碍/变性在很大程度上仍然未知。虽然星形胶质细胞条件培养基（ACM） 来源于小鼠SOD 1突变体模型或来源于人ALS患者脑星形胶质细胞能够基本上 调节MN的健康和存活，ACM中调节ALS条件下MN存活的星形胶质细胞因子 基本上还没有被确认。外泌体（直径50-150 nm），是一种主要类型的分泌性细胞外 囊泡（EV），从多泡体（MVB，一种中间内体结构）释放， 核内体成熟已经出现了从中枢神经系统（CNS）细胞类型分泌的EV和外来体 作为一种重要的细胞间途径，与神经退行性疾病的发病机制有关 包括ALS。星形胶质细胞来源的外泌体如何影响ALS中的（运动）神经元存活，特别是轴突特性 大部分尚未开发。在这个项目中，我们打算研究新的刺激和保护作用， 星形胶质细胞外泌体，特别是外泌体粘附分子HepaCAM信号传导到神经元，促进轴突生长， 生长、功能和（运动）神经元存活。我们还将确定功能失调的HepaCAM信号传导是否 从SOD 1G 93 A星形胶质细胞外泌体到（运动）神经元有助于MN轴突功能障碍和变性 在ALS中。 根据我们的初步结果，我们提出了以下目标在这个项目中：目标1：确定 星形胶质细胞外泌体对（运动）神经元轴突生长和功能影响;目的2：研究功能丧失 ALS模型中星形胶质细胞外泌体信号传导至（运动）神经元;目的3：阐明HepaCAM介导的 ALS模型中星形胶质细胞外泌体向（运动）神经元的信号传导;我们已经产生了大量的 初步数据，以支持我们的理由，并证明拟议目标的可行性。我们会委聘 小鼠遗传学，原代神经元和星形胶质细胞培养，分子生物学，病毒注射，各种成像， 生物化学方法来完成这些目标。该项目的结果将揭示一种新的星形胶质细胞 外泌体HepaCAM介导的调节（运动）神经元轴突生长和存活的机制。它还将 提供了关于ALS中星形胶质细胞外泌体功能丧失效应的重要知识。这些研究 将大大推进我们对ALS中星形胶质细胞功能障碍的理解，并有助于开发新的星形胶质细胞- 神经保护策略。

Abstract Dysfunctions of motor neuron (MN) axons that form both descending motor tracts and peripheral nerve tracts are widely observed in amyotrophic lateral sclerosis (ALS), which often precede MN soma (and axon) degeneration and significantly contribute to disease pathology. Although astroglial adhesion signals are essential for proper axon growth and guidance, whether and how glial adhesion molecules play a role in axon dysfunction/degeneration in ALS remains largely unknown. Although astroglia conditioned medium (ACM) derived from the mouse SOD1 mutant model or from human ALS patient brain astroglia is able to substantially modulate health and survival of MNs, astroglial factors in ACM that modulate MN survival in ALS conditions remain essentially unidentified. Exosomes (50-150 nm in diameter), a major type of secreted extracellular vesicles (EVs), are released from multivesicular bodies (MVBs, an intermediate endosome structure) during endosome maturation. EVs and exosomes secreted from central nervous system (CNS) cell types have emerged as an important intercellular pathway that is implicated in the pathogenesis of neurodegenerative diseases including ALS. How astroglia-derived exosomes affect (motor) neuron survival especially axon properties in ALS remains largely unexplored. In this project, we intend to investigate novel stimulatory and protective roles of astroglial exosomes, especially exosomal adhesion molecule HepaCAM signaling to neurons, in promoting axon growth, functions, and (motor) neuron survival. We will also determine whether dysfunctional HepaCAM signaling from SOD1G93A astroglial exosomes to (motor) neurons contributes to MN axon dysfunction and degeneration in ALS. Based on our preliminary results, we propose the following aims in this project: Aim 1: Determine the effect of astroglial exosomes on (motor) neuron axon growth and functions; Aim 2: Investigate loss-of-function of astroglial exosome signaling to (motor) neurons in ALS models; Aim 3: Elucidate HepaCAM-mediated astroglial exosome signaling to (motor) neurons in ALS models; We have generated a large amount of preliminary data to support our rationales and to demonstrate feasibility for proposed aims. We will employ mouse genetics, primary neuron and astroglial cultures, molecular biology, virus injections, various imaging, and biochemical approaches to complete these aims. Outcomes from this project will reveal a new astroglial exosomal HepaCAM-mediated mechanism in modulating (motor) neuron axon growth and survival. It will also provide important knowledge about the loss-of-functional effect of astroglial exosomes in ALS. These studies will significantly advance our understanding of the astroglial dysfunction in ALS and help develop new astroglia- based neuroprotective strategies.