Oligodendroglial Dysfunction in C9orf72 ALS and FTD

肌萎缩侧索硬化症(ALS)是一种进行性、致命性疾病，其临床和病理特征是 运动神经元进行性衰弱和变性。一部分患者也可以有额颞部 痴呆症合并皮质损伤。虽然ALS的症状是由于神经元退化造成的，但广泛的研究 研究表明，中枢神经系统中的支持细胞，包括小胶质细胞、星形胶质细胞和最近的少突胶质细胞， 会导致运动神经元退化。我们的实验室和其他实验室已经表明，少突胶质细胞在 ALS和功能障碍的少突胶质细胞可能通过功能衰竭导致运动神经元变性 对神经元的新陈代谢支持。少突胶质细胞功能障碍在散发性ALS中被发现，但也在家族性ALS中被发现 与超氧化物歧化酶突变相关的ALS。重要的是，我们实验室的研究表明 少突胶质细胞在SOD1小鼠神经退行性变中起关键作用，因为移除了突变的SOD1 特别是来自少突胶质前体细胞(OPC)和少突胶质细胞的细胞显著延长了小鼠的寿命 肌萎缩侧索硬化症的小鼠模型。在过去的几年里，许多研究小组都在关注最近的 在C9orf72中发现了六核苷酸重复扩张(HRE)，这是家族性心脏病最常见的原因 肌萎缩侧索硬化症也是额颞叶痴呆的常见原因。这些研究已经确定， 神经毒性可能是由于RNA和二肽重复序列(DPR)蛋白介导的事件。完全相同的 这些事件产生毒性的机制尚不清楚，但我们实验室和 另一些研究表明，神经元的核质转运和核孔蛋白被破坏。 C9orf72HREs的表达和这一关键细胞功能的恢复会导致神经元毒性的减弱。 到目前为止，只有一项关于C9orf72HRE在少突胶质细胞中的作用的研究。在这项提案中，我们 将深入研究C9orf72HREs在OPC和少突胶质细胞中的作用及其对 细胞和小鼠模型中的细胞功能障碍和变性。我们假设少突胶质细胞 是C9orf72 ALS的功能障碍，核质转运的改变导致 少突胶质细胞损伤和OPC分化能力降低。 具体地说，我们建议确定是否存在少突胶质细胞变性和OPC ALS患者的增殖，以及C9orf72 HRE的动物模型。我们的初步研究表明 C9ORF72在少突胶质细胞中高表达，而少突胶质细胞在C9orf72患者中功能障碍。到时候我们会的 确定C9orf72 ALS中OPC是否未能分化和/或少突胶质细胞变性 重复扩增对少突胶质细胞的直接作用或神经元毒性的间接作用。 利用少突胶质细胞单一培养及与C9orf72 iPS细胞和C9BAC来源的神经元联合培养 小鼠，连同适当的对照组，我们将评估OPC和少突胶质细胞的增殖、分化， 神经元的存活、髓鞘形成和支持。我们还将评估其对体内少突胶质细胞的影响 通过病毒载体选择性地在少突胶质细胞或神经元中表达HREs。为了更好地理解 少突胶质细胞损伤的机制，我们将确定OPC或少突胶质细胞是否有 ALS患者和C9orf72HREs BAC转基因小鼠核质转运功能障碍。 最后，希望使用这些模型系统来减轻损伤，我们将确定功能障碍 OPC和少突胶质细胞的核质转运可通过遗传和 药理学技术，包括反义寡核苷酸和核运输调节剂。

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease characterized clinically and pathologically by progressive weakness and degeneration of motor neurons. A subset of patients can also have frontotemporal dementia with cortex injury. Though the symptoms of ALS are due to neuron degeneration, extensive research has shown that support cells in the CNS, including microglia, astrocytes, and recently oligodendrocytes, contribute to motor neuron degeneration. Our lab and others have shown that oligodendrocytes degenerate in ALS and that dysfunctional oligodendrocytes contribute to motor neuron degeneration, perhaps through failure of metabolic support to neurons. Oligodendrocyte dysfunction has been found in sporadic ALS, but also familial ALS associated with mutations in superoxide dismutase. Importantly, research from our laboratory has shown that oligodendrocytes play a critical role in neurodegeneration in SOD1 mice, since removing mutant SOD1 specifically from oligodendrocyte precursor cells (OPCs) and oligodendrocytes significantly prolongs lifespan in this mouse model of ALS. In the last several years, many research groups have focused on the recently discovered hexanucleotide repeat expansions (HREs) in C9orf72, which is the most common cause of familial ALS and also a common cause of frontotemporal dementia. These studies have determined that the neurotoxicity is likely due to both RNA- and dipeptide repeats (DPR) protein-mediated events. The exact mechanism by which these events produce toxicity is unknown, but published work by our laboratory and others has demonstrated that nucleocytoplasmic transport and nuclear pore proteins are disrupted in neurons expressing C9orf72HREs and restoration of this critical cell function leads to attenuation of neuronal toxicity. To date, there has been only one study on the role of C9orf72HREs in oligodendrocytes. In this proposal, we will thoroughly investigate the role of C9orf72HREs in OPCs and oligodendrocytes and their contribution to cellular dysfunction and degeneration in cellular and mouse models. We hypothesize that oligodendrocytes are dysfunctional in C9orf72 ALS and that alterations of nucleocytoplasmic transport lead to oligodendrocyte injury and reduced capacity for OPC differentiation. Specifically we propose to determine whether there is oligodendrocyte degeneration and OPC proliferation in ALS patients, and animal models with C9orf72 HREs. Our preliminary studies suggest C9orf72 is highly expressed in oligodendrocytes, which are dysfunctional in C9orf72 patients. We will then determine whether OPCs fail to differentiate and/or oligodendrocytes degenerate in C9orf72 ALS due to direct effect of repeat expansion on oligodendrocytes or an indirect effect from neuronal toxicity. Using oligodendrocyte monocultures and co-cultures with neurons derived from C9orf72 iPS cells and C9BAC mice, along with appropriate controls, we will evaluate OPC and oligodendrocyte proliferation, differentiation, survival, myelination, and support of neurons. We will also evaluate the impact on oligodendrocytes in vivo through viral vectors expressing HREs selectively in oligodendrocytes or neurons. To better understand the mechanism of oligodendroglial injury, we will determine whether OPCs or oligodendrocytes have dysfunctional nucleocytoplasmic transport in ALS patients and C9orf72HREs BAC transgenic mice. Finally, in hopes of using these model systems to mitigate injury, we will determine whether dysfunctional nucleocytoplasmic transport in OPCs and oligodendrocytes can be attenuated through genetic and pharmacologic techniques, including antisense oligonucleotides and nuclear transport modulators.