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，其临床和病理特征为： 运动神经元的进行性虚弱和退化。一部分患者也可以有额颞叶 痴呆伴皮层损伤虽然ALS的症状是由于神经元变性，但广泛的研究表明， 已经表明，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，以及最近的少突胶质细胞， 导致运动神经元退化我们的实验室和其他实验室已经证明， 肌萎缩侧索硬化症和功能失调的少突胶质细胞导致运动神经元退化，可能是通过失败 对神经元的代谢支持。少突胶质细胞功能障碍已发现在散发性ALS，但也有家族性 ALS与超氧化物歧化酶突变相关。重要的是，我们实验室的研究表明， 少突胶质细胞在SOD 1小鼠的神经变性中起关键作用，因为去除突变的SOD 1 特别是来自少突胶质细胞前体细胞（OPCs）和少突胶质细胞的， 这个ALS的小鼠模型。在过去的几年里，许多研究小组都集中在最近的 在C9 orf 72中发现了六核苷酸重复扩增（HRE），这是家族性 ALS也是额颞叶痴呆的常见原因。这些研究表明， 神经毒性可能是由于RNA和二肽重复（DPR）蛋白介导的事件。的确切 这些事件产生毒性的机制尚不清楚，但我们实验室发表的工作， 其他人已经证明，核质转运和核孔蛋白在神经元中被破坏 表达C9 orf 72 HRE并恢复该关键细胞功能导致神经元毒性的减弱。 迄今为止，只有一项关于C9 orf 72 HRE在少突胶质细胞中的作用的研究。在本提案中，我们 将彻底研究C9 orf 72 HRE在OPCs和少突胶质细胞中的作用，以及它们对 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 在C9 orf 72 ALS中功能失调，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。 具体来说，我们建议确定是否有少突胶质细胞变性和OPC ALS患者和具有C9 orf 72 HRE的动物模型中的增殖。我们的初步研究表明 C9 orf 72在少突胶质细胞中高度表达，少突胶质细胞在C9 orf 72患者中功能失调。然后我们将 确定OPCs是否不能分化和/或C9 orf 72 ALS中的少突胶质细胞退化， 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。 使用少突胶质细胞单一培养物和与源自C9 orf 72 iPS细胞和C9 BAC的神经元的共培养物 小鼠，沿着适当的对照，我们将评估OPC和少突胶质细胞增殖，分化， 神经元的存活、髓鞘形成和支持。我们还将评估对体内少突胶质细胞的影响 通过在少突胶质细胞或神经元中选择性表达HRE的病毒载体。更好地了解 少突胶质细胞损伤的机制，我们将确定OPCs或少突胶质细胞是否具有 ALS患者和C9 orf 72 HREs BAC转基因小鼠中的核质转运功能障碍。 最后，希望使用这些模型系统来减轻伤害，我们将确定是否功能失调， OPCs和少突胶质细胞的核质转运可以通过遗传和 药理学技术，包括反义寡核苷酸和核转运调节剂。

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.