Retromer dysfunction in amyotrophic lateral sclerosis

项目摘要 这个博士前奖的建议提交，以支持解开机制的长期目标，通过 哪些特定的神经元亚群会在神经退行性疾病中死亡 (ALS)是一种无法治愈的成人发病的致命麻痹性疾病。这将通过研究 非神经元细胞在ALS中运动神经元（MN）死亡中的作用，这一点现在已得到广泛认可。有关这个 Przedborski实验室发现，从突变型超氧化物歧化酶1（mSOD 1）制备的星形胶质细胞 ALS的转基因小鼠模型通过毒性事件杀死培养的MN，其性质仍有待确定。 随后的研究支持ALS星形胶质细胞毒性可能是由于异常的 淀粉样前体蛋白（APP）的运输由于逆转录缺陷。虽然功能障碍的 retromer，导致APP的异常加工，已经在几种神经退行性疾病中提出 然而，星形胶质细胞中的逆转录功能障碍是否以及如何可能导致ALS中的MN变性， 不知道。因此，在这个项目中，试图阐明逆转录酶在ALS中的作用，我们提出了三个 目标。在目的1中，为了深入了解逆转录酶缺乏导致非细胞凋亡的机制， 自主性MN死亡，我们将在体外星形胶质细胞中表征逆转录病毒运输功能障碍的影响 通过使用针对Vps 35的RNA干扰-逆转录酶的核心成分-并检查i） 通过免疫印迹的APP片段水平，ii）通过共聚焦和实时成像的APP运输，以及iii）MN 通过免疫细胞化学检测毒性。在目标2中，我们将评估行为和神经病理 小鼠脑中的改变，特别注意脊髓MN通路，在星形胶质细胞特异性 retromer缺陷。对于该实验，Vps 35 Fl/Fl突变小鼠将与转基因Aldhlll-Cre小鼠杂交， 提供Vps 35的星形胶质细胞特异性缺失。最后，在目标3中，我们将使用小分子R33， 稳定逆转录聚合物复合物，以确定这种临床前治疗策略是否可以改变逆转录聚合物复合物。 在转基因mSOD 1小鼠中观察到ALS样表型。这个项目的重点是作用的retromer在 星形胶质细胞，这是以前没有做过的，使用一种独特的小鼠系和小分子，并研究ALS。 如果该项目成功，我们的结果可能对我们的理解产生深远的影响， 和治疗这种可怕的神经退行性疾病。

PROJECT SUMMARY This Predoctoral award proposal is submitted to support the long-term goal of unraveling the mechanisms by which specific subsets of neurons die in neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), an incurable adult-onset fatal paralytic disorder. This will be achieved by studying the involvement of non-neuronal cells in the death of motor neurons (MNs) in ALS which is now well-recognized. Relevant to this goal, the Przedborski lab has found that astrocytes prepared from a mutant superoxide dismutase 1 (mSOD1) transgenic mouse model of ALS kill cultured MNs through a toxic event whose nature remains to be defined. Subsequent investigations support the notion that ALS astrocyte toxicity might result from the aberrant trafficking of the amyloid precursor protein (APP) due to a retromer defect. Although dysfunction of the retromer, leading to an aberrant processing of APP, has been proposed in several neurodegenerative disorders, whether and how retromer dysfunction in astrocytes may contribute to MN degeneration in ALS is not known. Therefore, in this project, which seeks to elucidate the role of retromer in ALS, we propose three Aims. In Aim 1, to gain insights into the mechanism by which retromer deficiency results in non-cell autonomous MN death, we will characterize the effect of retromer trafficking dysfunction in astrocytes in vitro by using RNA interference against Vps35—a core component of the retromer—and examining changes in i) APP fragment levels through immunoblotting, ii) APP trafficking through confocal and live imaging, and iii) MN toxicity through immunocytochemistry. In Aim 2, we will assess the behavioral and neuropathological alterations in the mouse brain, with specific attention to the spinal MN pathway, following an astrocyte-specific retromer defect. For this experiment, Vps35Fl/Fl mutant mice will be crossed with transgenic Aldh1l1-Cre mice to provide an astrocyte-specific deletion of Vps35. Finally, in Aim 3, we will use the small molecule R33, which stabilizes the retromer complex, to determine whether such a preclinical therapeutic strategy can modify the ALS-like phenotype seen in transgenic mSOD1 mice. This project focuses on the role of the retromer in astrocytes, which has not been done before, uses a unique mouse line and small molecule, and studies ALS. Should the project be successful, our results may have far-reaching implications for both our understanding and treatment of this dreadful neurodegenerative disorder.