Mechanisms of disruption of axon transport of autophagic vesicles and lysosomes in C9orf72 ALS

项目摘要 肌萎缩侧索硬化症（Amyotrophic Lateral Sclerosis，ALS）是一种以退行性变为特征的神经退行性疾病 上、下运动神经元变性。最常见的已知原因的家族性和散发性 ALS和额颞叶痴呆（FTD）的一种形式是GGGGCC六核苷酸重复扩增 (HRE)在C9 ORF 72（C9）中。我们的实验室和其他人最近发现，受损的核质 NCT是C9-ALS中需要应激颗粒形成的基本和早期致病事件。 然而，受损的NCT的下游影响尚不清楚。最近的研究表明，自噬体 生物合成发生在远端轴突中，随后自噬囊泡（AV）逆行转运到轴突。 索马，提供了一个潜在的联系轴突运输（AT）和自噬，两种机制 已知参与ALS病理生理学的早期。在表达30个GGGGCC重复序列的果蝇中 (30R)，我们已经发现p62和溶酶体的积累，表明受损的调节， 自噬和溶酶体可能是C9-ALS的致病机制。此外，我们发现初步 30 R果蝇中逆行自噬体运输减少的证据。与此相一致， 在来自C9-ALS患者的iPS运动神经元（iPSN）中的初步实验显示， 轴突中溶酶体的积累。具体目标1将进一步表征轴突运输的多种货物， 30 R果蝇和C9 iPSN，使用活细胞成像方法。具体目标2将研究 轴突运输，自噬和溶酶体功能之间的关系，并确定拯救自噬是否可以拯救 AV轴突运输缺陷。最后，初步的果蝇数据显示，Mitf/TFEB，一种转录因子， 调节自噬和溶酶体，在30 R果蝇中错误定位于细胞质，表明 受损的核质转运可导致受损的自噬和溶酶体调节。具体目标 3将解决核质转运受损是受损轴突转运上游的假设 自噬的缺陷和破坏。通过在果蝇中使用强大的平行方法， AT和自噬的遗传操作，以及来自C9-ALS患者的iPSN， 实验操纵人类细胞的疾病，这项建议将调查详细的机制 C9-ALS中轴突运输途径和自噬和溶酶体的调节。这些研究的结果 这不仅有助于我们理解ALS的发病机制和治疗策略，而且还将有助于我们理解ALS的发病机制和治疗策略。 进一步我们的理解轴突生物学的自噬，重要的是在所有神经退行性疾病。

PROJECT SUMMARY Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by dying-back degeneration of upper and lower motor neurons. The most common known cause of familial and sporadic forms of ALS as well as frontotemporal dementia (FTD) is the GGGGCC hexanucleotide repeat expansion (HRE) in C9ORF72 (C9). Our laboratory and others recently discovered that impaired nucleocytoplasmic transport (NCT) is a fundamental and early pathogenic event in C9-ALS that requires stress granule formation. However, downstream effects of impaired NCT are unclear. Recent studies have shown that autophagosome biosynthesis occurs in the distal axon followed by retrograde transport of autophagic vesicles (AVs) to the soma as they mature, providing a potential link between axon transport (AT) and autophagy, two mechanisms well known to be involved early in ALS pathophysiology. In Drosophila expressing 30 GGGGCC repeats (30R), we have found an accumulation of p62 and lysosomes, suggesting that impaired regulation of autophagy and lysosomes may be a pathogenic mechanism for C9-ALS. Further, we have found preliminary evidence of a reduction in retrograde autophagosome transport in 30R Drosophila. Consistent with this, preliminary experiments in iPS motor neurons (iPSNs) derived from patients with C9-ALS showed an accumulation of lysosomes in axons. Specific Aim 1 will further characterize axon transport of multiple cargo in 30R Drosophila and C9 iPSNs using live cell imaging methods. Specific Aim 2 will examine the interrelation between axon transport, autophagy and lysosomal function and determine if rescuing autophagy can rescue axon transport deficits of AVs. Finally, preliminary fly data shows that Mitf/TFEB, a transcription factor regulating autophagy and lysosomes, is mislocalized to the cytoplasm in 30R Drosophila, indicating that impaired nucleocytoplasmic transport may lead to impaired autophagy and lysosome regulation. Specific Aim 3 will address the hypothesis that impaired nucleocytoplasmic transport is upstream of impaired axon transport defects and disruptions in autophagy. By using powerful parallel approaches in Drosophila, allowing precise genetic manipulation of AT and autophagy, and iPSNs derived from patients with C9-ALS, allowing experimental manipulation of human cells with the disease, this proposal will investigate detailed mechanistic pathways of axon transport and regulation of autophagy and lysosomes in C9-ALS. Results from these studies will not only aid our understanding of the pathogenesis and treatment strategies of ALS, but they will also further our understanding of the axonal biology of autophagy, important in all neurodegenerative diseases.