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

项目总结 肌萎缩侧索硬化症(ALS)是一种以消亡为特征的破坏性神经退行性疾病 上、下运动神经元变性。家族性和散发性最常见的已知原因 形式的肌萎缩侧索硬化症以及额颞性痴呆(FTD)是GGGGGCC六核苷酸重复序列的扩张 (HRE)在C9ORF72(C9)中。我们实验室和其他人最近发现，受损的核细胞质 转运(NCT)是C9-ALS的基础和早期致病事件，需要应激颗粒的形成。 然而，受损的NCT的下游影响尚不清楚。最近的研究表明，自噬小体 生物合成发生在远端轴突，随后自噬小泡(AVs)逆行运输到 胞体成熟时，在轴突运输(AT)和自噬之间提供了潜在的联系，这两种机制 众所周知，它早期参与了ALS的病理生理学。在果蝇中表达30个GGGGCC重复 (30R)，我们发现了p62和溶酶体的积累，这表明调节受损 自噬和溶酶体可能是C9-ALS的致病机制之一。此外，我们还发现了初步的 30R果蝇自噬小体逆行转运减少的证据。与此一致的是， 对C9-ALS患者来源的iPS运动神经元(IPSN)的初步实验表明， 轴突中溶酶体的堆积。具体目标1将进一步描述多个货物的轴突运输 30R果蝇和C9 iPSNs使用活细胞成像方法。《特定目标2》将考察两者之间的相互关系 轴突运输、自噬和溶酶体功能之间的关系，并确定自噬是否可以挽救 动静脉畸形的轴突运输缺陷。最后，Fly的初步数据显示，转录因子MITF/TFEB 在30R果蝇中，调节自噬和溶酶体的基因错误地定位于细胞质，表明 核质转运受损可能导致自噬和溶酶体调节受损。特定目标 3将解决核质转运受损位于轴突转运受损上游的假设。 自噬中的缺陷和干扰。通过在果蝇中使用强大的并行方法，允许精确地 来自C9-ALS患者的AT和自噬以及iPSNs的基因操作，允许 对人类细胞的实验操作与疾病，这一建议将调查详细的机制 C9-ALS轴突运输途径及自噬和溶酶体的调节。这些研究的结果 不仅有助于我们了解肌萎缩侧索硬化症的发病机制和治疗策略，而且 进一步加深我们对自噬的轴突生物学的理解，自噬在所有神经退行性疾病中都很重要。

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.