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

C9orf72 ALS 中自噬囊泡和溶酶体轴突运输破坏的机制

• 批准号: 10040769
• 负责人: Sarah Hatch Berth
• 金额: $ 19.93万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10040769
• 关键词: ALS patients; Address; Affect; Amyotrophic Lateral Sclerosis; Anabolism; Autophagocytosis; Autophagosome; Axon; Axonal Transport; Back; Biochemical; Biogenesis; Biology; C9ORF72; Cells; Cytoplasm; Data; Defect; Development; Disease; Disease model; Distal; Drosophila genus; Event; Foundations; Fractionation; Frontotemporal Dementia; Functional disorder; Genes; Genetic; Goals; Human; Image; Impairment; In Vitro; Inherited; Label; Laboratories; Lead; Link; Lysosomes; Membrane; Methods; Microtubules; Modeling; Motor; Motor Neurons; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear Pore; Nuclear Pore Complex; Organelles; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pharmacology; Phenotype; Phosphotransferases; Post-Translational Protein Processing; Process; Proteins; RNA; Regulation; Testing; Time; Training; Tubulin; Vesicle; Work; career; career development; experimental study; fly; genetic manipulation; imaging modality; improved; in vivo; induced pluripotent stem cell; insight; live cell imaging; motor neuron degeneration; neuronal cell body; nucleocytoplasmic transport; overexpression; protein aggregation; proteostasis; retrograde transport; stress granule; therapeutic evaluation; therapeutic target; tool; transcription factor; treatment strategy

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.

项目摘要 肌萎缩性侧索硬化症（ALS）是一种毁灭性的神经退行性疾病，特征在于死亡 上和下运动神经元的变性。家族和零星的最常见原因 ALS以及额颞痴呆（FTD）的形式是GGGGCC六核苷酸重复膨胀 （HRE）在C9orf72（C9）中。我们的实验室和其他人最近发现核细胞质受损 运输（NCT）是C9-ALS中的一个基本和早期致病事件，需要压力颗粒形成。 但是，NCT受损的下游效应尚不清楚。最近的研究表明自噬体 生物合成发生在远端轴突中，然后自噬囊泡（AVS）逆行转运至 躯体成熟时，提供了轴突传输（AT）和自噬之间的潜在联系，这是两个机制 众所周知，早期参与了ALS病理生理学。在果蝇中表达30 GGGGCC重复 （30R），我们发现P62和溶酶体的积累，这表明调节的调节受损 自噬和溶酶体可能是C9-ALS的致病机制。此外，我们发现了初步 30R果蝇中逆行自噬体转运减少的证据。与此一致 来自C9-ALS患者的IPS运动神经元（IPSN）的初步实验显示 轴突中溶酶体的积累。特定的目标1将进一步表征多个货物的轴突运输 30R果蝇和C9 IPSN使用活细胞成像方法。特定目标2将检查相互关系 在轴突运输，自噬和溶酶体功能之间，并确定救援自噬是否可以营救 AVS的轴突运输缺陷。最后，初步飞行数据表明MITF/TFEB是转录因子 调节自噬和溶酶体，在30R果蝇中被错误定位于细胞质，表明 核细胞质转运受损可能导致自噬和溶酶体调节受损。具体目标 3将解决以下假设，即受损的核细胞质转运是轴突转运受损的上游 自噬的缺陷和中断。通过在果蝇中使用强大的平行方法，允许精确 AT和自噬的基因操纵，以及来自C9-ALS患者的IPSN，允许 该疾病对人类细胞进行实验性操纵，该提案将研究详细的机理 C9-ALS自噬和溶酶体的轴突运输和调节的途径。这些研究的结果 不仅将帮助我们理解ALS的发病机理和治疗策略，还将 进一步，我们对自噬的轴突生物学的理解在所有神经退行性疾病中都很重要。