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

基本信息

批准号：
10647723
负责人：
Sarah Hatch Berth
金额：
--
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2023-08-07
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10647723
关键词：
Affect Amyotrophic Lateral Sclerosis Anabolism Autophagocytosis Autophagosome Axon Axonal Transport Back Binding Biochemical Biogenesis Biology C9ALS 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 Induced pluripotent stem cell derived neurons Inherited Label Laboratories Link Lysosomes Membrane Methods Microtubules Modeling Motor Motor Neurons Mutation Nerve Degeneration Neurodegenerative Disorders Nuclear Pore Nuclear Pore Complex Organelles Pathogenesis Pathogenicity Pathway interactions Patients 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 pharmacologic protein aggregation proteostasis retrograde transport stress granule therapeutic evaluation therapeutic target tool transcription factor treatment strategy

项目摘要

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.

项目概要肌萎缩侧索硬化症 (ALS) 是一种毁灭性的神经退行性疾病，其特征是死亡上、下运动神经元变性。家族性和散发性最常见的已知原因 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 C9ORF72 (C9) 中。我们的实验室和其他人最近发现核细胞质受损转运 (NCT) 是 C9-ALS 中的一个基本的早期致病事件，需要应激颗粒的形成。然而，NCT 受损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，然后将自噬囊泡 (AV) 逆行转运至体细胞成熟时，提供轴突运输（AT）和自噬这两种机制之间的潜在联系众所周知，其早期参与了 ALS 病理生理学。在果蝇中表达 30 个 GGGGCC 重复序列 (30R)，我们发现 p62 和溶酶体的积累，表明 p62 和溶酶体的调节受损自噬和溶酶体可能是 C9-ALS 的致病机制。此外，我们初步发现 30R 果蝇逆行自噬体转运减少的证据。与此相一致的是，对源自 C9-ALS 患者的 iPS 运动神经元 (iPSN) 进行的初步实验表明，溶酶体在轴突中积累。具体目标 1 将进一步表征多种货物的轴突运输使用活细胞成像方法的 30R 果蝇和 C9 iPSN。具体目标 2 将检查相互关系轴突运输、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 的轴突运输缺陷。最后，初步的飞行数据显示 Mitf/TFEB（一种转录因子）调节自噬和溶酶体，在 30R 果蝇中错误定位到细胞质，表明核质运输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：受损的核细胞质运输是受损的轴突运输的上游自噬的缺陷和破坏。通过在果蝇中使用强大的并行方法，可以实现精确的对 AT 和自噬以及源自 C9-ALS 患者的 iPSN 进行基因操作，允许对患有这种疾病的人类细胞进行实验操作，该提案将研究详细的机制 C9-ALS 中轴突运输以及自噬和溶酶体调节的途径。这些研究的结果不仅有助于我们了解 ALS 的发病机制和治疗策略，而且还将帮助我们了解 ALS 的发病机制和治疗策略。进一步加深我们对自噬轴突生物学的理解，自噬在所有神经退行性疾病中都很重要。