Mechanisms of autophagic dysfunction in progranulin-related neurodegeneration

颗粒体蛋白前体相关神经变性中自噬功能障碍的机制

• 批准号: 10327305
• 负责人: Michael Fernandopulle
• 金额: $ 5.18万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10327305
• 关键词: Address; Affect; Affinity; Aging; Amyotrophic Lateral Sclerosis; Annexins; Area; Autophagocytosis; Autophagosome; Axon; Binding; Binding Proteins; Binding Sites; Biochemical; Blood; Brain Diseases; Calcium; Calcium Channel Agonists; Capsid Proteins; Cell division; Cells; Characteristics; Chimeric Proteins; Co-Immunoprecipitations; Complex; Data; Defect; Degenerative Disorder; Dementia; Dendrites; Disease; Dissection; Endoplasmic Reticulum; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Frontotemporal Dementia; Functional disorder; Gene Mutation; Generations; Genes; Genetic; Glycoproteins; Homeostasis; Impairment; Incidence; Induced pluripotent stem cell derived neurons; Infection; Label; Lead; Lysosomes; Mediating; Medical Genetics; Membrane; Microscopy; Mitotic; Molecular; Monitor; Mutation; Nerve Degeneration; Nervous system structure; Neurocognitive; Neurodegenerative Disorders; Neuromuscular Diseases; Neurons; Organelles; PGRN gene; Pathologic; Pathway interactions; Patients; Process; Protein Biosynthesis; Protein Export Pathway; Proteins; Proteomics; Quality Control; Reactive Oxygen Species; Research; Site; Skin; Starvation; Structure; Synapses; Techniques; Testing; Time; Work; age related neurodegeneration; base; cell age; cell injury; dementia risk; effective therapy; familial amyotrophic lateral sclerosis; frontotemporal lobar dementia-amyotrophic lateral sclerosis; functional loss; genetic manipulation; inhibitor; loss of function mutation; member; misfolded protein; new therapeutic target; overexpression; oxidative damage; prevent; protein misfolding; recruit; stem cells; stressor

PROJECT SUMMARY As cells age, protein quality control becomes increasingly important. Accumulated stressors such as starvation, oxidative damage, and infections lead to organelle dysfunction and protein misfolding. Stem cells, such as those present in the skin, gut, and blood, can dilute these insults through cell division. Neurons and other post-mitotic cells, however, must confront them directly. Autophagy, or lysosome-mediated degradation, is the primary mechanism for clearing large dysfunctional entities within the cell. Neuronal autophagy must be especially robust for two main reasons: 1) the high rates of protein synthesis and ATP generation in neurons entail a higher incidence of misfolded proteins and reactive oxygen species, both of which damage the cell, and 2) high spatial separation of multiple specialized regions (e.g. axons, dendrites, synapses) demands local clearance of damage in those areas to prevent key functional loss. Predictably, errors in autophagy often affect the aging nervous system. Frontotemporal dementia (FTD), a progressive neurocognitive disease, is associated with several single-gene mutations involved in autophagy and broader protein quality control. Many of these genes overlap with those implicated in amyotrophic lateral sclerosis (ALS), a common neuromuscular disease. Progranulin (PGRN), a monogenic cause of FTD and risk modifier for ALS, is a lysosomal glycoprotein that causes defects in autophagy through an unknown mechanism. Recent work by our lab has demonstrated that another ALS- associated protein, annexin A11 (ANXA11), shows decreased recruitment to the lysosome in PGRN deficient neurons. ER exit site (ERES) proteins, which regulate protein export from the endoplasmic reticulum, also show decreased lysosomal recruitment in PGRN deficiency. ANXA11 is known to bind members of ERESs, as well as to associate physically with the lysosome. Furthermore, ERESs may be involved in more than just protein export. Our collaborators recently discovered a phenomenon where lysosomes directly engulf and degrade ERESs bearing misfolded proteins—a clear example of autophagic involvement. I propose to test the hypothesis that PGRN regulates ERES autophagy through ANXA11 action by addressing the following specific aims: 1) determine how PGRN regulates ANXA11 interaction with lysosome-organelle contact sites in iPSC-derived neurons, and 2) determine how PGRN and ANXA11 jointly regulate autophagic activity. I will use a combination of sophisticated microscopy techniques, biochemical protein identification, and targeted genetic manipulation to complete these aims. Uncovering the mechanism of PGRN-related neurodegeneration could lead to a better understanding of the shared pathophysiology of FTD and ALS, providing new drug targets for these incurable and universally devastating diseases of aging.

项目概要 随着细胞老化，蛋白质质量控​​制变得越来越重要。累积的压力源，例如饥饿， 氧化损伤和感染会导致细胞器功能障碍和蛋白质错误折叠。干细胞，例如那些 存在于皮肤、肠道和血液中，可以通过细胞分裂稀释这些损伤。神经元和其他有丝分裂后 然而，细胞必须直接面对它们。自噬或溶酶体介导的降解是主要的 清除细胞内大型功能障碍实体的机制。神经元自噬必须特别强大 有两个主要原因：1）神经元中蛋白质合成和 ATP 生成的高速率需要更高的 错误折叠蛋白质和活性氧的发生率，两者都会损害细胞，2）高空间 多个专门区域（例如轴突、树突、突触）的分离需要局部清除损伤 在这些区域，以防止关键功能丧失。可以预见的是，自噬的错误常常会影响衰老的神经 系统。额颞叶痴呆 (FTD) 是一种进行性神经认知疾病，与多种疾病有关 涉及自噬和更广泛的蛋白质质量控​​制的单基因突变。其中许多基因重叠 与肌萎缩侧索硬化症（ALS）（一种常见的神经肌肉疾病）有关的患者。颗粒体蛋白前体 (PGRN) 是 FTD 的单基因原因和 ALS 的风险调节剂，是一种导致缺陷的溶酶体糖蛋白 通过未知的机制进行自噬。我们实验室最近的工作表明，另一种 ALS- 相关蛋白膜联蛋白 A11 (ANXA11) 在 PGRN 缺陷中表现出溶酶体募集减少 神经元。内质网出口位点 (ERES) 蛋白调节内质网蛋白的输出，也显示 PGRN 缺乏时溶酶体募集减少。已知 ANXA11 可以结合 ERES 的成员，以及 与溶酶体发生物理联系。此外，ERES 可能不仅仅涉及蛋白质输出。 我们的合作者最近发现了溶酶体直接吞噬并降解ERES的现象 携带错误折叠的蛋白质——自噬参与的一个明显例子。我建议检验以下假设 PGRN 通过 ANXA11 作用调节 ERES 自噬，实现以下具体目标：1) 确定 PGRN 如何调节 ANXA11 与 iPSC 衍生的溶酶体细胞器接触位点的相互作用 神经元，2) 确定 PGRN 和 ANXA11 如何共同调节自噬活性。我将使用一个组合 先进的显微镜技术、生化蛋白质鉴定和靶向基因操作 完成这些目标。揭示 PGRN 相关神经退行性变的机制可能会带来更好的结果 了解 FTD 和 ALS 共同的病理生理学，为这些无法治愈的疾病提供新的药物靶点 以及普遍毁灭性的衰老疾病。

项目成果

Development and Comparative Evaluation of Endolysosomal Proximity Labeling-Based Proteomic Methods in Human iPSC-Derived Neurons.

• DOI: 10.1021/acs.analchem.0c03107
• 发表时间: 2020-12-01
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Frankenfield AM;Fernandopulle MS;Hasan S;Ward ME;Hao L
• 通讯作者: Hao L

Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons.

• DOI: 10.1002/cpcb.51
• 发表时间: 2018-06
• 期刊: Current protocols in cell biology
• 作者: Fernandopulle MS;Prestil R;Grunseich C;Wang C;Gan L;Ward ME
• 通讯作者: Ward ME

RNA transport and local translation in neurodevelopmental and neurodegenerative disease.

• DOI: 10.1038/s41593-020-00785-2
• 发表时间: 2021-05
• 期刊: Nature neuroscience
• 影响因子: 25
• 作者: Fernandopulle MS;Lippincott-Schwartz J;Ward ME
• 通讯作者: Ward ME

SNX19 restricts endolysosome motility through contacts with the endoplasmic reticulum.

• DOI: 10.1038/s41467-021-24709-1
• 发表时间: 2021-07-27
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Saric A;Freeman SA;Williamson CD;Jarnik M;Guardia CM;Fernandopulle MS;Gershlick DC;Bonifacino JS
• 通讯作者: Bonifacino JS