Mechanisms of autophagic dysfunction in progranulin-related neurodegeneration

项目总结 随着细胞的老化，蛋白质质量控制变得越来越重要。累积的应激源，如饥饿， 氧化损伤和感染导致细胞器功能障碍和蛋白质错误折叠。干细胞，比如那些 存在于皮肤、肠道和血液中，可以通过细胞分裂来稀释这些侮辱。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失调实体的机制。神经元自噬必须特别强大 主要有两个原因：1)神经元中蛋白质合成和三磷酸腺苷生成的高速率需要更高的 错误折叠的蛋白质和活性氧物种的发生率，这两者都会损害细胞，以及2)高空间 分离多个特化区域(例如轴突、树突、突触)需要局部清除损伤 在这些领域防止关键功能丧失。可以预见的是，自噬的错误通常会影响老化的神经症患者。 系统。额颞叶痴呆(FTD)是一种进行性神经认知疾病，与几种 单基因突变涉及自噬和更广泛的蛋白质质量控制。这些基因中有许多是重叠的 与肌萎缩侧索硬化症(ALS)有关的患者，这是一种常见的神经肌肉疾病。普罗米林 (PGRN)是FTD的单基因致病因子，也是ALS的风险修饰物，是一种导致缺陷的溶酶体糖蛋白 通过一种未知的机制进行自噬。我们实验室最近的研究表明，另一种ALS- 相关蛋白Annexin A11(ANXA11)在PGRN缺乏的患者中对溶酶体的募集减少 神经元。调节内质网蛋白质输出的内质网退出位点(ERES)蛋白也显示 PGRN缺乏症患者溶酶体募集减少。已知ANXA11结合ERES成员以及 与溶酶体发生物理联系。此外，ERES可能参与的不仅仅是蛋白质出口。 我们的合作者最近发现了一种现象，即溶酶体直接吞噬并降解ERES 携带错误折叠的蛋白质--这是自噬参与的一个明显例子。我建议检验一下这个假设 PGRN通过ANXA11作用调节ERES自噬，具体目的如下：1) 确定PGRN如何调节ANXA11与IPSC来源的溶酶体-细胞器接触部位的相互作用 以及2)决定PGRN和ANXA11如何共同调节自噬活动。我将使用组合 先进的显微技术、生化蛋白质鉴定和有针对性的基因操作 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 了解FTD和ALS的共同病理生理机制，为这些无法治愈的疾病提供新的药物靶点 以及普遍存在的破坏性衰老疾病。

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.