Mechanisms of autophagic dysfunction in progranulin-related neurodegeneration

项目摘要 随着细胞老化，蛋白质质量控制变得越来越重要。不断积累的压力，比如饥饿， 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，例如那些 存在于皮肤、肠道和血液中的蛋白质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 错误折叠的蛋白质和活性氧的发生率，这两者都损害细胞，和2）高空间 多个专门区域（如轴突、树突、突触）的分离需要局部清除损伤 以防止关键功能丧失。可以预见的是，自噬的错误通常会影响衰老的神经系统， 系统额颞叶痴呆（FTD）是一种进行性神经认知疾病， 涉及自噬和更广泛的蛋白质质量控制的单基因突变。这些基因中有许多是重叠的 与肌萎缩侧索硬化症（ALS）有关，ALS是一种常见的神经肌肉疾病。颗粒蛋白 （PGRN）是FTD的单基因病因和ALS的风险修饰因子，是一种溶酶体糖蛋白， 通过一种未知的机制进行自噬我们实验室最近的工作表明，另一种ALS- 相关蛋白，膜联蛋白A11（ANXA 11），显示减少募集到溶酶体中PGRN缺乏 神经元内质网出口位点（ERES）蛋白，调节蛋白质从内质网的输出，也显示了 PGRN缺乏症中溶酶体募集减少。已知ANXA 11结合ERES成员，以及 与溶酶体结合此外，ERES可能不仅仅参与蛋白质输出。 我们的合作者最近发现了溶酶体直接吞噬和降解ERES的现象 带有错误折叠的蛋白质--一个明显的自噬作用的例子。我想验证这个假设， PGRN通过ANXA 11作用调节ERES自噬，具体目标如下：1） 确定PGRN如何调节ANXA 11与iPSC衍生的细胞中溶酶体-细胞器接触位点的相互作用。 神经元，和2）确定PGRN和ANXA 11如何共同调节自噬活性。我会用一个组合 复杂的显微镜技术，生化蛋白质鉴定，和有针对性的遗传操作， 完成这些目标。揭示PGRN相关神经退行性变的机制可能会导致更好的 了解FTD和ALS的共同病理生理学，为这些无法治愈的疾病提供新的药物靶点。 和普遍破坏性的衰老疾病。

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.