Elucidating the role of Adaptor Protein complex-4 in regulating axonal autophagic and lysosomal pathways

自噬和溶酶体途径（ALP）从细胞中清除错误折叠的蛋白质和受损的细胞器。 因此，它们的功能对于神经元等长寿细胞特别重要。ALP功能障碍是 与阿尔茨海默病（AD）的各个阶段相关。这包括大量积累 细胞外Aβ沉积物周围营养不良轴突中的自噬体和溶酶体样细胞器（淀粉样蛋白 斑块），这是在人类阿尔茨海默病脑组织中观察到的标志性病理学特征， 在阿尔茨海默病的转基因小鼠模型中重现。淀粉样斑块的形成直接影响到 与分泌酶对淀粉样前体蛋白（APP）的异常/淀粉样蛋白原性蛋白水解加工有关。 然而，是否这些细胞器的异常运输和积累携带这些蛋白质货物 引发阿尔茨海默氏病的这一关键致病事件，尚未得到实验解决。此外，本发明还提供了一种方法， 关于轴突中调节ALP的机制和特定分子组分的信息仍然有限。 阐明这些机制并鉴定分子组分可能使治疗性调节 神经元ALP减少阿尔茨海默病中淀粉样斑块负荷和毒性Aβ肽产生。本 最后，我们提出的研究旨在了解a）适配器复合物AP-4如何调节轴突 自噬体和溶酶体的生物发生、成熟和转运; B）AP-4的缺失如何有助于淀粉样蛋白 体内斑块形成以及潜在地鉴定促进最佳逆行轴突生长的新AP-4货物 溶酶体转运、APOE代谢和突触活性。这些拟议研究的核心是我们的 AP-4缺失导致ALP细胞器在轴突鞘中异常积累的初步数据提示， 包括APP裂解蛋白BACE 1和PSEN 2的积累。这个和我们的初步数据 表明AD小鼠脑中AP-4水平降低，并且前额叶中AP-4的年龄依赖性损失 甚至野生型小鼠的皮质使我们假设AP-4依赖的轴突自噬体和溶酶体 成熟和运输保护神经元免于淀粉样蛋白生成APP加工，从而免于淀粉样斑块 发展此外，我们还使用超分辨率显微镜观察了轴索中的ALP细胞器， 作为分离的轴突和细胞器上的蛋白质组学在AP-4复合物丢失后， ALP中间体在这些病理条件下的积累及其对构建的相对贡献- APP加工机械。我们提出的努力，解剖出AP-4介导的轴突ALP运输， 成熟也可能带来新的治疗机会，重点是动员这些轴突ALP细胞器 以限制Aβ的产生和轴突病理。此外，神经元自噬的细胞生物学的新见解 我们的研究揭示的溶酶体途径可能与其他神经退行性疾病有更广泛的相关性。 具有溶酶体成分的疾病，如帕金森病、遗传性痉挛性 截瘫和额颞叶痴呆

The autophagic and lysosomal pathways (ALP) clear misfolded proteins and damaged organelles from cells. Their function is therefore particularly critical for long-lived cells such as neurons. Dysfunction in the ALP is associated with various stages of Alzheimer’s disease (AD). This includes the robust accumulation of autophagosomes and lysosome-like organelles in dystrophic axons around extracellular Aβ deposits (Amyloid plaques), which are hallmark pathological features observed in human Alzheimer’s disease brain tissue and recapitulated in transgenic mouse models of Alzheimer’s disease. Amyloid plaque formation has been directly linked to aberrant/amyloidogenic proteolytic processing of amyloid precursor protein (APP) by secretases. However, whether abnormal trafficking and accumulation of these organelles bearing these protein cargoes trigger this critical pathogenic event in Alzheimer’s disease, has not been experimentally addressed. In addition, information on mechanisms and specific molecular components regulating ALP in axons remains limited. Elucidating these mechanisms and identifying molecular components might enable therapeutic modulation of neuronal ALP to reduce amyloid plaque burden and toxic Aβ peptide production in Alzheimer’s disease. To this end, our proposed research seeks to understand a) how the adaptor complex, AP-4, regulates axonal autophagosome and lysosome biogenesis, maturation, and transport; b) how loss of AP-4 contributes to amyloid plaque formation in vivo as well as potentially identify new AP-4 cargo that facilitate optimal retrograde axonal lysosome transport, APOE metabolism, and synaptic activity. Central to these proposed studies is our preliminary data that AP-4 loss causes abnormal accumulation of ALP organelles in axonal swellings reminiscent of AD pathology, including build-up of APP cleaving proteins BACE1 and PSEN2. This and our preliminary data demonstrating reduced AP-4 levels in AD mouse brain and an age-dependent loss of AP-4 in the pre-frontal cortex of even wild type mice lead us to hypothesize that AP-4-dependent axonal autophagosome and lysosome maturation and transport protects neurons from amyloidogenic APP processing and thus, from amyloid plaque development. Additionally, our use of super-resolution microscopy on ALP organelles in axonal swellings as well as proteomics on isolated axons and organelles upon loss of AP-4 complex, will yield novel insight into the kinds of ALP intermediates accumulating under these pathological conditions and their relative contributions to build- up of APP processing machinery. Our proposed efforts to dissect out AP-4 mediated axonal ALP transport and maturation could also lead to new therapeutic opportunities that focus on mobilizing these axonal ALP organelles to limit Aβ production and axonal pathology. Furthermore, new insights into cell biology of neuronal autophagic and lysosomal pathways revealed by our studies could have broader relevance to other neurodegenerative diseases that have a lysosome component to their pathology such as Parkinson’s disease, Hereditary Spastic Paraplegia, and fronto-temporal dementia.