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

自噬和溶酶体途径(ALP)清除细胞中错误折叠的蛋白质和受损的细胞器。 因此，它们的功能对神经元等长寿细胞尤为关键。碱性磷酸酶功能障碍是 与阿尔茨海默病(AD)的不同阶段有关。这包括强劲的积累 细胞外Aβ沉积(淀粉样蛋白)周围营养不良轴突中的自噬小体和溶酶体样细胞器 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特征 在阿尔茨海默病转基因小鼠模型上进行了总结。淀粉样斑块的形成已经直接 与分泌酶对淀粉样前体蛋白(APP)的异常/淀粉样蛋白分解过程有关。 然而，这些细胞器携带这些蛋白质货物的异常贩运和积累是否 在阿尔茨海默病中触发这一关键的致病事件，尚未得到实验解决。此外, 关于调节轴突内碱性磷酸酶的机制和特定分子成分的信息仍然有限。 阐明这些机制并确定分子组成可能使治疗调节成为可能 神经元碱性磷酸酶可减少阿尔茨海默病患者的淀粉样斑块负荷和有毒Aβ多肽的产生。对这件事 最后，我们提出的研究试图了解a)适配器复合体AP-4如何调节轴突 自噬小体和溶酶体的生物发生、成熟和运输；b)AP-4缺失对淀粉样蛋白的作用 在体内形成斑块以及潜在地鉴定促进最佳逆行轴突的新的AP-4载体 溶酶体运输、载脂蛋白E代谢和突触活性。这些拟议研究的核心是我们的 AP-4缺失导致轴突肿胀中碱性磷酸酶细胞器异常堆积的初步数据让人想起 AD病理，包括APP裂解蛋白BACE1和PSEN2的积聚。这和我们的初步数据 显示AD小鼠脑内AP-4水平降低和前额叶AP-4的年龄相关性丢失 即使是野生型小鼠的大脑皮层，我们也推测AP-4依赖的轴突自噬小体和溶酶体 成熟和转运保护神经元免受淀粉样变性APP的处理，从而保护神经元免受淀粉样斑块的影响 发展。此外，我们还对轴突肿胀中的碱性磷酸酶细胞器进行了超分辨显微镜观察。 由于在分离的轴突和细胞器上丢失AP-4复合体的蛋白质组学，将对这种类型产生新的见解 在这些病理条件下积累的碱性磷酸酶中间体及其对构建- 应用程序处理机器的运行。我们提出的研究AP-4介导的轴突ALP运输和 成熟还可能带来新的治疗机会，专注于动员这些轴突碱性磷酸酶细胞器。 以限制Aβ的产生和轴突病理。此外，对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变有更广泛的相关性 病理中含有溶酶体成分的疾病，如帕金森氏症、遗传性痉挛 截瘫和额颞部痴呆。

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.