Dysfunction of the Autophagy-Lysosomal Pathway as a Common Mechanism of Neurodegeneration

资助项目摘要 错误折叠的蛋白质聚集体在CNS中的积累是许多神经退行性疾病的共同特征。 疾病，包括阿尔茨海默病（AD）、肌萎缩侧索硬化（ALS）、额颞叶痴呆（frontotemporal dementia (FTD)、亨廷顿病（HD）和帕金森病（PD）。神经变性的统一机制 在这些疾病中，通过自噬-溶酶体途径（ALP）的蛋白质清除受损， 毒性蛋白质的积累导致神经元功能障碍并最终导致神经元死亡。我们假设 这里要测试的是，虽然ALP可能在每种疾病中受损，但细胞功能障碍的细微差异， ALP有助于每种疾病的神经病理学和临床症状的独特变化。 我们将使用机器人显微镜（RM）研究不同神经退行性疾病的ALP， 延时成像技术，这是一个公正的，敏感的和定量的方法，使我们能够识别 干扰ALP，并确定这些干扰如何与神经变性。我们使用RM来 在ALS、FTD、HD和PD的人类模型中监测单个神经元的变性，其中来自 患有这些疾病的家族性或散发性形式的患者分化为神经元（I-神经元）。的情况下 在ALS、HD和PD中，神经元在每种疾病中最脆弱（运动神经元- ALS;纹状体神经元- HD; 多巴胺能神经元- PD）比来自对照志愿者的类似神经元具有更大的死亡风险。因为 人模型在生理水平表达内源水平的突变致病蛋白 并显示自发性疾病表型，我们可以使用RM来研究细胞机制， 与每种疾病中神经变性最直接相关ALP和蛋白质清除受损。 我们在RM中集成了一种称为光学脉冲标记（OPL）的新技术， 蛋白质与纵向单细胞分析，使我们能够直接测量的流量致病 蛋白质和清除途径在单个神经元中的高通量。我们做了OPL检测来监测 亨廷顿蛋白、TDP 43、α-突触核蛋白、LRRK 2和tau的代谢，以及蛋白酶体和自噬的代谢。 清除途径，包括线粒体自噬。我们还使RM适应高吞吐量视频和Z- 投影成像，使我们能够捕捉细胞内形态和运输的动态变化， 细胞器如线粒体和溶酶体。将这些ALP测定与RM结合，使我们能够预测 ALP的紊乱如何影响细胞的命运，预先确定哪些细胞群更有可能 并更好地了解抵抗退化的神经元群体 补偿受损的ALP以存活。 我们的最终目标是将RM/OPL、人类神经元疾病模型与我们的专利化合物沿着 增加了错误折叠蛋白质和常见分子靶点的周转， 全基因组测序，以确定新的治疗策略来治疗这些神经退行性疾病。

ABSTRACT OF FUNDED PROJECT Accumulation of misfolded protein aggregates in the CNS is a common feature of many neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD) and Parkinson's disease (PD). A unifying mechanism of neurodegeneration in these diseases is impaired protein clearance via the autophagy-lysosome pathway (ALP) which results in the buildup of toxic proteins that cause neuronal dysfunction and ultimately neuronal death. Our hypothesis to be tested here is that while ALP may be impaired in each disease, subtle differences in the dysfunction of the ALP contribute to unique variations in neuropathology and clinical symptoms of each disease. We will investigate ALP across different neurodegenerative diseases using robotic microscopy (RM), a time- lapse imaging technology, which is an unbiased, sensitive and quantitative method that enables us to identify perturbations in ALP and determine how those perturbations relate to neurodegeneration. We used RM to monitor degeneration of single neurons in human models of ALS, FTD, HD and PD in which iPSCs from patients with familial or sporadic forms of these diseases are differentiated to neurons (i-neurons). In the case of ALS, HD and PD, neurons most vulnerable in each disease (motor neurons- ALS; striatal neurons- HD; dopaminergic neurons- PD) have a greater risk of dying than similar neurons from control volunteers. Because the human models express endogenous levels of the mutant disease-causing proteins at physiological levels and display spontaneous disease phenotypes, we can use RM to investigate the cellular mechanisms that impair ALP and protein clearance that are most directly relevant to neurodegeneration in each disease. We integrated a new technology into RM called optical pulse-labeling (OPL) that combines photoswitchable proteins with longitudinal single-cell analysis, enabling us to directly measure the flux of disease-causing proteins and clearance pathways in single neurons in high-throughput. We made OPL assays to monitor the metabolism of huntingtin, TDP43, α-synuclein, LRRK2 and tau, and of the proteasomal and autophagic clearance pathways, including mitophagy. We have also adapted RM to high-throughput video and Z- projection imaging, enabling us to capture dynamic changes in intracellular morphology and trafficking of organelles such as mitochondria and lysosomes. Combining these ALP assays with RM, enables us to predict how disturbances in the ALP may affect cellular fate, predetermine which cell populations are more likely to degenerate in each disease and better understand how neuronal populations that are resistant to degeneration compensate for impaired ALP to survive. Our ultimate aim combines RM/OPL, human neuron disease models along with our proprietary compounds that increase turnover of misfolded proteins and common molecular targets that we found with family based whole genome sequencing, to identify new therapeutic strategies to treat these neurodegenerative disorders.