Pharmacologic Lysosomal Flux Activators to Ameliorate Alzheimer's Disease and Related Dementias

大多数神经退行性疾病都存在共同的分子表型，包括蛋白质和/或蛋白质- RNA聚集，血脂水平紊乱，线粒体功能障碍，溶酶体功能障碍，以及神经- 发炎。文献提供的证据表明，阿尔茨海默病的致病标志和 相关痴呆可通过溶酶体通量-1的遗传和药物激活而正常化 这样做的机制被称为巨型自噬。为了产生机械多样化的溶酶体通量激活剂， 我们通过基于细胞的表型筛选筛选了940,000个小分子，以识别108个有效的小分子 分子撞击加速了脂滴的清除。大多数已知的溶酶体通量激活剂通过 抑制mTOR，它抑制免疫系统，使本已脆弱的老年人口处于 感染疾病的风险更高。在这项建议中，我们寻求非依赖mTOR的溶酶体通量激活剂。在……里面 目标1我们使用传统的和新的分析方法来识别我们的HITS的目标以及它们的机制 行动。这些化合物是否诱导大范围的自噬(一种细胞成分循环途径)或 自噬的特殊形式将在包括Aim 1的拟议化验中揭示。我们还将探索 溶酶体通量激活是否通过其他机制发生，如转录重编程，还是通过 新的机制。在目标1中产生的数据将指导最好的溶酶体通量激活剂的优先顺序 适用于改善阿尔茨海默氏症和相关痴呆症。Aim 2活动将仔细检查溶酶体 通量激活剂在诱导多能干细胞来源的神经元、星形胶质细胞中的剂量效应和给药方案 和遗传性阿尔茨海默病患者的神经胶质细胞，以及从这些细胞衍生的脑器官中， 在缺乏这些突变的脑细胞和器官中也是如此。因为自噬循环利用蛋白质、核酸 酸、低聚糖和脂类进入它们的积木中重复使用，这是一个潜在的风险，提高 溶酶体通量可以降解关键的细胞成分，从而导致机制上的毒性。 有机化合物非常适合于测试避免诱导的溶酶体通量激活剂多次给药方案。 细胞毒性或细胞应激，同时也使阿尔茨海默病相关的致病因素正常化 出现了表型。我们将使用基于质谱学的蛋白质组学和代谢组学/脂质组学来 用20-30种优先的溶酶体通量激活剂分析治疗后的有机物，以了解如何给药，以便 以避免细胞毒性，同时使阿尔茨海默病相关的病理生物学表型正常化。此外 进行多个生物复制和适当的统计分析，另一种方式确保 重复性和严谨性是我们已经将我们的溶酶体通量激活剂分发给了多个外部 合作者在阿尔茨海默氏症模型中进行独立的细胞分析和疗效评估 疾病。我们希望提供一套经过验证的、机械上多样化的、低风险的溶酶体通量激活剂作为 治疗阿尔茨海默病的候选人。

A common molecular phenotype exists for most neurodegenerative diseases, including protein and/or protein- RNA aggregation, lipid level perturbations, mitochondrial dysfunction, lysosomal dysfunction, and neuro- inflammation. The literature provides evidence that this pathogenic signature of Alzheimer’s disease and related dementias can be normalized by genetic and pharmacologic activation of lysosomal flux–one mechanism to do this is called macroautophagy. To generate mechanistically diverse lysosomal flux activators, we screened 940,000 small molecules by a cell-based phenotypic screen to identify 108 validated small molecule hits that hastened lipid droplet clearance. Most known lysosomal flux activators function through inhibition of mTOR, which suppresses the immune system, putting the already vulnerable elderly population at higher risk for infectious disease. In this proposal we seek mTOR-independent lysosomal flux activators. In Aim 1 we employ traditional and novel assays to identify the targets of our hits, as well as their mechanisms of action. Whether these compounds induce macroautophagy (a cell component recycling pathway) or a specialized form of autophagy will be revealed by the proposed assays comprising Aim 1. We will also explore whether lysosomal flux activation occurs by other mechanisms, such as transcriptional reprogramming, or by a novel mechanism. The data generated in Aim 1 will guide prioritization of lysosomal flux activators that are best suited for ameliorating Alzheimer’s disease and related dementias. Aim 2 activities will scrutinize lysosomal flux activator dosing efficacy and dosing regimens in induced pluripotent stem cell-derived neurons, astrocytes and glial cells from hereditary Alzheimer’s disease patients and in brain organoids derived from these cells, as well as in brain cells and organoids lacking these mutations. Because autophagy recycles proteins, nucleic acids, oligosaccharides and lipids into their building blocks for reuse, it is a potential risk that enhancing lysosomal flux could degrade critical cellular components and therefore lead to on-mechanism toxicity. Organoids are well-suited for testing lysosomal flux activator multidosing regimens that avoid inducing cytotoxicity or cellular stress, while also normalizing the pathogenic Alzheimer’s disease-associated phenotypes present. We will use mass spectrometry-based proteomics and metabolomics / lipidomics to analyze the organoids after treatment by 20-30 prioritized lysosomal flux activators to learn how to dose so as to avoid cytotoxicity while normalizing the Alzheimer’s disease-relevant pathobiological phenotypes. Besides carrying out multiple biological replicates and appropriate statistical analyses, another way to ensure reproducibility and rigor is that we have distributed our lysosomal flux activators to multiple outside collaborators to carry out independent cellular assays and and efficacy assessments in models of Alzheimer’s disease. We hope to deliver a validated, mechanistically diverse, de-risked set of lysosomal flux activators as candidates to treat Alzheimer’s disease.