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

大多数神经退行性疾病存在一种常见的分子表型，包括蛋白质和/或蛋白质- RNA聚集、脂质水平扰动、线粒体功能障碍、溶酶体功能障碍和神经元损伤。 炎症文献提供的证据表明，阿尔茨海默病的这种致病特征和 相关的痴呆症可以通过遗传和药理学激活溶酶体通量-1而正常化 这种机制被称为巨自噬。为了产生机制上不同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， 分子撞击加速脂滴清除。大多数已知的溶酶体通量激活剂通过 抑制mTOR，抑制免疫系统，使本已脆弱的老年人口， 感染疾病的风险更高。在该提案中，我们寻求mTOR非依赖性溶酶体通量激活剂。在 目的1我们采用传统的和新的检测方法来确定我们的命中目标，以及他们的机制， 行动上这些化合物是否诱导巨自噬（一种细胞成分再循环途径）或 自噬的特殊形式将通过所提出的包含Aim 1的测定来揭示。我们还将探索 无论溶酶体通量激活是通过其他机制发生的，如转录重编程，还是通过 新机制目标1中生成的数据将指导最佳溶酶体通量激活剂的优先级排序。 适用于改善阿尔茨海默病及相关痴呆症。Aim 2活性将仔细检查溶酶体 诱导多能干细胞衍生的神经元、星形胶质细胞中的通量激活剂给药功效和给药方案 以及来自遗传性阿尔茨海默病患者的神经胶质细胞和来自这些细胞的脑类器官， 以及缺乏这些突变的脑细胞和类器官。因为自噬会破坏蛋白质， 酸，寡糖和脂质进入其结构单元以供再利用，这是一个潜在的风险， 溶酶体流可降解关键的细胞组分，因此导致机制上的毒性。 类器官非常适合于测试避免诱导溶酶体通量激活剂的多剂量方案。 细胞毒性或细胞应激，同时也使阿尔茨海默病相关的致病性正常化 表型存在。我们将使用基于质谱的蛋白质组学和代谢组学/脂质组学， 分析20-30种优先溶酶体通量激活剂治疗后的类器官，以了解如何给药， 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。除了 进行多次生物重复和适当的统计分析，这是确保 我们已经将我们的溶酶体通量激活剂分布到多个外部环境中， 合作者进行独立的细胞分析和阿尔茨海默氏症模型的疗效评估 疾病我们希望提供一套经过验证的、机制多样的、无风险的溶酶体通量激活剂， 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.