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

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.