STUDYING NEURONAL AUTOPHAGY IN HUMAN AGEING USING INDUCED NEURONS DIRECTLY REPROGRAMMED FROM ADULT HUMAN DERMAL FIBROBLASTS

Age is the greatest risk factor for neurodegenerative diseases (NDDs), yet the molecular links between physiological aging and NDD pathogenesis remain poorly understood. Autophagy- a lysosomal degradation pathway essential for maintaining cytoplasmic homeostasis- declines with age and contributes to neuronal dysfunction. To investigate aging alters autophagic flux in human neurons, we utilized direct reprogramming of human dermal fibroblasts, generating induced neurons (iNs), a method that retains donor-specific genetic and epigenetic signatures. We generated iNs from a cohort of 54 healthy donors ranging in age from 24 to 86 years. Neuronal identity was verified by immunocytochemistry and high-content automated microscopy using neuronal (TAU) and autophagy-specific markers (BECLIN1, LC3, P62, LAMP1) under both basal and stress-induced conditions. Our results indicate donor specific alterations in autophagy. We identified distinct clusters of stress-induced autophagic flux in the case of both young and old donor groups. These variations were observed in autophagosome formation, turnover, as well as in the lysosomal degradation step. These findings suggest different trajectories of age-related decline in neuronal homeostasis, which may contribute to increased vulnerability to age-related neurodegeneration. Currently, we are validating these findings across the full donor cohort. In parallel, we are conducting multi-omic autophagy profiling using a range of molecular assays including genome-wide DNA methylation arrays, bulk RNA-sequencing, mass spectrometry, and metabolomics to compare young and old iNs. Our long-term goal is to identify key regulators of autophagy and explore rejuvenation strategies. This approach could inform future therapies for NDDs, where impaired autophagy and accelerated aging are often observed.