A Dual Therapeutic Strategy for CLN3 Disease: Intracellular TRPML-1–Mediated Stimulation of Lysosomal Exocytosis in CLN3-Deficient Neurons and extracellular Microglial Clearance of Glycerophosphodiesters

Abstract

CLN3 disease is a fatal paediatric neurodegenerative disorder characterized by lysosomal dysfunction due and the intracellular accumulation of autofluorescent ceroid lipopigments, specifically glycerophosphodiesters (GPDs). Caused by a mutation in the CLN3 gene, the disease remains poorly understood and currently has no effective treatment. This proposal presents a dual therapeutic approach that targets both intracellular and extracellular GPD accumulation. The first strategy involves activating the Transient receptor potential mucolipin-1 (TRPML-1) channel in CLN3-deficient neurons, aimed at stimulating lysosomal exocytosis and thereby lysosomal clearance of GPDs. The second strategy aims to harness the phagocytic role of healthy microglia to clear GPDS from the extracellular environment of GPDs. To test this approach, CLN3-deficient iPSC-derived neurons with doxycycline-inducible TRPML-1 overexpression will be co-cultured with microglia, derived from healthy human induced pluripotent stem cells (hiPSCs). Lysosomal function and metabolite clearance will be assessed using lysosome immunoprecipitation (lyso-IP), immunofluorescent microscopy, western blotting, and mass spectrometry-based metabolomics, lysosomal function will be assessed. We hypothesize that this dual approach will reduce intracellular GPD accumulation via TRPML-1 mediated neuronal lysosomal exocytosis, and promote extracellular GPD clearance by microglia, ultimately restoring cellular homeostasis in the CLN3 disease model.