Investigating the role of lysosomal exocytosis in Alzheimer’s disease

Background Many studies have implicated the endosomal‐lysosomal system in the production of beta‐amyloid (Aβ), but the actual compartments involved in Aβ secretion have never been visualized. Although lysosomes are expected to be a site of Aβ clearance, studies have also shown them to be a site of Aβ accumulation. Lysosomal exocytosis is well recognized as a major secretory pathway in non‐neuronal cells but is virtually unstudied in neurons. This project investigates the role and mechanism of lysosomal secretion in neurons. We hypothesize that lysosomal exocytosis is a pathway for Aβ release and could be modulated by Rab27b in iPSC‐derived human neurons. Method To study lysosomal exocytosis in a neuronal system, our lab established normal and mutant APP Swedish expressing iPSC‐neurons. The involvement of lysosome‐related secretory proteins (Rab27b, Munc13‐4 and synaptotagmin‐7) were examined using siRNA, dominant‐negative or shRNA constructs. Cells were co‐transduced with a pH‐sensitive dual‐fluorescent lysosomal marker (pHluorin‐LAMP1‐mAppleFP) to identify and track lysosomes. Cells were also loaded with fluorescent exogenous Aβ or a dye to mark endogenous Aβ, and then treated with ionomycin to induce exocytosis. Total internal reflection fluorescence (TIRF) microscopy was performed to capture videos of molecular events occurring within 250nm from the plasma membrane. Imaris software was used to analyze co‐localization of LAMP‐1 with Aβ, and the number and timing of lysosomal secretory events. Result In untreated cells, loaded Aβ accumulated in lysosomes. After treatment with ionomycin, a dramatic decrease of lysosomal Aβ was observed. In live cells, TIRF microscopy captured lysosomes docking and fusing with the plasma membrane over 150 seconds after ionomycin treatment. This was visualized by pHluorin‐LAMP1 (which is normally dark in an acidic pH) fluorescing green upon lysosomal fusion with the plasma membrane. Overexpression of Rab27b N133I mutant significantly increased lysosomal exocytosis of Aβ compared to wild‐type, and Rab27b shRNA dramatically reduced lysosomal exocytosis and Aβ secretion. Conclusion Our results demonstrate that lysosomal exocytosis is a novel secretion pathway in human neurons and identify potential pharmacological targets that regulate clearance of toxic Aβ.