Investigating lysosomal exocytosis as a pathway of beta‐amyloid release from neurons

Abstract

AbstractBackgroundThe endosomal‐lysosomal system has long been linked to the production of beta‐amyloid (Aβ), but the specific intracellular compartments involved in Aβ secretion remain contentious. Conventionally, lysosomes are associated with Aβ degradation, yet studies have also shown that lysosomes are a site of Aβ accumulation. Lysosomal exocytosis is a major secretory pathway in non‐neuronal cells, but few studies have investigated this pathway in neurons. This project examined the role and mechanism of lysosomal exocytosis in neurons, and we hypothesized that lysosomal exocytosis is a pathway for Aβ secretion from neurons.MethodsWe have established human iPSC‐derived neurons in culture, including cells bearing the APP Swedish mutation. Cells were transduced with pH sensitive fluorescent (pHluorin/pHmScarlet) tagged compartment markers to examine neurosecretory granules (synaptophysin), early endosomes (Rab5), late endosomes (Rab9) and lysosomes (LAMP1). Cells were loaded with fluorescent exogenous Aβ or Amytracker to label endogenous Aβ. We then stimulated secretion with ionomycin and used live cell Total Internal Reflection Fluorescence (TIRF) microscopy to examine molecular events occurring within 250 nm to the plasma membrane. shRNA against proteins known to be involved with lysosomal secretion (Rab27b, Munc13‐4 and synaptotagmin‐7) were used as controls. Imaris software was used to analyze the number and timing of lysosomal secretory events and the secretion of Aβ.ResultsAs previously reported, exogenous Aβ in wild‐type cells and endogenous Aβ in APP Swedish cells accumulates in lysosomes, but not in early endosomes. TIRF microscopy captured videos of lysosomes approaching and fusing with the plasma membrane. Fusion appears as a reduction in normal fluorescent LAMP1 signal and an increase in pH sensitive fluorescent protein marker signal (reflecting the loss of luminal acidity due to fusion). Intracellular Aβ was released from neurons as lysosomes fused with the plasma membrane, seen as a simultaneous decrease in fluorescent Aβ labelling. Silencing of Rab27b significantly reduced lysosomal exocytosis and Aβ secretion, which confirmed that this is a lysosomal process.ConclusionOur results demonstrate that lysosomal exocytosis is a novel secretion pathway for Aβ in human neurons and identify potential pharmacological targets that regulate clearance of Aβ.