Lysosomal proteomics reveals mechanisms of neuronal APOE4-associated lysosomal dysfunction

APOE4 is the primary risk factor for Alzheimer disease (AD). Early AD pathological events first affect the neuronal endolysosomal system, which in turn causes neuronal protein aggregation and cell death. Despite the crucial influence of lysosomes upon AD pathophysiology, and that APOE4 localizes to lysosomes, the influence of APOE4 on lysosomal function remains unexplored. We find that expression of APOE4 in neuronal cell lines results in lysosomal alkalinization and impaired lysosomal function. To identify driving factors for these defects, we performed quantitative lysosomal proteome profiling. This revealed that APOE4 expression results in differential regulation of numerous lysosomal proteins, correlating with APOE allele status and disease severity in AD brains. In particular, APOE4 expression results in the depletion of lysosomal LGALS3BP and the accumulation of lysosomal TMED5. We additionally validated that these lysosomal protein changes can be targeted to modulate lysosomal function. Taken together, this work thereby reveals that APOE4 causes widespread lysosomal defects through remodeling the lysosomal proteome, with the lysosomal TMED5 accumulation and LGALS3BP depletion manifesting as lysosomal alkalinization in APOE4 neurons.<b>Abbreviation</b>: Aβ-β-amyloid; AD-Alzheimer disease; APMS-affinity purification mass spectrometry; APOE-apolipoprotein E; APP-amyloid beta precursor protein; BafA1-bafilomycin A1; CMA-chaperone-mediated autophagy; COP-coatomer; ER-endoplasmic reticulum; ERAD-endoplasmic reticulum-associated degradation; ERGIC-endoplasmic reticulum-Golgi intermediate compartment; FCCP-carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GABA-gamma-aminobutyric acid; GFP-green fluorescent protein; GPI-glycerophosphoinositol; GPN-glycyl-L-phenylalanine 2-naphthylamide; HA-hemagglutinin protein tag; iPSC-induced pluripotent stem cells; LAMP1-lysosomal associated membrane protein 1; LGALS3BP-galectin 3 binding protein; LysoIP-lysosomal immunoprecipitation; MAPT-microtubule associated protein tau; MR-CTSB-magic red cathepsin B; MS-mass spectrometry; NFT-neurofibrillary tangles; PCA-principal component analysis; PLA-proximity ligation assay; PSEN1-presenilin 1; PSEN2-presenilin 2; p-MAPT/Tau-phospho-MAPT/tau; ROS-reactive oxygen species; RT-qPCR-reverse transcriptase quantitative polymerase chain reaction; SEM-standard error of the mean; siRNA-short interfering RNA; SRCR-scavenger receptor cysteine-rich domain; TMED-transmembrane p24 trafficking protein; TMEM192-transmembrane protein 192.