Abnormalities in the endosomal-autophagic-lysosomal (EAL) system are an early event in Alzheimer's disease (AD) pathogenesis. However, the mechanisms underlying these abnormalities are unclear. The transient receptor potential channel mucolipin 1(TRPML1), a vital endosomal-lysosomal Ca2+ channel whose loss of function leads to neurodegeneration, has not been investigated with respect to EAL pathogenesis in late onset AD (LOAD). Here, we identify pathological hallmarks of TRPML1 dysregulation in LOAD neurons, including increased perinuclear clustering and vacuolation of endolysosomes. We reveal that iPSC human cortical neurons expressing APOE e4, the strongest genetic risk factor for LOAD, have significantly diminished TRPML1-induced endolysosomal Ca2+ release. Furthermore, we found that blocking TRPML1 function in primary neurons by depleting the TRPML1 agonist PI(3,5)P2. via PIKfyve inhibition, recreated multiple features of EAL neuropathology evident in LOAD. This included increased endolysosomal Ca2+ content, enlargement and perinuclear clustering of endolysosomes, autophagic vesicle accumulation, and early endosomal enlargement. Strikingly, these AD-like neuronal EAL defects were rescued by TRPML1 reactivation using its synthetic agonist ML-SA1. These findings implicate defects in TRPML1 in LOAD EAL pathogenesis and present TRPML1 as a potential therapeutic target.

Good’s buffers are commonly used for cell culture and, although developed to have minimal to no biological impact, they cause alterations in cellular processes such as autophagy and lysosomal enzyme activity. Using Chinese hamster ovary cells and induced pluripotent stem cell-derived neurons, this study explores the effect of zwitterionic buffers, specifically HEPES, on lysosomal volume and Ca2+ levels. Certain zwitterionic buffers lead to lysosomal expansion and reduced lysosomal Ca2+. Care should be taken when selecting buffers for growth media to avoid detrimental impacts on lysosomal function.

Background TRPML1 (transient receptor potential channel mucolipin 1) is an essential endosomal‐lysosomal Ca2+ channel whose loss of function can cause neurodegeneration. Defects in the endosomal‐autophagic‐lysosomal (EAL) are a primary feature of Alzheimer’s disease (AD) pathogenesis, and many late‐onset AD (LOAD) risk genes, including APOE4, functionally converge on the EAL system, although the underlying mechanisms remain unclear. Dysfunctional TRPML1 activity has been reported in genetic models of AD involving cells deficient in presenilin 1. Here, we hypothesised that TRPML1 malfunction underlies EAL defects in LOAD, and can be therapeutically targeted to remediate EAL defects in LOAD. Method Cell and molecular approaches were employed to investigate the integrity of endolysosomes in LOAD (n = 10) and matched control (n = 10) post‐mortem brain, and in APOE isoform expressing human cortical neurons derived from induced pluripotent stems cells. TRPML1 activity was manipulated in primary neurons to determine whether blocking TRPML1 could recreate EAL defects evident in LOAD and whether TRPML1 could be targeted therapeutically to protect against EAL defects in LOAD. Result We identified perinuclear clustering of enlarged LAMP‐1‐positive endolysosomes in post‐mortem LOAD hippocampal neurons, indicative of TRMPL1 dysregulation. Our results reveal for the first time that TRPML1 activity was significantly reduced in APOE4 expressing human cortical neurons derived from induced pluripotent stems cells, resulting in elevated endolysosomal Ca2+ content. Furthermore, depletion of the TRPML1 agonist, PI(3,5)P2 in primary neurons using YM201636, recreated many features of AD endolysosomal neuropathology. This included endolysosomal swelling, accumulation of autophagic vacuoles (AVs), and increased endolysosomal Ca2+ accumulation. Strikingly, the re‐activation of TRPML1 in YM201636‐treated cultures, using the synthetic small molecule agonist ML‐SA1, restored endolysosomal Ca2+ pool to its normal size, mitigated endolysosomal swelling, prevented the accumulation of AVs, and increased levels of non‐pathogenic alpha‐secretase derived C‐terminal fragments of the amyloid precursor protein (APP). Conclusion Together, these finding implicate defects in TRPML1 in LOAD and present TRPML1 as a novel potential therapeutic target to remediate endolysosomal pathology in AD.

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