Fig 2: Schematic illustrating the CD38-LRRK2-TFEB signaling pathway in B-lymphocytes and macrophages. The CD38-LRRK2 complex exists on the plasma membrane and activation of CD38 results in the internalization of the CD38-LRRK2 complex and its targeting to the endolysosomal system. CD38 promotes generation of an NAADP-dependent lysosomal calcium signal, which is dependent on LRRK2 kinase activity. This results in the activation of PPP3/calcineurin, which dephosphorylates TFEB at Ser211 and allows its nuclear translocation. The pathogenic kinase overactive LRRK2G2019S mutant aberrantly activates TFEB via NAADP-TPCN2-dependent calcium signaling and stabilizes TFEB by promoting its C-terminal phosphorylation. Solid arrows indicate known relationships, while dashed arrows indicated hypothesized connections.

Fig 3: CD38 calcium signaling and nuclear localization of TFEB is dependent on LRRK2. Immunoblot of endogenous TFEB after nuclear fractionation of WT and lrrk2 KO (A) B-lymphocytes and (B) BMDMs treated with 5 µg/ml anti-CD38 antibody (clone 90) or an isotype control (Iso) for 1 h. (C) Quantification of western blots from A (upper, n = 4) and B (lower, n = 3). (D) Intracellular calcium response of purified WT (black) and lrrk2 KO (red) B-lymphocytes stimulated with anti-CD38 (clone 90, 5 and 10 µg/ml) antibody. (E) Quantification of calcium response (area under curve) from (D) to 5 min (n = 4). (F) Intracellular calcium response of purified WT (black) and LRRK2G2019S KI (blue) B-lymphocytes stimulated with anti-CD38 (clone 90, 5 and 10 µg/ml) antibody. (G) Quantification of calcium response from (F) to 5 min (n = 4). (H) Intracellular calcium response of purified WT (solid lines) or LRRK2G2019S KI (dashed lines) B-lymphocytes stimulated with anti-CD38 (clone 90 10 µg/ml, black) antibody with or without Ned-19 (100 µM, blue) or Gly-Phe-ß-naphthylamide (200 µM, green). (I) Quantification of calcium responses (area under curve) from (H) to 5 min (n = 4). (J) Purified wild type (WT, black) or lrrk2 KO (KO, red) B-lymphocytes stimulated with NAADP-AM (3 µM). (K) Quantification of calcium responses (area under curve) from (H) to 5 min (n = 6). Partial calcium response curve is shown (to 3 min) as fluorescent counts (iLm1). For western data, histone H3 and GAPDH are shown as fractionation and loading controls. Western bands are quantified, normalized to the loading control, and presented relative to the control lane. (# – not significant; *p < 0.05; **p < 0.002, ***p < 0.0001, Student’s t-test or one-way ANOVA with post hoc Tukey’s HSD or Fisher’s LSD).

Fig 4: TFEB nuclear translocation was decreased in T2DM mice and HG-cultured HT22 cells, which was related to decreased TFEB activity and expression. Nucleus (A) and cytosol TFEB (B) were measured via subcellular fractionation experiments by western blotting in the hippocampal of mice, n = 6; Nucleus and cytosol TFEB (C) were measured via subcellular fractionation experiments by western blotting in HT22 cells, n = 3; GAPDH was used as the loading control of cytosol TFEB, Histone-H3 was used as the loading control of nucleus TFEB. HT22 cells were treated with or without high glucose. After immunostaining with TFEB (green) and DAPI (blue), cells were examined by fluorescence microscopy, scale bar: 10 μm. The colocalization of TFEB and DAPI was calculated and analyzed, n = 3. TFEB, p-TFEB, mTOR and p-mTOR were measured via western blotting in the hippocampal of mice (E), n = 6, and HT22 cells (F), n = 3, GAPDH was used as the loading control. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001