Fig 2: ASM secreted during cell injury associates with the PM and is proteolytically modulated.(A) Effect of protease inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca2+ media containing (green) or not (blue) a protease inhibitor cocktail and incubated at 37°C for the indicated time. As controls, cells not treated with SLO (NT) were incubated with (purple) or without (red) inhibitors. Supernatant samples were placed on ice, protease inhibitors were added and ASM activity was assayed. Similar results were obtained with HeLa cells (not shown). (B) Effect of inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca2+-containing media with 100 µM E64, 100 µM pepstatin-A (PEP-A) or no inhibitors, and incubated at 37°C for 30 s. The data represent the mean +/- SD of triplicate assays. * P = 0.039, Student’s t test. (C) Detection of cell-associated ASM during the first seconds after SLO wounding. NRK cells were permeabilized with SLO (100 ng/ml), incubated at 37°C in Ca2+-containing media for the indicated time, washed and immunofluorescence was performed with rabbit anti-ASM antibodies, followed by imaging under identical settings. (D) Detection of the active 65 kDa form of ASM in supernatant and membrane-associated fractions of wounded cells. NRK cells were permeabilized or not with SLO (100 ng/ml) with or without 100 µM E64 or 100 µM pepstatin-A for the indicated periods of time, and samples of the supernatant or of material removed from the cell surface by an acid wash were analyzed by Western blot with rabbit anti-ASM antibodies. The data are representative of at least three independent experiments. (E) Detection of the 65 kDa ASM form in the supernatant and membrane-associated fractions of wounded cells depleted or not in cathepsin D (CATD). NRK cells were permeabilized or not with SLO and analyzed as described in (D). The data are representative of at least three independent experiments.

Fig 3: NHE6-null endolysosomal model in neurons. A, Schematic representation of endosomal maturation and trafficking in WT neurons. Newly synthesized CatD enzymes are trafficked through the endocytic pathway by M6PRs until they reach the highly acidic lysosome lumen to assist in degradation, ensuring proper lysosome functioning. B, Loss of NHE6 leads to overacidification of both the endosomal and lysosomal lumen that ultimately results in lysosome dysfunction. CatD becomes prematurely active in hyperacidified endosomal compartments yet is less likely to be trafficked and active in lysosomes, likely due in part to impaired endosome-lysosome fusion. Trafficking of M6PRs, which are responsible for delivering newly synthesized CatD to lysosomes, is also disrupted as they accumulate in endosomes and are unable to be transported back to the TGN. Endolysosomal trafficking is further altered as MVBs are more likely to fuse with the PM, resulting in enhanced exosome release.

Fig 4: Correlation analysis of PSAP and a-Syn (A) and CTSD and a-Syn (B), obtained from Western blots from 15 and 10 PD patients, respectively.

Fig 5: MGRN1 does not affect lysosomal competence. (a) HeLa cells treated with irrelevant (GFP) or MGRN1 siRNAs were fixed and immunostained with anti-CD63 antibody. Enlarged views of the areas within the white boxes (insets) are also shown. Scale bar, 5 µm. (b) Graph plotting data from (a) shows significant increase (~1.5 folds) in average diameter of CD63 positive vesicles for MGRN1-depleted cells, where the vesicles diameter are ~0.78 µm and diameter of vesicles in control cells are ~0.51 µm. Note the presence of intra-luminal vesicles in both the samples, except that the ones with MGRN1 depletion are bigger in size. Approximately 240 vesicles were counted from three independent experiments. ***P=0.001, using Student's t-test. Error bars, ±S.E.M. (c) HeLa cells treated with irrelevant (GFP) or MGRN1 siRNAs subjected to Alexa-Fluor 488 EGF uptake. Cells were washed, fixed at 40 min time point, immunostained for CD63 and imaged. Enlarged views of the areas within the white boxes (insets) are also shown. Insets reveal enlarged views of multiple Alexa-Fluor 488-labeled EGF positive puncta (green) on CD63 positive vesicles with intra-luminal membranes (red). Scale bar, 5 µm. (d) Cell lysates treated with indicated siRNAs were analyzed for the levels of CTSD. The levels of ß-tubulin serve as loading control. The low and dark exposures of the CTSD blot indicate the different processed forms of the enzyme. Efficiency of knockdown was confirmed by immunoblotting with anti-MGRN1. The blots are representative of at least three experiments. (e) Histograms plotting data from (d) show significant (~1.5 folds) but similar increase in the levels of mature and immature CTSD, as normalized against the protein levels of ß-tubulin (top left) and (bottom left) with the depletion of MGRN1. However, note that the fold change in the mature and immature forms of CTSD are comparable between control and MGRN1 siRNA-treated samples (right). Graph representing three independent experiments. **P=0.05, n.s., not significant (P=0.75) using Student's t-test. Error bars, ±S.E.M. (f) Cell lysates were analyzed for CTSD activity. Histogram plotted for the activity of this enzyme in MGRN1 and GFP siRNA-treated samples. To block CTSD activity, cells were either treated with vehicle control or pepstatin A, as indicated. Graph represents average of three independent experiments, performed in triplicate for each cell concentration. Error bars, ±S.E.M.; RFU, relative fluorescence units. (g) HeLa cells treated with irrelevant (GFP) or MGRN1 siRNAs were fixed and immunostained with anti-CTSD antibody. Two fields for each condition are shown. (h) Graph plotting the average diameter of vesicles showed significant increase (~1.4 folds) when MGRN1 is depleted, imaged in (g). Approximately 205 vesicles were counted from three independent experiments. ***P=0.001, using Student's t-test. Error bars, ±S.E.M. (i) Lysosomal pH values were measured ratiometrically using LysoSensor yellow/blue DND-160–Dextran. In control cells, the average lysosomal pH was detected as 4.62±0.02, while in cells treated with MGRN1 siRNAs the pH was 4.66±0.01; n.s., not significant (P=0.9), using Student's t-test. Graph represents average of three independent experiments. Error bars, ±S.E.M.