Lysosomal storage causes cellular dysfunction in mucolipidosis II skin fibroblasts

Abstract

Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed ~2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 ± 0.08 versus 4.79 ± 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated ~3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH(4)Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates.

FIGURE 1.

Total lysosomal enzymes are supplied to ML-II skin fibroblasts. A, multiple lysosomal enzymes were excreted into culture supernatant in NH₄Cl-treated normal cells and untreated ML-II cells. Activities of 10 major lysosomal enzymes were measured in culture supernatants of normal cells, ML-II cells, and normal cells treated with 20 mm NH₄Cl for 7 days. Relative activities as compared with those of normal cells (means ± S.D.) are shown. B, activities of major lysosomal enzymes were measured in skin fibroblasts of normal, ML-II, and enzyme-treated ML-II cells. Measurements were carried out in triplicate for each sample, and means ± S.D. are indicated. Units are nmol/h/mg of protein. Relative activities (%) as compared with normal cells are shown above each bar. C, to elucidate whether this enzyme uptake was mediated by the M6P receptor, we performed enzyme treatment with various concentrations of M6P (0, 5, and 10 mm). ML-II skin fibroblasts were collected before treatment (pre), and at 48 and 72 h after treatment, and major enzyme activities were measured. Means are indicated, and units are nmol/h/mg of protein. Additional data are available for B and C (supplemental Fig. 1). D, micrographs showing localization of enzymes within normal, ML-II, and enzyme-treated ML-II skin fibroblasts evaluated by cathepsin B and LAMP-2 immunocytochemistry co-staining. Merging of cathepsin B and LAMP-2 signals indicates that incorporated enzymes were targeted to lysosomes. Scale bars, 20 μm. We also confirmed these results by immunocytochemical co-staining using cathepsin D and LAMP-2 (data not shown).

FIGURE 2.

Phospholipid and cholesterol are accumulated in ML-II cells. A, storage materials were measured as phospholipid and cholesterol in normal, ML-II, and enzyme-treated ML-II skin fibroblast total lysates (n = 3). Enzyme treatment of ML-II cells decreased phospholipid and cholesterol contents. *, p < 0.01, **, p < 0.005, ***, p < 0.001. B and C, LBPA (B) and filipin (C) staining micrographs of normal, ML-II, and enzyme-treated ML-II cells are shown. Accumulation of LBPA and cholesterol in endosomes and lysosomes of ML-II cells was corrected by enzyme treatment. Scale bars, 20 μm.

FIGURE 3.

Lysosomal acidification is impaired in ML-II. A, lysosomal pH was measured in skin fibroblasts of normal, ML-II, and enzyme-treated ML-II cells (n = 3). ML-II cells showed a significant increase in lysosomal pH value, and the pH value was normalized by enzyme treatment. *, p < 0.001. B, to elucidate whether storage materials affect the acidifying function, we loaded the cholesterol concentration in culture medium of normal cells. As a result, cholesterol increased lysosomal pH dose dependently in normal cells.

FIGURE 4.

Lysosomes are proliferated in ML-II skin fibroblasts. A, LysoTracker and DAPI fluorescent micrographs of normal, ML-II, and enzyme-treated ML-II cells, indicating a reduction in total lysosomal amount. Scale bar, 20 μm. B, to quantify the amount of lysosomes in one cell, the ratio of LysoTracker/DAPI fluorescence was measured with a plate reader after dual staining of a living suspension of normal, ML-II, and enzyme-treated ML-II cells (n = 3). The lysosomal amount was estimated to be accumulated ∼3-fold in ML-II cells. *, p < 0.05, **, p < 0.005.

FIGURE 5.

Intracellular logistics are impaired in ML-II. A, endocytic targeting of ceramide in normal, ML-II, and enzyme-treated ML-II cells was evaluated by BODIPY-ceramide targeting. Micrographs show fluorescence of living cells using confocal microscopy after treatment with BODIPY-ceramide for 30 min. Scale bars, 20 μm. B, micrographs showing results of MP6 receptor antibody uptake testing of normal, ML-II, and enzyme-treated ML-II cells. In normal and enzyme-treated ML-II cells, M6P receptors appear to be cycling and distributing broadly. Arrows indicate the Golgi pattern of M6P receptor localization in ML-II cells. Scale bars, 20 μm.

FIGURE 6.

Enzyme treatment improves autophagic and mitochondrial status. A, LC3 immunoblotting of total lysates of normal, ML-II (non), and enzyme-treated ML-II cells indicates a decrease in the specific autophagosome marker, LC3-II, after enzyme treatment of ML-II cells. B and C, LC3 (B) and MitoTracker (C) staining micrographs of normal, ML-II, and enzyme-treated ML-II cells. Scale bars, 20 μm. Enzyme treatment of ML-II cells decreased the number of LC3-positive large vesicles and showed a remarkable recovery of mitochondrial structure.

FIGURE 7.

Inclusion bodies disappear after enzyme treatment in ML-II. A, phase-contrast micrographs of ML-II and enzyme-treated ML-II cells. Scale bars, 20 μm. B, electron micrographs of ML-II and enzyme-treated ML-II cells. Scale bars, 5 μm and 500 nm (inset). Micrographs show accumulation of inclusion bodies in ML-II cells and their reduction following enzyme treatment.