Impaired autophagic and mitochondrial functions are partially restored by ERT in Gaucher and Fabry diseases

Abstract

The major cellular clearance pathway for organelle and unwanted proteins is the autophagy-lysosome pathway (ALP). Lysosomes not only house proteolytic enzymes, but also traffic organelles, sense nutrients, and repair mitochondria. Mitophagy is initiated by damaged mitochondria, which is ultimately degraded by the ALP to compensate for ATP loss. While both systems are dynamic and respond to continuous cellular stressors, most studies are derived from animal models or cell based systems, which do not provide complete real time data about cellular processes involved in the progression of lysosomal storage diseases in patients. Gaucher and Fabry diseases are rare sphingolipid disorders due to the deficiency of the lysosomal enzymes; glucocerebrosidase and α-galactosidase A with resultant lysosomal dysfunction. Little is known about ALP pathology and mitochondrial function in patients with Gaucher and Fabry diseases, and the effects of enzyme replacement therapy (ERT). Studying blood mononuclear cells (PBMCs) from patients, we provide in vivo evidence, that regulation of ALP is defective. In PBMCs derived from Gaucher patients, we report a decreased number of autophagic vacuoles with increased cytoplasmic localization of LC3A/B, accompanied by lysosome accumulation. For both Gaucher and Fabry diseases, the level of the autophagy marker, Beclin1, was elevated and ubiquitin binding protein, SQSTM1/p62, was decreased. mTOR inhibition did not activate autophagy and led to ATP inhibition in PBMCs. Lysosomal abnormalities, independent of the type of the accumulated substrate suppress not only autophagy, but also mitochondrial function and mTOR signaling pathways. ERT partially restored ALP function, LC3-II accumulation and decreased LC3-I/LC3-II ratios. Levels of lysosomal (LAMP1), autophagy (LC3), and mitochondrial markers, (Tfam), normalized after ERT infusion. In conclusion, there is mTOR pathway dysfunction in sphingolipidoses, as observed in both PBMCs derived from patients with Gaucher and Fabry diseases, which leads to impaired autophagy and mitochondrial stress. ERT partially improves ALP function.

Fig 1. Autophagy-lysosomal complex is affected in GD and FD.

(A) PBMCs from healthy subjects (control), patients with GD (types 1 and 3) and FD were stained with Cyto-ID autophagy detection kit. The graph shows the relative levels of autophagosome vesicle number in control, GD and FD groups. Values were normalized to the control group. Each dot represents a patient sample.*p<0.05 One-way ANOVA, p = 0.016 ANOVA with Bartlett’s multiple comparisons test. (B) GD, FD and control PBMCs were stained with LysoTrackerRed to measure lysosomal accumulation. Values were normalized to the control group. GD (n = 5), FD (n = 6) and control (n = 10). *p<0.05. (C) Quantification of Beclin 1 and LAMP1 protein level from control (n = 10), GD1 (n = 5), GD3 (n = 4) and FD (n = 8) samples after western blot. The protein level was measured relative to total protein level. Values were normalized to the control group. *p<0.05 Student’s T-Test. (D) SQSTM1/p62 level in GD type 1 and type 3 (n = 11), FD (n = 5) and control (n = 5) samples were measured with a SQSTM1/p62 ELISA kit. SQSTM1/p62 level was measured in triplicates, on three biological repeats. Values normalized to control group. *p<0.05 Student’s T-Test. (E) Representative western blot showing LC3-I and LC3-II levels in PBMCs from GD (Type 1 and 3), FD and control samples. (F) Q-PCR was performed to determine relative LAMP1, LC3A/B and SQSTM1/p62 mRNA expression levels in GD and FD PBMCs. RNA level was measured in triplicates, n = 5 or 10 samples analysed each group. All data were expressed as S.E.M. and * p<0.05 vs control.

Fig 2. The effects of mTOR inhibitor rapamycin on autophagy in GD and FD.

(A-C) PBMCs from healthy controls (A), GD (B), and FD (C) were treated with the 10 nM rapamycin (RAP) alone or in combination with 20 μM 3MA for 3h. Then, PBMCs were stained with Cyto-ID autophagy detection kit. Samples were measured in triplicates, control group n = 5, GD1 n = 11, GD3 n = 3, FD n = 5. All data were expressed as S.E.M. and * p<0.05 vs control. (D) Representative western blot showing Beclin1 and LAMP1 protein expression in GD (N370S/L444P and L444P/L444P mutations), FD, and control samples after 3h treatment with 10 nM rapamycin. (E) Quantification of relative levels of Beclin1 and LAMP1 normalized to actin from separate experiments in which the control were untreated PBMCs from the same group. n = 5–7 samples analysed each patient group. Membranes were re-probed with actin or total protein for normalization.* p<0.05 vs untreated samples. (F) Q-PCR was performed to determine relative DDIT3 and HSPA5 mRNA expression levels in GD and FD samples. The RNA level was measured in triplicates, n = 5 or 6 samples analysed each group. All data are expressed as S.E.M. and * p<0.05 vs untreated samples.

Fig 3. Mitochondrial function is affected in GD and FD.

(A) Effect of rapamycin (RAP) treatment on ATP levels. PBMCs from healthy subjects, GD and FD were treated for 3h with the 10 nM RAP alone, 20 μM 3MA or a combination. ATP levels were measured using CellTiter-Glo assay. Samples were measured in triplicates, control group n = 4, GD1 n = 6, GD3 n = 3, FD n = 4. All data were expressed as S.E.M. and * p<0.05 vs untreated control. (B) A scatter diagram comparing autophagosome volume to ATP levels in response to 10 nM RAP treatment for 3h. (C) Quantification of Tfam protein level from control (n = 10), GD1 (n = 10), GD3 (n = 3) and FD (n = 6) samples after detection by western blotting. Quantification of relative level of Tfam normalized to actin. Insert: representative western blot showing Tfam protein expression in healthy subjects (control), GD1 and FD samples. *p<0.05 control vs GD (Student’s T test, One-way ANOVA with Kruskal-Wallis test) and p = 0.037 controls vs. GD (Dunn’s multiple comparisons test). (D) Analysis of mitochondrial biogenesis. PBMCs from healthy subjects (Control), GD1, GD3 and FD were treated with the 10 nM rapamycin for 3h. The data is the ratio of the mitochondrial encoded gene ND1 to nuclear-encoded GAPDH as determined by qPCR. Untreated PBMCs from healthy subjects served as the control and an average of their values was set as 1. Data were expressed as S.E.M. and * p<0.05 (E) Q-PCR was performed to determine relative Tfam, CytC and NRF1 mRNA expression levels in GD1 and FD samples. RNA level was measured in triplicates, n = 3–4 samples analysed each group. All data were expressed as S.E.M. and * p<0.05 vs control group. (F). Fluorescence images of ΔѰm. The mitochondria of freshly isolated PBMCs from patients with GD, FD and healthy controls were stained with Mito-ID Membrane Potential reagent, and visualized by fluorescence microscopy. Green fluorescence color stain mitochondria with a low membrane potential, highly polarized mitochondria exhibit red color.

Fig 4. Assessment of autophagy and mitochondrial functions after ERT.

(A, B and C) Autophagy vacuole number, lysosome, cellular ATP and mitochondrial activity (MitoTracker) levels were measured immediately following ERT treatment. Blood was collected before (Pre) and after (Post) ERT infusion and PBMCs were isolated from GD1 (A), GD3 (B) and FD (C) patients. Values represent post-infusion to pre-infusion levels. Samples were measured in triplicates, n = 3. All data were expressed as S.E.M. and * p<0.05 vs pre-infusion. (D) Representative western blot (30 μg WCE) shows GBA, LAMP1, LC3-I/II and Tfam protein expression in PBMCs of healthy and GD1 subjects before and after ERT infusion. LC3 western blot representing protein expression in untreated (-) or treated with 100 μM ambroxol (AMB) PBMC derived from healthy subjects (control) and GD1 samples before and after ERT infusion. (E) Quantification of LAMP1, total level of LC3I and Tfam normalized to protein bands on Ponceau staining from n = 3–4 separate experiments in which control were PBMCs from healthy control group. * p<0.05 vs pre-infusion samples (Student’s T test). # p = 0.013 vs pre-infusion samples (F test to compare variance). (F) Relative amount of mitochondria in GD1 patient pre- and post-infusion. Shown is the ratio of the mitochondrial encoded gene ND1 to nuclear-encoded GAPDH as determined by qPCR. Samples were measured in triplicates, n = 4. All data were expressed as S.E.M. and * p<0.05 vs pre-infusion samples (Student’s T test).

Fig 5. Effect of recombinant enzyme on the ALP pathway and ATP levels.

(A-B) Autophagy in PBMC (A) or lymphocytes (B) following recombinant GCase treatment. PBMCs or lymphocytes from healthy subjects (control, black bar) and GD1 (grey bar) patients were treated for 3h with 10 μg/ml rhGCase and stained with Cyto-ID autophagy detection kit. (C) Autophagosome staining of PBMCs and lymphocytes (from healthy subjects) incubated with or without 10 μg/ml rhGCase. Staining was performed using Cyto-ID autophagy kit (green color). (D and E) Effect of recombinant GCase and α-Gal A on lysosomes. PBMCs from healthy (black bar) and GD1 (grey bar) individuals were incubated for 3h with the 10 μg/ml rhGCase or α-Gal A and stained with lysosomal dye, LysoTracker Red. (F) ATP level in healthy (control) and GD type 1 PBMCs before and after 3h rhGCase treatment. (G) ATP level in control and FD PBMCs before and after 3h α-Gal A treatment. All samples were measured in triplicates, n = 3–4 patients from each group. All data were expressed as S.E.M. and * p<0.05 vs untreated samples.

Fig 6. A working model of ALP dysfunction in GD and FD disrupt mTOR signalling pathway.

(A) mTOR signalling pathway in normal cells. Nutrient availability and intracellular concentration of ATP are important for activation/inactivation of mTOR. Active form of mTOR is localized on lysosome, inhibits TFEB, autophagosome-lysosome fusion, regulates mitochondrial metabolism and activates UPR pathway. During nutrient-depletion, pH in lysosomes decreased and mTOR translocates to cytoplasm in inactive phase. TFEB migrates to the nucleus and activates expression of lysosomal genes. Transcription factor 4E-BP activates mitochondrial genes. Rapamycin inhibits mTOR activity (without releasing it from the lysosomal surface) and activates autophagosome-lysosome fusion. (B). mTOR signaling pathway in GD cells. Chronic elevation of lysosomal pH in GD prevents mTOR translocation to lysosomal surface and activation of the mTOR pathway. Moreover, lysosomal acidification obstructs autophagosome-lysosome fusion. Inhibition of mitochondrial metabolism leads to an imbalance in energy metabolism. Also, we postulate that TFEB migrates to the nucleus and activates the transcription of lysosomal genes and nuclear encoded mitochondrial genes. (C) mTOR signaling pathway in FD. In FD, α-Gal A deficiency leads to accumulation of Gb3 in lysosomes, partial inhibition of mTOR activity and deregulation of autophagosome-lysosome function and intracellular ATP balance.