Substrate Reduction Therapy Reverses Mitochondrial, mTOR, and Autophagy Alterations in a Cell Model of Gaucher Disease

Abstract

Substrate reduction therapy (SRT) in clinic adequately manages the visceral manifestations in Gaucher disease (GD) but has no direct effect on brain disease. To understand the molecular basis of SRT in GD treatment, we evaluated the efficacy and underlying mechanism of SRT in an immortalized neuronal cell line derived from a Gba knockout (Gba^-/-) mouse model. Gba^-/- neurons accumulated substrates, glucosylceramide, and glucosylsphingosine. Reduced cell proliferation was associated with altered lysosomes and autophagy, decreased mitochondrial function, and activation of the mTORC1 pathway. Treatment of the Gba^-/- neurons with venglustat analogue GZ452, a central nervous system-accessible SRT, normalized glucosylceramide levels in these neurons and their isolated mitochondria. Enlarged lysosomes were reduced in the treated Gba^-/- neurons, accompanied by decreased autophagic vacuoles. GZ452 treatment improved mitochondrial membrane potential and oxygen consumption rate. Furthermore, GZ452 diminished hyperactivity of selected proteins in the mTORC1 pathway and improved cell proliferation of Gba^-/- neurons. These findings reinforce the detrimental effects of substrate accumulation on mitochondria, autophagy, and mTOR in neurons. A novel rescuing mechanism of SRT was revealed on the function of mitochondrial and autophagy-lysosomal pathways in GD. These results point to mitochondria and the mTORC1 complex as potential therapeutic targets for treatment of GD.

Keywords: cell biology; glucosylceramide; glucosylsphingosine; lysosomal storage disorders; neurons.

Figure 1

Glucosylceramide (GC) and glucosylsphingosine (GS) analysis by LC/MS-MS in neurons and isolated mitochondria. (A) GC and GS levels in Gba^-/- neurons were significantly reduced by 300 nM GZ452 treatment. (B) In the isolated mitochondria, GC level in GZ452-treated Gba^-/- neurons was reduced to WT (Gba^+/+) level. Triplicate experiments (n ≥ 2–4 samples). One-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001). ns, not statistically significant.

Figure 2

GZ452 treatment improved mitochondrial function. (A) Seahorse analysis of oxygen consumption rate (OCR) in Gba^+/+, Gba^-/-, and GZ452-treated Gba^-/- neurons. (B) Quantitation of OCR data showed ATP production and maximal respiration was increased in GZ452-treated Gba^-/- neurons (triplicate experiments, n = 4 per experiment). (C,D) Mitochondrial membrane potential (MMP) assay. GZ452 treatment significant improved MMP in Gba^-/- neurons (C). CCCP treatment disrupted MMP in neurons as a negative control (D) (triplicate experiments, n = 4 per experiment). (E) MitoTracker-stained mitochondria (scale bar = 50 µM). (F) Quantitation of MitoTracker density in cells showed reduced signals in GZ452-treated Gba^-/- neurons compared with untreated Gba^-/- neurons (n ≥ 6 images from triplicate experiments). (G) VDAC and β-actin were measured by WB. “>” points a non-specific band. (H) Quantitation data showed GZ452 treatment reduced VDAC protein (normalized by β-actin) level in Gba^-/- neurons (duplicate experiments). One-way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). ns, not statistically significant.

Figure 3

GZ452 treatment protected lysosome and autophagy in Gba^-/- neurons. (A) LysoTracker-stained lysosomes (scale bar = 50 µM). (B) Quantitation of LysoTracker density in cells (n ≥ 10 images). (C) Lamp1 and β-actin were measured by WB in neurons. (D) Quantitation of Lamp1 protein level after normalized with β-actin showed reduction in Lamp1 in GZ452-treated Gba^-/- neurons (duplicate experiments). (E) Autophagy flux was analyzed in neurons by CYTO-ID^® autophagy kit. Increased fluorescence unit indicates that autophagy flux was blocked in Gba^-/- neurons. GZ452 treatment improved autophagy flux from 2.4-fold to 1.3-fold above normal level (triplicate experiments, n = 8 per experiment). (F) LC3 and β-actin were measured by WB. After normalized with β-actin controls, quantitation data showed LC3-II level was increased in Gba^-/- neurons (2.4-fold) compared with Gba^+/+ and reduced to 1.5-fold in GZ452-treated Gba^-/- neurons (duplicate experiments). (G) Gba^-/- neurons contain large-size vacuoles (arrows), shown by representative EM images. Large vacuoles were not present in GZ452-treated Gba^-/- neurons. (H) Quantitation of vacuoles with contents in neurons (n ≥ 9 cells). One-way ANOVA analysis (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). ns, not statistically significant.

Figure 4

GZ452 treatment improved proliferation and reduced hyperactivity of mTOR pathway proteins in Gba^-/- neuron. (A) Cell proliferation was measured by MTT assay and presents as absorbance unit at 490 nm (triplicate experiments, n = 8 per experiments). (B) Protein levels of phospho-mTOR, phospho-S6 ribosomal protein (S6-RP), phospho-4E-BP1, mTOR, S6-RP, 4E-BP1, and β-actin were measured by WB. (C) Quantitation data (normalized by β-actin) of the protein levels of phospho-mTOR, phospho-S6-RP and phospho-4E-BP1. (D) Quantitation data of the protein levels of mTOR, S6-RP, and 4E-BP1. Experiments were repeated two or three times (n = 3 samples). One-way ANOVA analysis (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). ns, not statistically significant.

Figure 5

WB analysis of mTOR pathway proteins in mouse and human brains. (A) Protein was analyzed by WB. Gba^{-/-(NestinCre)} mice brains (Gba^-/-) (29 days) were compared with WT mice (35 days). Human GD2 brain (15 months) and GD3 hippocampus (11.5 years) were compared with healthy control hippocampus (12 years). (B) Fold change of WT level after normalization by β-actin. Phospho-S6-RP (arrow)/S6-RP, phospho-4E-BP1/4E-BP1, and phospho-mTOR were increased in Gba^{-/-(NestinCre)} mice brains compared with WT mice. Student’s t-test (replicate assays, n = 2 mice brains). In human samples, phospho-S6-RP/S6-RP, phospho-4E-BP1/4E-BP1, and mTOR were increased in GD2 brain and GD3 hippocampus compared with healthy control hippocampus. Phospho-mTOR was undetectable (U.D.) in human brains (replicate experiments, n = 1 human brain). Student’s t-test (* p < 0.05; ** p < 0.01). ns, not statistically significant.