Acid ceramidase inhibition ameliorates α-synuclein accumulation upon loss of GBA1 function

Abstract

GBA1 encodes the lysosomal enzyme β-glucocerebrosidase (GCase) which converts glucosylceramide into ceramide and glucose. Mutations in GBA1 lead to Gaucher's disease and are a major risk factor for Parkinson's disease (PD) and Dementia with Lewy bodies (DLB), synucleinopathies characterized by accumulation of intracellular α-synuclein. In this study, we examined whether decreased ceramide that is observed in GCase-deficient cells contributes to α-synuclein accumulation. We demonstrated that deficiency of GCase leads to a reduction of C18-ceramide species and altered intracellular localization of Rab8a, a small GTPase implicated in secretory autophagy, that contributed to impaired secretion of α-synuclein and accumulation of intracellular α-synuclein. This secretory defect was rescued by exogenous C18-ceramide or chemical inhibition of lysosomal enzyme acid ceramidase that converts lysosomal ceramide into sphingosine. Inhibition of acid ceramidase by carmofur resulted in increased ceramide levels and decreased glucosylsphingosine levels in GCase-deficient cells, and also reduced oxidized α-synuclein and levels of ubiquitinated proteins in GBA1-PD patient-derived dopaminergic neurons. Together, these results suggest that decreased ceramide generation via the catabolic lysosomal salvage pathway in GCase mutant cells contributes to α-synuclein accumulation, potentially due to impaired secretory autophagy. We thus propose that acid ceramidase inhibition which restores ceramide levels may be a potential therapeutic strategy to target synucleinopathies linked to GBA1 mutations including PD and DLB.

Figure 1.

Characterization of GCase-deficient cells. (A) Schematic diagram of human GBA1 gene structure and target sequence of GBA1-CRISPR/Cas9 construct. The GBA1-CRISPR construct targets four out of five human GBA1 isoforms. (B) Cell lysates from wild-type (WT) and GCase-deficient (KO) HEK293-FT cells were subjected to immunoblot analysis using an N-terminal or C-terminal GCase antibody. (C) Triton X-100 soluble cell lysates were prepared from wild-type or GCase-deficient cells. GCase activity in 7.5 μg of cell lysates was measured in the presence or absence of CBE. The detailed GCase assay is described in the Materials and Methods section. GCase activity was measured in triplicate. (D) Cells were fixed with 4% formaldehyde in PBS and immuno-stained with mouse anti-GluCer antibody and DAPI. Representative images are shown. Data represent mean ± S.E.M. N = 30 microscopic fields for wild-type cells, n = 25 microscopic fields for GCase-deficient cells, two-tailed unpaired t-test, ***P < 0.001 compared with wild-type cells. n.s. = not significant.

Figure 2.

Loss of GBA1 leads to α-synuclein and autophagy substrate accumulation. (A) Cells were lysed with 2× SDS sample buffer and cell lysates were analyzed with immunoblot analysis using indicated antibodies. Blot band intensities were normalized to tubulin, and compared with wild-type cells. Graphs show normalized band intensities of intracellular α-synuclein. N = 3, two-tailed unpaired t-test, ***P < 0.001 compared with wild-type cells. (B) Cells were fixed with 4% formaldehyde in PBS for 10 min and subsequently fixed again with cold methanol for 5 min, and immuno-stained with anti-p62 antibody. Representative images are shown. Data represent mean ± S.E.M. n = 10 microscopic fields for wild-type, n = 7 microscopic fields for GCase-deficient cells. Two-tailed unpaired t-test, ***P < 0.001 compared with wild-type cells. (C) LAMP2 and p62 levels are increased in GCase-deficient cells. Cells were prepared as in (B), and immuno-stained with mouse anti-Lamp2 antibody and rabbit anti-LC3B antibody. Representative images are shown. Data represent mean ± S.E.M. n = 10 microscopic fields for wild-type, n = 10 microscopic fields for GCase-deficient cells. Two-tailed unpaired t-test, ***P < 0.001, **P < 0.01 compared with wild-type cells. (D, E) Cells were lysed with 2× SDS sample buffer, and cell lysate samples were analyzed with immunoblot analysis using indicated antibodies. Band intensities were normalized to tubulin, and compared with wild-type cells. Graphs show normalized band intensities. Data represent mean ± S.E.M. N = 4, two-tailed unpaired t-test, ***P < 0.001 compared with wild-type cells.

Figure 3.

GCase-deficient cells exhibit defective Baf-A1-induced extracellular secretion of α-synuclein, p62 and mature cathepsin-D. (A–D) Stimulation (starvation or CCCP)-induced degradative autophagic process in GCase-deficient cells. (A) Representative immunoblot blot from cell lysates from wild-type and GCase-deficient cells starved for 5 h with Hanks’ balanced salt solution. (B) Quantification of tubulin normalized LC3 II levels (n = 4). (C) Representative immunoblot blot data from cell lysates from wild-type and GCase-deficient cells treated with 5 μm CCCP for indicated times. Cell lysates were immunoblotted with antibodies against GCase, p62, Mfn2, and tubulin. (D) Band intensities were normalized to tubulin and compared with 0 h wild-type sample. Data represent mean ± S.E.M. n = 3. (E, F) GCase-deficient cells exhibit defective extracellular secretion of α-synuclein and p62. Wild-type and GCase-deficient cells were treated with 300 nm Baf-A1 for the indicated times. Both intracellular fractions and extracellular media fractions from each timepoint were collected as described in the Materials and Methods section. Protein samples from intracellular lysates and extracellular media fractions were analyzed with immunoblot analysis using indicated antibodies. (E) Representative immunoblot data are shown. (F) Graph shows secretion indexes (extracellular level/intracellular level at 5 h) of α-synuclein, and p62 and sortilin. Data represent mean ± S.E.M. N=4 independent experiments. Two-tailed paired t-test, ***P < 0.001, compared with wild-type cells. (G, H) GCase-deficient cells show defective extracellular secretion of mature cathepsin-D. Wild-type and GCase-deficient cells were treated with 300 nm Baf-A1 for the indicated times. Both intracellular fractions and extracellular media fractions from each timepoint were collected. Protein samples from intracellular lysates and extracellular media fractions were analyzed with immunoblot analysis using cathepsin-D antibodies. (G) Representative immunoblot data are shown. (H) Graph shows secretion indexes (extracellular level/intracellular level at 5 h) of mature cathepsin-D (mCat-D) and immature cathepsin-D (iCat-D). Data represent mean ± S.E.M. N = 4 independent experiments. Two-tailed paired t-test, *P < 0.05, compared with wild-type cells. n.s. = not significant.

Figure 4.

Rab8a levels and distribution are altered by loss of GBA1. (A) Rab8a protein levels are elevated in GCase-deficient cells. Total cell lysates from wild-type and GCase-deficient cells were subjected with immunoblot analysis using indicated antibodies. Band intensities were normalized to tubulin, and compared with wild-type cells. Data represent mean ± S.E.M. N = 4, two-tailed unpaired t-test, ***P < 0.001, compared with wild-type cells. (B) Representative confocal images of Rab8a in wild-type and GCase-deficient cells. Cells were seeded onto PDL-coated glass coverslip with the cell density of ∼40 000 cells per coverslip. Three days later, cells were fixed with 4% formaldehyde in PBS for 12 min, and subsequently fixed with cold methanol for 5 min. The fixed cells were then stained for Rab8a and nuclear DNA. (C) Acid ceramidase expression in wild-type cells increases Rab8a protein levels. Wild-type HEK293-FT cells were transfected with either empty pCAG vector or pCAG-ASAH1 vector. Two days later, total cell lysates from transfected cells were subjected to immunoblot analysis using indicated antibodies. Band intensities were normalized to tubulin, and compared with empty pCAG-transfected cells. Data represent mean ± S.E.M. N = 6, two-tailed unpaired t-test, ***P < 0.001, compared with pCAG-transfected cells.

Figure 5.

Exogenous C18-Cer treatment rescues defective extracellular α-synuclein and p62 secretion and Rab8a mislocalization upon loss of GBA1. (A, B) Exogenous C18-Cer treatment corrects defective secretion of α-synuclein and p62 in GCase-deficient cells. GCase-deficient cells were pre-treated with either DMSO or 4 μm C18-Cer for ∼18 h. Cells were then treated with 300 nm Baf-A1 for the indicated times. Both intracellular fraction and extracellular media fraction from each timepoint were collected as described in the Materials and Method section. Protein samples from intracellular lysates and extracellular media fractions were analyzed with immunoblot analysis using indicated antibodies. (A) Representative immunoblot data are shown. (B) Graph shows secretion indexes (extracellular level/intracellular level at 5 h) of α-synuclein, and p62 and sortilin. Data represent mean ± S.E.M. N = 3, two-tailed paired t-test, **P < 0.01, *P < 0.05, compared with DMSO-treated cells. n.s = not significant. (C) Exogenous C18-Cer treatment reverts altered Rab8a localization in GCase-deficient cells. Wild-type and GCase-deficient cells were seeded onto poly-d-lysine coated glass coverslip with the cell density of ∼40 000 cells per coverslip. Two days later, cells were treated with either DMSO or 4 μm C18-Ceramide for ∼20 h. Cells were then fixed with 4% formaldehyde in PBS for 12 min, and subsequently fixed with cold methanol for 5 min. The fixed cells were then stained for Rab8a and nuclear DNA. Representative confocal images are shown.

Figure 6.

Acid ceramidase expression phenocopies loss of GBA1. (A, B) Protein levels of acid ceramidase are elevated in GCase-deficient cells. Total cell lysates from wild-type and GCase-deficient cell were subjected to immunoblot analysis using indicated antibodies. Immunoblot data is shown in (A). (B) Band intensities were normalized to tubulin, and compared with wild-type cells. Graph represents mean ± S.E.M; n = 3, two-tailed unpaired t-test, **P < 0.01; compared with wild-type cells. (C, D) Acid ceramidase expression phenocopies GCase-deficient cells. HEK293-FT cells were transiently transfected either with empty pCAG vector or pCAG-ASAH1 vector. Two days after transfection, total cell lysates were prepared with 2× SDS sample buffer, and subjected to immunoblot analysis using indicated antibodies. Representative immunoblot data are shown in (C). (D) Protein levels were normalized to tubulin, and compared with empty vector transfected cells. Graph represents mean ± S.EM. n = 6, two-tailed unpaired t-test, **P < 0.01; ***P < 0.001; compared with wild-type cells.

Figure 7.

Carmofur treatment reverts reduced ceramide levels and increase GluSph levels in GCase-deficient cells. Wild-type and GCase-deficient cells were treated with either DMSO or 7.5 μm carmofur for ∼20 h. After drug treatments, total lipids were extracted and measured as described in the Materials and Methods section. Lipid analysis results were expressed as lipid levels of picomoles (pmol)/three million cells. Levels of GluSph, GluCer species and dihydroceramide (A), and ceramide species (B) are shown. For each group, three independent samples were analyzed. Data represent mean ± S.E.M. Two-tailed unpaired t-test, ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 8.

Galactosylceramide (GalCer) levels are reduced in and mature GALC levels are elevated in GCase-deficient cells. (A) Reduced galactosylceramide (GalCer) levels in in GCase-deficient cells. Wild-type and GCase-deficient cells were treated with either DMSO or 7.5 μm carmofur for ∼20 h. After drug treatments, total lipids were extracted and measured as described in the Materials and Methods section. Lipid analysis results were expressed as lipid levels of picomoles (pmol)/three million cells. Levels of galactosylceramide species. For each group, three independent samples were analyzed. Data represent mean ± S.E.M. Two-tailed unpaired t-test, ***P < 0.001. (B) Mature GALC levels are elevated in GCase-deficient cells. Total cell lysates from wild-type and GCase-deficient cell were subjected to immunoblot analysis using antibody against GALC, GCase or tubulin. Immunoblot data is shown in the left. Band intensities of the mature form of galactosylceramidase (m-GALC) and the immature form of galactosylceramidase (i-GALC) were normalized to tubulin, and compared with wild-type cells. Graph represents mean ± S.E.M. n = 3, two-tailed unpaired t-test, **P < 0.01; compared with wild-type cells. n.s. = not significant. (C) Levels of Sph and Sph-1-P. For each group, three independent samples were analyzed. Data represent mean ± S.E.M. Two-tailed unpaired t-test, **P < 0.01, *P < 0.05.

Figure 9.

Carmofur reduces intracellular α-synuclein and autophagic substrate accumulation in GCase-deficient cells. (A) Carmofur reduces intracellular α-synuclein levels in a dose-dependent manner. Wild-type HEK cells were treated with DMSO or the indicated concentration of carmofur for 18 or 45 h. Total cell lysates were analyzed with immunoblotting with α-synuclein and tubulin antibody. Band intensities of α-synuclein were normalized to tubulin levels. (B, C) Carmofur reduces α-synuclein, Rab7 and autophagic substrates in GCase-deficient cells. Wild-type cells were treated with DMSO, and GCase-deficient cells were treated with either DMSO or 7.5 μm carmofur for ∼18 h. Total cell lysates were analyzed with immunoblotting using indicated antibodies. (B) Representative immunoblot data are shown. (C) Blot band intensities were normalized to tubulin, and compared with DMSO-treated wild-type cells. Graphs show normalized band intensities of intracellular α-synuclein (n = 4), LC3B-II (n = 4), p62 (n = 4), ubiquitinated proteins (n = 4), LAMP2 (n = 4) and Rab7 (n = 4). Data represent mean ± S.E.M. Two-tailed unpaired t-test, *P < 0.05, compared with DMSO-treated GCase-deficient cells. n.s = not significant.

Figure 10.

Carmofur promotes Baf-A1-induced α-synuclein and p62 extracellular secretion in GCase-deficient cells. (A, B) Carmofur in GCase-deficient cells ameliorates intracellular accumulation of α-synuclein and autophagic substrates (p62 and LC3II). Wild-type cells were pre-treated with DMSO for ∼18 h, and GCase-deficient cells were pre-treated with either DMSO or 7.5 μm carmofur for ∼18 h. Cells were then treated with 300 nm Baf-A1 for the indicated times. Total cell lysates were analyzed with immunoblot analysis using indicated antibodies. (A) Representative immunoblot data are shown. (B) Blot band intensities were normalized to tubulin, and compared with DMSO-treated GCase-deficient cells. Graphs show normalized band intensities of intracellular α-synuclein (N = 3), LC3B-II (N = 4) and p62 (N = 4). Data represent mean ± S.E.M. Two-tailed paired t-test, *P < 0.05; **P < 0.01; compared with DMSO-treated GCase-deficient cells. (C, D) Carmofur promotes extracellular secretion of α-synuclein and p62 in GCase-deficient cells. GCase-deficient cells were pre-treated with either DMSO or 7.5 μm carmofur for ∼18 h. Cells were then treated with 300 nm Baf-A1 for the indicated times. Both intracellular fractions and extracellular media fractions from each time point were collected as described in the Materials and Methods section. Protein samples from intracellular lysates and extracellular media fractions were analyzed with immunoblot analysis using indicated antibodies. (C) Representative immunoblot data are shown. (D) Graph shows secretion indexes (extracellular level/intracellular level at 5 h) of α-synuclein, and p62, and sortilin. N = 3 independent experiments. Data represent mean ± S.E.M. Two-tailed paired t-test, ***P < 0.001, *P < 0.05, compared with DMSO-treated cells. n.s = not significant.

Figure 11.

Carmofur reduces oxidized α-synuclein and ubiquitinated proteins in PD patient-derived dopaminergic neurons harboring a heterozygous GBA1-c.84dupG mutation or heterozygous GBA1-N370S mutation. (A) iPSCs derived from a PD patient carrying heterozygous GBA1-c.84dupG mutation were differentiated into dopaminergic neurons for 40 days. Dopaminergic neurons were treated with either DMSO or 7.5 μm carmofur for 2 days. Total cellular lysates were analyzed with immunoblotting using antibodies against ubiquitinated proteins (Ub), tubulin, TH, oxidized α-synuclein (Syn303), or total α-synuclein (C20). Saturated pixels in blot images are shown in red. (B) Band intensities were normalized to tubulin, and compared with DMSO-treated control. Data represent mean ± S.E.M. N = 6 from two independent differentiations into dopaminergic neurons, two-tailed unpaired t-test, **P < 0.01, *P < 0.05 compared with DMSO control. (C) iPSCs (clone PD267) derived from a PD patient carrying heterozygous GBA1-N370S mutation were differentiated into dopaminergic neurons for 40 days and treated with either DMSO or 7.5 μm carmofur for 2 days. Total lysates were analyzed with immunoblotting using antibodies against ubiquitinated proteins (Ub), tubulin, TH, oxidized α-synuclein (Syn303), or α-synuclein (C20). Saturated pixels in blot images are shown in red. (D) Band intensities were normalized to tubulin, and compared with DMSO-treated controls. Data represent mean ± S.E.M. N = 6, two-tailed unpaired t-test, *P < 0.05, compared with DMSO control. (E) Control neurons were differentiated for 40 days and treated with either DMSO or 7.5 μm carmofur for 2 days. Total lysates were analyzed with immunoblotting using antibodies against ubiquitinated proteins (Ub), tubulin, TH, oxidized α-synuclein (Syn303), or α-synuclein (C20). Saturated pixels in blot images are shown in red. (F) Band intensities were normalized to tubulin, and compared with DMSO-treated control. Data represent mean ± S.E.M. N = 6, two-tailed unpaired t-test, n.s. = not significant, compared with DMSO control.