Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease

Abstract

Glucocerebrosidase 1 (GBA) mutations responsible for Gaucher disease (GD) are the most common genetic risk factor for Parkinson's disease (PD). Although the genetic link between GD and PD is well established, the underlying molecular mechanism(s) are not well understood. We propose that glucosylsphingosine, a sphingolipid accumulating in GD, mediates PD pathology in GBA-associated PD. We show that, whereas GD-related sphingolipids (glucosylceramide, glucosylsphingosine, sphingosine, sphingosine-1-phosphate) promote α-synuclein aggregation in vitro, glucosylsphingosine triggers the formation of oligomeric α-synuclein species capable of templating in human cells and neurons. Using newly generated GD/PD mouse lines of either sex [Gba mutant (N370S, L444P, KO) crossed to α-synuclein transgenics], we show that Gba mutations predispose to PD through a loss-of-function mechanism. We further demonstrate that glucosylsphingosine specifically accumulates in young GD/PD mouse brain. With age, brains exhibit glucosylceramide accumulations colocalized with α-synuclein pathology. These findings indicate that glucosylsphingosine promotes pathological aggregation of α-synuclein, increasing PD risk in GD patients and carriers.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is a prevalent neurodegenerative disorder in the aging population. Glucocerebrosidase 1 mutations, which cause Gaucher disease, are the most common genetic risk factor for PD, underscoring the importance of delineating the mechanisms underlying mutant GBA-associated PD. We show that lipids accumulating in Gaucher disease, especially glucosylsphingosine, play a key role in PD pathology in the brain. These data indicate that ASAH1 (acid ceramidase 1) and GBA2 (glucocerebrosidase 2) enzymes that mediate glucosylsphingosine production and metabolism are attractive therapeutic targets for treating mutant GBA-associated PD.

Keywords: GBA; glucosylsphingosine; α-synuclein.

Figure 1.

Sphingolipids accumulating in GD promote the acceleration of α-synuclein aggregation into different pathologic species. A, Spatial topography of GlcCer metabolism in GD with reference to subcellular compartmentalization of enzymes and sphingolipids. Red upward arrows indicate upstream and downstream sphingolipids that accumulate peripherally due to GBA deficiency. Green zone represents lysosome. B, Summary of circular dichroism results obtained by incubating α-synuclein with the indicated lipids for 5 d. For all circular dichroism data, experimental lipids were added at 34.5 μm and α-synuclein added at 13.8 μm [(total lipid PC + experimental lipid liposome): protein = 10:1]. C, Circular dichroism spectra of WT α-synuclein at day 0. Unfolded proteins have minima at 195 nm, whereas α-helical conformations have minima at 205 and 222 nm. D, Circular dichroism spectra of WT α-synuclein at day 5. Proteins with β-sheeted conformations have minima at 217 nm. E, Time course of β-sheet conformation acquisition of WT α-synuclein in presence of GD sphingolipids. The percentage β-sheet conformation was calculated using DichroWeb. p values relative to PC for GlcCer = 0.005, GlcSph = 0.016, Sph = 0.024, S1P = 0.028, and Cer = 0.353. F, Time course of α-helical conformation acquisition in the presence of GM1 and BMP. p values relative to PC for BMP = 0.003, GM1 = 6.778 × 10⁻⁵. G, Time course of β-sheet conformation acquisition of mutant A53T α-synuclein in presence of same sphingolipids. p values relative to PC for GlcCer = 0.320, GlcSph = 0.003, Sph = 0.002, S1P = 0.049, Cer = 0.262. F, Time course of β-sheet conformation acquisition of mutant A30P α-synuclein in presence of same sphingolipids. p values relative to PC for GlcCer = 0.369, GlcSph = 0.012, Sph = 0.003, S1P = 8.0319 × 10⁻⁴, Cer = 0.481. E–H, Data are mean ± SEM. n = 3 experiments per sample, *p < 0.05 versus PC (one-tailed Student's t test). **p < 0.01 versus PC (one-tailed Student's t test). ***p < 0.001 versus PC (one-tailed Student's t test).

Figure 2.

GlcSph and Sph promote oligomeric α-synuclein aggregates. A, Electron microscopy images of day 5 circular dichroism samples. Left panels, Images of monomeric and aggregated α-synuclein are shown for reference. Scale bar, 50 nm. B, AFM images of α-synuclein aggregated with Gaucher sphingolipids. Scale bar, 500 nm. Height of images = 50 nm. N = 3 independent experiments. C, Frequency distribution of size of α-synuclein species formed by aggregation in vitro as analyzed by AFM. Control conditions are in the absence of lipids, but aggregated for longer durations. The GD sphingolipids used are denoted. There are three images/condition. D, Median length of α-synuclein species formed.

Figure 3.

Oligomeric GlcSph and Sph α-synuclein species promote α-synuclein seeding. A, Intracellular α-synuclein aggregation assay in HEK293T cells, in which α-synuclein species aggregated in the presence of various lipids were added to cell media with a bioporter. Arrow indicates aggregated α-synuclein-GFP. Quantification shown for percentage of HEK293T cells with intracellular aggregates when templated with GD-related sphingolipid preformed α-synuclein species. N = 3 experiments with ≥241 cells per condition were assayed. p values relative to PC: Cer = 1.000, GM1 = 0.423, GlcCer = 0.301, GlcSph = 0.014, Sph = 4.900 × 10⁻⁴, S1P = 0.298. B, Intracellular α-synuclein aggregation assay as performed in human neurons. Quantification of relative aggregation, higher molecular weight species normalized to monomeric α-synuclein. N = 2 experiments. p values relative to PC: Cer = 0.894, GM1 = 0.544, GlcCer = 0.910, GlcSph = 0.021, Sph = 5.500 × 10⁻⁴, S1P = 0.867. *p < 0.05 (two-tailed Student's t test). ***p < 0.001 (two-tailed Student's t test).

Figure 4.

Generation and characterization of GD/PD mice. A, Schematic depicting design of Gba KO and mutant mouse lines: gray represents WT; black represents KO; green represents mutant. Ai, Homozygous Gba KO mice were produced through insertion of a Neo cassette between exons 7 and 8. Gba KO was rescued in skin using Cre recombinase, preventing lethality in these lines. Aii, Viable Gba mutant mice (Gba^N370S/KO and Gba^L444P/KO) were generated with one copy of Gba N370S or L444P and one copy of Gba KO with a rescue in skin, as noted above, to create “homozygous” mutants. B, Summary of all 14 genotypes assessed. C, Positive PCR-based genotyping of all 7 GD mouse lines. D, Quantitative Western blotting of GCase1 protein levels in brains of GD/PD mice. N = 2 experiments. p values versus WT: L444P/WT = 0.127, N370S/WT = 0.626, KO/WT = 0.230, L444P/KO = 0.002, N370S/KO = 0.003, KO/KO = 0.001. p values versus N370S/KO: L444P/KO = 0.010, KO/KO = 0.001. E, GCase1 enzymatic activity. Heterozygous Gba^L444P/WT, Gba^N370S/WT, and Gba^KO/WT were grouped as Gba ^/WT, whereas homozygous Gba^L444P/KO, Gba^N370S/KO, and Gba^KO/KO were grouped as Gba ^/KO. N ≥ 17/genotype. p values versus WT: Het = 0.008, KO = 3.658 × 10⁻¹³; p values versus Het: KO = 2.577 × 10⁻¹⁹. F, Relative GCase1 activity in young (3-months) and old (12-months) brains. N ≥ 6 per genotype. p values versus corresponding young cohort: WT = 0.170, Het = 0.003, KO = 0.753. G, GD mice are viable and exhibit normal weights at 4 months of age. D–F, *p < 0.05 (two-tailed Student's t test). **p < 0.01 (two-tailed Student's t test). ***p < 0.001 (two-tailed Student's t test).

Figure 5.

Characterization of GD/PD mice. A, Representative images of motor neurons in the ventral horn of L3 spinal cords. All PD lines showed loss of motor neurons in the lumbar spinal cord, consistent with previous literature (Chandra et al., 2005). B, Quantification of motor neuron loss in Gba ^WT/WT, heterozygous Gba ^/WT, and homozygous Gba ^/KO with and without SNCA^Tg. Gba^L444P/WT, Gba^N370S/WT, and Gba^KO/WT were grouped as Gba ^/WT, whereas Gba^L444P/KO, Gba^N370S/KO, and Gba^KO/KO were grouped as Gba ^/KO. N and p values relative to WT/WT: WT/WT: N = 3; /WT: N = 2, p = 0.489; /KO: N = 5, p = 0.144; SNCA^Tg: N = 2, p = 0.002; /WT SNCA^Tg: N = 2, p = 0.009; /KO SNCA^Tg: N = 3, p = 0.001. C, Representative images of microglial staining in T10 spinal cords. All PD lines showed significant recruitment of microglia relative to WT. D, Quantification of microglia levels in Gba ^WT/WT, Gba ^/WT, Gba ^/KO, SNCA^Tg, and Gba ^/KOSNCA^Tg. N and p values relative to WT/WT: WT/WT: N = 5; /WT: N = 2, p = 0.315; /KO: N = 1, no p value; SNCA^Tg: N = 2, p = 0.005; /KO SNCA^Tg: N = 3, p = 0.001. B, D, **p < 0.01, relative to Gba^WT/WT (two-tailed Student's t test). Hanging grip test was used as an indicator of declining motor function in (E) Gba L444P, (F) Gba N370S, and (G) Gba KO lines. All PD lines exhibit motor deficits by 10 months. Both sexes were used; N is noted in parentheses after each genotype. H, Graph of months elapsed between onset of motor phenotype measured by the hanging grip test and death. GD/PD mice dying earlier than 14 months (GD/PD Early Death) show accelerated decline in motor function relative to PD mice. p value relative to PD = 1.64 × 10⁻⁴. **p < 0.001 (two-tailed Student's t test).

Figure 6.

GlcSph accumulates early in mouse brain: A, GlcCer; B, GlcSph; C, Sph; D, S1P levels in GD/PD brains. N = 2–6 per genotypes, except Gba^KO/KO (N = 1); age of mice = 2–3 months. p value relative to WT/WT for GlcSph levels: L444P/WT = 0.234, N370S/WT = 0.313, KO/WT = 0.608, L444P/KO = 3.350 × 10⁻⁴, N370S/KO = 0.002. *p < 0.05. **p < 0.01. ***p < 0.001. E, AFM images of α-synuclein incubated with 0.1, 1, and 10 μm GlcSph with PC control on day 0 and 7. Scale bars, 1 μm. F, Percentage β-sheeted content of α-synuclein in the presence of 0.1, 1, and 10 μm GlcSph on day 0 and day 7. N = 3 for all samples. p value relative to PC: GlcSph-0.1 = 0.243, GlcSph-1 = 0.472, GlcSph-10 = 0.004. **p < 0.01 (two-tailed Student's t test). G, Subcellular fractionation showing lysosomes on the top fraction (F1) and mitochondria on the bottom fraction between 27% and 23% density interfaces (F2). Two smaller bands are seen between F1 and F2 (not taken). H, Western blot shows enrichment of lysosomes in F1. Cathepsin D, ATP6V1a, and LAMP1 were used as lysosomal markers; Cathepsin D and ATP6V1a can be seen in the homogenate, supernatant, and lysosomal fraction, but not in the mitochondrial fraction. LAMP1 can be seen predominantly in the lysosomal fraction. I, Relative protein levels in lysosomal fractions were determined by BCA. GBA KO lysosomes (11.38 mg/ml) were found to have almost 3 times as much protein as WT lysosomes (4.27 mg/ml), a likely indicator of expected lysosomal enlargement and dysfunction in the GBA KO mice. p value /KO relative to WT = 0.031. *p < 0.05 (two-tailed Student's t test). J, Dot blot shows relative GlcCer levels using a GlcCer antibody (Glycobiotech). Blotted GlcCer lipid shows that the dot blot method is robust and quantitative, with a standard curve with R² = 0.99. K, GlcCer levels from brain homogenate, lysosomal fraction, and mitochondrial fraction were analyzed using the dot blot method in J. No difference between WT and GBA KO was found in any of the fractions, including total brain homogenates confirming the lipidomic results in A. However, there was a clear enrichment of GlcCer in the mitochondrial fraction relative to the others.

Figure 7.

GD/PD mice exhibit α-synuclein pathology and GlcCer accumulation. A, Histograms of age of death of SNCA^Tg mice (N = 12), heterozygous Gba ^/WTSNCA^Tg (N = 36), and homozygous Gba ^/KOSNCA^Tg (N = 18). Gaussian curve fitted to SNCA^Tg histogram superimposed over all three histograms, showing differences in distributions; 1, 2, and 3 SDs from the mean shown through progressively darker shading under Gaussian curve. B, Quantification of GlcCer levels in the CA3 region of hippocampus in homozygous Gba ^/KOSNCA^Tg brain of early death and anticipated death mice (N = 3 mice per group). N and p values relative to anticipated death: anticipated death: N = 3; early death: N = 3, p = 0.013. C, Quantification of GlcCer levels in the CA3 region of hippocampus in GD/PD mice cohorts. Values normalized to 1 for Gba^WT/WT. N and p values relative to WT/WT: WT/WT: N = 3; SNCA^Tg: N = 3, p = 0.624; /WT SNCA^Tg: N = 4, p = 0.783; /KO SNCA^Tg: N = 3, p = 0.010. D, Relative Ser129 phosphorylated α-synuclein levels in the CA3 region of the hippocampus in the same brains. N and p values relative to WT/WT: WT/WT: N = 3; SNCA^Tg: N = 3, p = 0.010; /WT SNCA^Tg: N = 4, p = 0.036; /KO SNCA^Tg: N = 3, p = 0.010. E, Correlation of GlcCer and Ser129 phosphorylation α-synuclein levels across the different genotypes. r² = 0.827, Pearson's product moment-correlation p = 1.618 × 10⁻⁵. N = 3 or 4 per genotype; age of mice = 8–11 months. F, Representative brain sections stained with antibodies to GlcCer and Ser129 phosphorylated α-synuclein. Scale bar, 250 μm. G, Western blot of soluble fraction of mouse brain homogenate following differential detergent extract in age-matched Gba^WT/WT, SNCA^Tg, and Gba^/KOSNCA^Tg exhibiting early death, probing for phosphorylated α-synuclein S129. H, Quantification of G, focusing on expected bands at 16 and 37 kDa. 16 kDa: N and p values versus WT/WT: SNCA^Tg: N = 3, p = 4.120 × 10⁻⁴; /KO SNCA^Tg: N = 3, p = 0.008. 37 kDa: N and p values versus WT/WT: SNCA^Tg: N = 3, p = 0.090; /KO SNCA^Tg: N = 3, p = 0.002. I, Dot blot of soluble fraction of mouse brain homogenate following differential detergent extract in age-matched Gba^WT/WT, SNCA^Tg, and Gba^/KOSNCA^Tg exhibiting early death, using anti-oligomer antibody A11. J, Quantification of ELISA assay probing for the presence of aggregated α-synuclein in total brain homogenate from age-matched Gba^WT/WT, SNCA^Tg, and Gba^/KOSNCA^Tg exhibiting early death. N and p values relative to WT/WT: WT/WT: N = 3; SNCA^Tg: N = 3, p = 0.016; /KO SNCA^Tg: N = 3, p = 0.014; p values versus SNCA^Tg: /KO SNCA^Tg = 0.034. B, C, Two-tailed Student's t test. D, H, J, One-tailed Student's t test. *p < 0.05. **p < 0.01.

Figure 8.

GBA2 and acid ceramidase levels in GD mice. A, Gba2 activity levels relative to Gba^WT/WT. Heterozygous Gba^L444P/WT, Gba^N370S/WT, and Gba^KO/WT were grouped as Gba ^/WT, whereas homozygous Gba^L444P/KO, Gba^N370S/KO, and Gba^KO/KO were grouped as Gba ^/KO. Activity is shown for both young (3-month) and old (12-month) mice. N and p values relative to young cohort: WT/WT: N = 8 3-month, 9 1-year mice, p = 0.507; /WT: N = 26 3-month, 9 1-year mice, p = 0.038; /KO: N = 16 3-month, 8 1-year mice, p = 0.398. *p < 0.05 (two-tailed Student's t test). B, Gba2 protein levels in Gba^WT/WT, Gba^L444P/WT, Gba^N370S/WT, Gba^KO/WT, Gba^L444P/KO, Gba^N370S/KO, and Gba^KO/KO in mouse brain. N = 2 per genotype. Age = 3-month mice. C, Gba2 protein levels of Gba^WT/WT, Gba ^/WT, and Gba ^/KO. Protein levels are shown for both young (3-month) and old (12-month) mice. N = 3 per genotype. p values relative to young cohort: WT/WT = 0.193, /WT = 0.021, /KO = 0.991. *p < 0.05 (two-tailed Student's t test). D, Acid ceramidase levels normalized to actin shown for all GD genotypes, determined via Western blot. N = 2 per genotype. Age = 3-month-old mice. E, Acid ceramidase levels of Gba^WT/WT, Gba ^/WT, and Gba ^/KO. Protein levels are shown for both young (3-month) and old (12-month) mice. N = 3 per genotype.

Figure 9.

Model of how GD-related sphingolipids impact α-synuclein pathology. Deletion or GD mutations in GBA leads to accumulation of GlcSph in the cytosol of neurons. GlcSph directly interacts with α-synuclein to promote its aggregation into distinct pathogenic oligomeric species. These pathogenic species further template intracellular α-synuclein aggregation and may have the capacity to spread to neighboring neurons. With age, GlcCer also accumulates; and there is a decrement of Gba2 and lysosomal enzymes, exacerbating α-synuclein pathology and proteostasis, leading to death of neurons.