α-Synuclein accumulation and GBA deficiency due to L444P GBA mutation contributes to MPTP-induced parkinsonism

Abstract

Background: Mutations in glucocerebrosidase (GBA) cause Gaucher disease (GD) and increase the risk of developing Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB). Since both genetic and environmental factors contribute to the pathogenesis of sporadic PD, we investigated the susceptibility of nigrostriatal dopamine (DA) neurons in L444P GBA heterozygous knock-in (GBA ^+/L444P ) mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a selective dopaminergic mitochondrial neurotoxin.

Method: We used GBA ^+/L444P mice, α-synuclein knockout (SNCA ^-/- ) mice at 8 months of age, and adeno-associated virus (AAV)-human GBA overexpression to investigate the rescue effect of DA neuronal loss and susceptibility by MPTP. Mitochondrial morphology and functional assay were used to identify mitochondrial defects in GBA ^+/L444P mice. Motor behavioral test, immunohistochemistry, and HPLC were performed to measure dopaminergic degeneration by MPTP and investigate the relationship between GBA mutation and α-synuclein. Mitochondrial immunostaining, qPCR, and Western blot were also used to study the effects of α-synuclein knockout or GBA overexpression on MPTP-induced mitochondrial defects and susceptibility.

Results: L444P GBA heterozygous mutation reduced GBA protein levels, enzymatic activity and a concomitant accumulation of α-synuclein in the midbrain of GBA ^+/L444P mice. Furthermore, the deficiency resulted in defects in mitochondria of cortical neurons cultured from GBA ^+/L444P mice. Notably, treatment with MPTP resulted in a significant loss of dopaminergic neurons and striatal dopaminergic fibers in GBA ^+/L444P mice compared to wild type (WT) mice. Levels of striatal DA and its metabolites were more depleted in the striatum of GBA ^+/L444P mice. Behavioral deficits, neuroinflammation, and mitochondrial defects were more exacerbated in GBA ^+/L444P mice after MPTP treatment. Importantly, MPTP induced PD-like symptoms were significantly improved by knockout of α-synuclein or augmentation of GBA via AAV5-hGBA injection in both WT and GBA ^+/L444P mice. Intriguingly, the degree of reduction in MPTP induced PD-like symptoms in GBA ^+/L444P α-synuclein (SNCA) ^-/- mice was nearly equal to that in SNCA ^-/- mice after MPTP treatment.

Conclusion: Our results suggest that GBA deficiency due to L444P GBA heterozygous mutation and the accompanying accumulation of α-synuclein render DA neurons more susceptible to MPTP intoxication. Thus, GBA and α-synuclein play dual physiological roles in the survival of DA neurons in response to the mitochondrial dopaminergic neurotoxin, MPTP.

Keywords: GBA; MPTP; Mitochondrial dysfunction; Parkinson’s disease; α-synuclein.

Fig. 1

L444P GBA heterozygous mutation leads to GBA abnormalities, accumulation of α-synuclein, and mitochondrial defects both in vitro and in vivo. a GBA enzymatic activities were measured using lysosome-enriched fraction samples of ventral midbrain (VMB) in WT or GBA^+/L444P mice. GBA enzyme activity was normalized against GBA enzyme activity of WT mice. b VMB lysates from WT and GBA^+/L444P were immunoblotted with anti-GBA and α-synuclein antibodies. c GBA and α-synuclein expression levels were normalized against β-actin. a, c Error bars represent the mean ± S.E.M (n = six mice per group). d Representative transmission electron microscopy (TEM) images of mitochondria in the SNpc of WT and GBA^+/L444P mice. e Mitochondrial length and f aspect ratio (mitochondrial major axis over minor axis) were measured from littermate WT control (sixty-four mitochondria from seven cells) and GBA^+/L444P mice (fifty mitochondria from seven cells) group and represented as graph. g Representative images of MitoTracker positive structure in WT and GBA^+/L444P primary cortical neurons (10 DIV). h The intensity of MitoTracker positive structure of WT and GBA^+/L444P primary cultured neurons (n = three mice per group). i Mitochondrial length and j aspect ratio (mitochondrial major axis over minor axis) are shown (thirty mitochondria from three different images of each group). k Reactive oxygen species (ROS) levels were measured in primary cortical neurons of WT and GBA^+/L444P using CM-H2DCFDA. l Mitochondrial complex I enzyme activity in WT and GBA^+/L444P primary cortical neurons. m, n Oxygen consumption rate (OCR) was determined by Seahorse assay in WT and GBA^+/L444P primary cortical neurons. k-n Error bars represent the mean ± S.E.M (n = six mice per group). Student’s t-test or *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT group

Fig. 2

Effect of L444P GBA heterozygous mutation on the susceptibility of mice to MPTP-induced PD-like symptoms. a Representative photomicrographs from coronal mesencephalon sections containing TH-positive neurons in WT and GBA^+/L444P mice treated with saline or MPTP, respectively (scale bar, 500 μm). b Stereology counts of TH and c Nissl-positive neurons in the SNpc region. Unbiased stereologic counting was performed for the SNpc region.) Representative photomicrograph of striatal sections stained for TH immunoreactivity with low (scale bar, 100 μm) and high magnification (scale bar, 50 μm). e Quantification of dopaminergic fiber densities in the striatum using Image J software (NIH). a-e Error bars represent the mean ± S.E.M (n = ten mice per group). Striatal DA and metabolites levels were measured by HPLC-ECD. Levels of f DA, g DOPAC, and h HVA in the striatum from WT and GBA^+/L444P mice treated with saline or MPTP were measured. i DA turnover [(DOPAC + HVA/DA)] in the striatum was calculated. f-i Error bars represent the mean ± S.E.M (n = four mice per group). j Pole test was conducted on the sixth day post MPTP injection. Maximum time to climb down the pole was limited to 60 s. Error bars represent the mean ± S.E.M (n = fifteen mice per group). k Representative images of immunohistochemistry data for glial fibrillary acidic protein (GFAP, astrocyte specific marker) with low (scale bar, 100 μm) and high magnification (scale bar, 50 μm). l Intensities of GFAP positive signals in the SNpc of WT and GBA^+/L444P mice treated with saline or MPTP were quantified and shown as a graph. k, l Error bars represent the mean ± S.E.M (ten mice per group). Two-way ANOVA was used for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. *P < 0.05, **P < 0.01, ***P < 0.001 vs. MPTP-treated WT group, or saline-treated WT and GBA^+/L444P group. n.s: not significant

Fig. 3

Effect of α-synuclein on susceptibility of GBA^+/L444P mice to MPTP-induced PD-like symptoms. a Representative photomicrographs from coronal mesencephalon sections containing TH-positive neurons in littermate WT control, GBA^+/L444P, SNCA^−/−, and SNCA^−/-GBA^+/L444P mice treated with saline or MPTP, respectively (scale bar, 500 μm). b Stereology counts of TH, and c Nissl-positive neurons in the SNpc region. Unbiased stereologic counting was performed for the SNpc region. d Representative photomicrograph of striatal sections stained for TH immunoreactivity with low (scale bar, 100 μm) and high magnification (scale bar, 50 μm). e Quantification of dopaminergic fiber densities in the striatum using Image J software (NIH). a-e Error bars represent the mean ± S.E.M (n = four mice per group). f Pole test was conducted on the sixth day post MPTP injection. g Grip strength test was conducted on the sixth day post MPTP injection. Behavioral abnormalities and susceptibility in pole test and grip strength test induced by MPTP injection were ameliorated in SNCA^−/− and SNCA^−/-GBA^+/L444P mice. Maximum time to climb down the pole was limited to 60 s. f, g Error bars represent means ± S.E.M (n = six or seven mice per group). Two-way ANOVA was used for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. *P < 0.05, **P < 0.01, ***P < 0.001 vs. MPTP-treated WT group, or MPTP-treated WT and GBA^+/L444P group. n.s: not significant

Fig. 4

Deficiency of α-synuclein leads to decreased susceptibility of GBA^+/L444P mice to MPTP-induced mitochondrial defects. a, b Relative quantity of mitochondrial DNA (mtDNA) in the ventral midbrain was measured using two different mtDNA markers (CYTB and COX) normalized to GAPDH. c Representative immunofluorescent images of TH (green), SDHA (red), and DAPI (blue). White dot line is shown in TH neurons. d Intensities of SDHA positive signals in the SNpc of mice treated with saline or MPTP were quantified and shown as a graph. e Immunoblots of SDHA, PDH, VDAC, TH, α-synuclein, and GBA. VMB lysates were immunoblotted with anti-SDHA, anti-PDH, anti-VDAC, anti-TH, anti-α-synuclein, and anti-GBA antibodies. f SDHA, g PDH, h VDAC, i TH, j α-synuclein, and k GBA expression levels were normalized against β-actin. a-k Error bars represent the mean ± S.E.M (n = three mice per group). Two-way ANOVA was used for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. *P < 0.05, **P < 0.01, ***P < 0.001 vs. saline-treated WT or saline-treated GBA^+/L444P or MPTP-treated WT group. n.s: not significant

Fig. 5

Effect of GBA overexpression on susceptibility of GBA^+/L444P mice to MPTP-induced PD-like symptoms. a Representative photomicrographs from coronal mesencephalon sections containing TH-positive neurons in AAV-Con injected WT, AAV-GBA injected WT, AAV-Con injected GBA^+/L444P, and AAV-GBA injected GBA^+/L444P mice treated with saline or MPTP, respectively (scale bar, 500 μm). b Stereology counts of TH, and c Nissl-positive neurons in the SNpc region. Unbiased stereologic counting was performed in the SNpc region. d Representative photomicrograph of striatal sections stained for TH immunoreactivity with low (scale bar, 100 μm) and high magnification (scale bar, 50 μm). e Quantification of dopaminergic fiber densities in the striatum using Image J software (NIH). a-e Error bars represent means ± S.E.M (n = five mice per group). f Pole test was conducted on the sixth day post MPTP injection. g Grip strength test was conducted on the sixth day post MPTP injection. Behavioral abnormalities and susceptibility in pole test and grip strength test induced by MPTP injection were ameliorated in WT and GBA^+/L444P mice with AAV-GBA. f, g Error bars represent the mean ± S.E.M (six or seven mice per group for behavioral studies). Two-way ANOVA was used for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. *P < 0.05, **P < 0.01, ***P < 0.001 vs. MPTP-treated WT group with AAV-Con or MPTP-treated GBA^+/L444P group with AAV-Con. n.s: not significant