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01.12.2018 | Research article | Ausgabe 1/2018 Open Access

Molecular Neurodegeneration 1/2018

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

Zeitschrift:
Molecular Neurodegeneration > Ausgabe 1/2018
Autoren:
Seung Pil Yun, Donghoon Kim, Sangjune Kim, SangMin Kim, Senthilkumar S. Karuppagounder, Seung-Hwan Kwon, Saebom Lee, Tae-In Kam, Suhyun Lee, Sangwoo Ham, Jae Hong Park, Valina L. Dawson, Ted M. Dawson, Yunjong Lee, Han Seok Ko
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s13024-017-0233-5) contains supplementary material, which is available to authorized users.

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.
Zusatzmaterial
Additional file 1: Figure S1. Activity of 20S proteasome was measured in WT and GBA +/L444P primary cultured neurons (n = three per each group). Student’s t-test was used to for statistical analysis. *P < 0.05, ***P < 0.001. (PDF 80 kb)
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Additional file 2: Figure S2. The schematic diagram depicts the time schedule of intervention and analyses performed. Numerals represent the days experiments were conducted. On 1th day we injected saline or MPTP (2 h interval, 4 times, 20 mg/kg free base) in 8 months WT, GBA +/L444P , SNCA−/−, GBA +/L444P SNCA−/− mice. On 6th day, the pole and grip strength were performed. On 7th day, mice were sacrificed for indicated studies. Following are animal numbers used for these studies: behavioral (n = 10), neurochemical (n = 5), immunohistochemistry (n = 5), and biochemical studies (n = 4) per each treatment group. (PDF 110 kb)
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Additional file 3: Figure S3. Effect of α-synuclein deficiency and GBA overexpression on susceptibility of GBA +/L444P mice to MPTP-induced gliosis. a, c Representative images of immunohistochemistry data for GFAP with low (scale bar, 500 μm) and high magnification (scale bar, 50 μm). b, d Intensities of GFAP positive signals in the SNpc of mice treated with saline or MPTP were quantified and shown as a graph. Error bars represent the mean ± S.E.M (n = four mice per group). Two-way ANOVA was used to test for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. *P < 0.05, ***P < 0.001 vs. MPTP-treated WT or GBA +/L444P with AAV5-Con or MPTP-treated GBA +/L444P with AAV5- Con. n.s: not significant. (PDF 3329 kb)
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Additional file 4: Figure S4. MPTP-induced α-synuclein oligomer. a Filter trap soluble-α- synuclein oligomer species assay from ventral midbrain of WT and GBA +/L444P mice with or without MPTP. Error bars represent the mean ± S.E.M. (n = four mice per group). Two-way ANOVA was used for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. ***P < 0.001 vs. MPTP-treated WT. N.D: not detection. b Filter trap insoluble-α-synuclein filament species assay from ventral midbrain of WT and GBA +/L444P mice with or without MPTP. α-Synuclein preformed fibril (PFF) is positive control. (PDF 574 kb)
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Additional file 5: Figure S5. Lysosomal calcium concentration. SH-SY5Y cells were transfected with indicated constructs for 48 h. The cells were then labeled with CellLight® Lysosome-RFP (LAMP1; red) and loaded with 0.1 mg/ml of lysosomal calcium indicator Oregon Green BAPTA-1 dextran (BAPTA-1; green) for 12 h. The Oregon Green BAPTA-1 signals that co-localized to lysosome (LAMP-1; red) were used for measuring lysosomal calcium concentration. [Ca2+]lys was measured using ratiometric methods via confocal microscopy. Two-way ANOVA was used to test for statistical analysis followed by post-hoc Bonferroni test for multiple group comparison. ***P < 0.001. (PDF 1122 kb)
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Additional file 6: Figure S6. The schematic diagram depicts the time AAV5 injection schedule of intervention and analyses performed. Numerals represent the days’ experiments were conducted. For stereotaxic injection of AAV5-GFP and AAV5-hGBA, 8-month-old mice of indicated genotypes were anesthetized with pentobarbital (60 mg/kg). An injection cannula (26.5 gauge) was stereotaxically applied to the substantia nigra pars compacta (SNpc). After AAV5 stereotaxic injection for 1 month, we injected saline or MPTP (2 h interval, 4 times, 20 mg/kg free base) in WT, and GBA +/L444P mice. On 6th day the pole and grip strength test were performed. On 7th day, mice were sacrificed for indicated studies. Following are animal numbers used for these studies: behavioral (n = 6-8), neurochemical (n = 4), immunohistochemistry (n = 4), and biochemical studies (n = 4) per each treatment group. (PDF 126 kb)
13024_2017_233_MOESM6_ESM.pdf
Additional file 7: Figure S7. Neuron specific AAV5-hGBA overexpression in SNpc region. a Vector design of AAV5 hGBA. b Representative immunofluorescent images of GFP (green, injection marker), GFAP (red, astrocyte, non-neuronal marker), Tuj1 (Violet, neuronal marker), and DAPI (Blue). (PDF 4671 kb)
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Additional file 8: Figure S8. GBA overexpression inhibits MPTP-reduced mitochondrial protein level in GBA +/L444P mice. a Immunoblots of SDHA, PDH, VDAC, TH, GBA, and α-synuclein from AAV5-Con injected WT, AAV5-hGBA injected WT, AAV5-Con injected heterozygous, and AAV5-hGBA injected heterozygous mice treated with saline or MPTP. VMB lysates were immunoblotted with anti-SDHA, anti-PDH, anti-VDAC, anti-TH, and anti-GBA antibodies. b SDHA, c PDH, d VDAC, e TH, f GBA, and g α-synuclein expression levels were normalized against β-actin. 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 with AAV-Con or saline-treated GBA +/L444P with AAV5-Con or MPTP-treated WT with AAV5-Con or MPTP-treated GBA +/L444P with AAV5-Con group. (PDF 683 kb)
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Additional file 9: Figure S9. L444P GBA heterozygous mutation leads to autophagy abnormality. a, b Effect of rapamycin on GBA +/L444P expression. Primary cortical neurons were cultured from WT and GBA +/L444P mice. After 10 DIV, primary neurons were treated with 20 nM of rapamycin (mTOR inhibitor for inducing autophagy) for 24 h. a Representative Immunoblots of GBA. b GBA expression levels were normalized against β-actin and the error bars represent the mean ± S.E.M (n = four per group). c Immunoblots of Autophagy marker proteins SQSTM1/p62, and LC3A/B-I/II. VMB lysates were immunoblotted with anti-SQSTM1/p62, and anti-LC3A/B-I/II antibodies. d SQSTM1/p62, and e LC3A/B-II expression levels were normalized against β-actin. Error bars represent the mean ± S.E.M (n = four 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. n.s: not significant. (PDF 338 kb)
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