Impact of Gba2 on neuronopathic Gaucher's disease and α-synuclein accumulation in medaka (Oryzias latipes)

Abstract

Homozygous mutations in the lysosomal glucocerebrosidase gene, GBA1, cause Gaucher's disease (GD), while heterozygous mutations in GBA1 are a strong risk factor for Parkinson's disease (PD), whose pathological hallmark is intraneuronal α-synuclein (asyn) aggregates. We previously reported that gba1 knockout (KO) medaka exhibited glucosylceramide accumulation and neuronopathic GD phenotypes, including short lifespan, the dopaminergic and noradrenergic neuronal cell loss, microglial activation, and swimming abnormality, with asyn accumulation in the brains. A recent study reported that deletion of GBA2, non-lysosomal glucocerebrosidase, in a non-neuronopathic GD mouse model rescued its phenotypes. In the present study, we generated gba2 KO medaka and examined the effect of Gba2 deletion on the phenotypes of gba1 KO medaka. The Gba2 deletion in gba1 KO medaka resulted in the exacerbation of glucosylceramide accumulation and no improvement in neuronopathic GD pathological changes, asyn accumulation, or swimming abnormalities. Meanwhile, though gba2 KO medaka did not show any apparent phenotypes, biochemical analysis revealed asyn accumulation in the brains. gba2 KO medaka showed a trend towards an increase in sphingolipids in the brains, which is one of the possible causes of asyn accumulation. In conclusion, this study demonstrated that the deletion of Gba2 does not rescue the pathological changes or behavioral abnormalities of gba1 KO medaka, and GBA2 represents a novel factor affecting asyn accumulation in the brains.

Keywords: GBA1; GBA2; Gaucher’s disease; Glucocerebrosidase; Parkinson’s disease; Sphingolipids; α-Synuclein.

Fig. 1

Phenotypes of gba2 KO medaka. a The Gba2 enzymatic activity in the brains of medaka. The Gba2 activity was almost eliminated in gba2 KO medaka and reduced by 42% in gba2^+/− medaka (n = 6 for each genotype). A one-way ANOVA with Tukey's multiple comparison test was performed. b A Kaplan–Meier plot showed no significant differences in the survival among genotypes (n = 24–33 for each genotype). A Log-rank (Mantel-Cox) test was performed. c Analysis of the spontaneous swimming movement. gba2 KO medaka showed a normal locomotor function compared with WT medaka at 6 mpf (n = 6 for each genotype). A two-tailed unpaired Student’s t-test was performed. The bottom images are representative movement tracks for each genotype. d Fertilization rate analysis showed no marked differences among genotypes at 6 pmf (n = 3 for each group). A one-way ANOVA with Tukey's multiple comparison test was performed. n.s. not significant, ***p < 0.001. The bars indicate the mean ± SEM

Fig. 2

Deletion of Gba2 in gba1 KO medaka did not rescue their phenotypes. a A Kaplan–Meier plot showed that the deletion of Gba2 in gba1 KO medaka shortened the lifespan (n = 15–41 for each genotype). A Log-rank (Mantel-Cox) test was performed. b Representative TH immunohistochemical staining of the middle diencephalic dopaminergic neurons in WT, gba1 KO, and gba1/gba2 DKO medaka at 3 mpf. c The number of TH-positive (TH⁺) neurons in the middle diencephalon at 3 mpf. The number of TH⁺ neurons was decreased in gba1/gba2 DKO medaka compared with WT medaka but did not markedly differ between gba1/gba2 DKO and gba1 KO medaka (n = 6–10 for each genotype). A one-way ANOVA with Tukey's multiple comparison test was performed. d The number of TH⁺ neurons in the locus coeruleus at 3 mpf. The number of TH⁺ neurons was decreased in gba1/gba2 DKO medaka compared with WT medaka but did not differ markedly between gba1/gba2 DKO and gba1 KO medaka (n = 4–10 for each genotype). A one-way ANOVA with Tukey's multiple comparison test was performed. e qRT-PCR analysis of TNF-α mRNA in the brains of 3 mpf medaka. TNF-α mRNA levels were normalized to β-actin mRNA. The TNF-α mRNA levels did not differ markedly between gba1/gba2 DKO and gba1 KO medaka (n = 6 for each genotype). Dunn’s multiple comparisons test was performed. f qRT-PCR analysis of ApoE-b mRNA in the brains of 3 mpf medaka. The ApoE-b mRNA levels were normalized to β-actin mRNA. The amount of ApoE-b mRNA was increased in gba1/gba2 DKO medaka compared with WT medaka but did not differ markedly between gba1/gba2 DKO and gba1 KO medaka (n = 6 for each genotype). A one-way ANOVA with Tukey's multiple comparison test was performed. n.s. not significant, ****p < 0.0001. The bars indicate mean ± SEM

Fig. 3

The amount of sphingolipids was altered in the brains of gba1 and gba2 mutant medaka. a The amount of GlcCer was increased in gba1/gba2 DKO medaka compared with gba1 KO medaka (n = 3 for each genotype). b The amount of GalCer did not differ markedly between gba1 KO and gba1/gba2 DKO medaka (n = 3 for each genotype). c The amount of ceramide did not differ among genotypes (n = 3 for each genotype). d The amount of sphingosine did not differ markedly between gba1 KO and gba2 KO medaka (n = 3 for each genotype). e The amount of GlcSph was increased in gba1/gba2 DKO compared with gba1 KO medaka (n = 3 for each genotype). f The amount of GalSph was increased in gba1 KO and gba1/gba2 DKO medaka compared with WT medaka (n = 3 for each genotype). A one-way ANOVA with Tukey's multiple comparison test was performed. N.D.: not detected (below the detection threshold). n.s. not significant, *p < 0.05, **p < 0.01 and ***p < 0.001. The bars indicate the mean ± SEM

Fig. 4

Gba1 and Gba2 deficiency caused asyn accumulation in the medaka brains. a, b Immunoblot analysis of asyn in the brains at 3 mpf. Asyn was increased in the gba1 KO, gba2 KO and gba1/gba2 DKO medaka compared with WT medaka (n = 6 for each genotype). NSE was used as a loading control. A one-way ANOVA Newman-Keuls multiple comparison test was performed. The bars indicate the mean ± SEM. n.s. not significant, *p < 0.05, c Asyn immunohistochemical staining of the diencephalon at 3 mpf. Asyn accumulations (arrowheads) were observed in gba1 KO and gba1/gba2 DKO medaka but not in gba2 KO medaka or WT medaka

Fig. 5

Autophagic dysfunction in gba1 KO and gba1/gba2 DKO medaka, but not in gba2 KO medaka. a, b Immunoblot analysis of p62 in the brains at 3 mpf. The expression of p62 was increased in the gba1/gba2 DKO and gba1 KO medaka but not in gba2 KO medaka (n = 6 for each genotype). a, c Immunoblot analysis of LC3-I and LC3-II in the brains at 3 mpf. The LC3-II/I ratio was increased in the gba1/gba2 DKO and gba1 KO medaka compared with the WT and gba2 KO medaka (n = 6 for each genotype). A one-way ANOVA with Tukey's multiple comparison test was performed. n.s. not significant, **p < 0.01, and ***p < 0.001. The bars indicate the mean ± SEM

Fig. 6

Proposed mechanisms of deletion of Gba1 and Gba2 leading to asyn accumulation. Deletion of Gba1 and Gba2 induces changes in the sphingolipid metabolism. These changes might induce conformational changes in asyn, resulting in asyn accumulation. Furthermore, the deletion of Gba1 causes autophagic dysfunction, resulting in the impairment of asyn degradation and leading to asyn accumulation. The solid and striped arrows indicate the downstream pathways of gba1 KO and gba2 KO, respectively