Limits...
Viable neuronopathic Gaucher disease model in Medaka (Oryzias latipes) displays axonal accumulation of alpha-synuclein.

Uemura N, Koike M, Ansai S, Kinoshita M, Ishikawa-Fujiwara T, Matsui H, Naruse K, Sakamoto N, Uchiyama Y, Todo T, Takeda S, Yamakado H, Takahashi R - PLoS Genet. (2015)

Bottom Line: Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease.Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD).Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

ABSTRACT
Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.

No MeSH data available.


Related in: MedlinePlus

Generation of GBA nonsense mutant medaka.(A) Upper panel: DNA and amino acid sequences of GBA nonsense mutant medaka. Lower panels: Sequence data for each genotype. A = green, T = red, G = black, and C = blue. (B) Relative GCase activity in medaka brains (n = 10). (C) Abnormal posture (‘bent spine’) in GBA-/- medaka at 3 months. (D) Survival curves for each genotype. PFD, post-fertilization day. (E) Quantification of glucocerebroside and galactocerebroside in medaka brains at 3 months with SFC/MS/MS (n = 3–4). For all analyses, data are the mean ± standard error of the mean (SEM). n.s. means not significant.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4383526&req=5

pgen.1005065.g001: Generation of GBA nonsense mutant medaka.(A) Upper panel: DNA and amino acid sequences of GBA nonsense mutant medaka. Lower panels: Sequence data for each genotype. A = green, T = red, G = black, and C = blue. (B) Relative GCase activity in medaka brains (n = 10). (C) Abnormal posture (‘bent spine’) in GBA-/- medaka at 3 months. (D) Survival curves for each genotype. PFD, post-fertilization day. (E) Quantification of glucocerebroside and galactocerebroside in medaka brains at 3 months with SFC/MS/MS (n = 3–4). For all analyses, data are the mean ± standard error of the mean (SEM). n.s. means not significant.

Mentions: We generated GBA nonsense mutant medaka to investigate the mechanisms by which GBA mutation leads to PD. To identify medaka GBA orthologs, we searched the medaka genome database (http://www.ensembl.org/Oryzias_latipes/Info/Index) with the basic local alignment search tool and found only one ortholog of human GBA. We cloned the medaka GBA with reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends and found that this gene has 11 exons encoding a protein of 522 amino acids. The amino acid sequence of medaka GBA showed 53% homology to that of human GBA (S1 Fig). Next, we screened a targeting-induced local lesions in genome (TILLING) library for medaka GBA using a high-resolution melting assay[17,24]. We identified a nonsense mutant (W337X) and generated the nonsense mutant medaka by in vitro fertilization (Fig. 1A). We examined GCase activity in the brains of GBA mutants after crossing with heterozygous mutants. GBAW337X/W337X (GBA-/-) medaka showed complete deficiency in GCase activity, and GBAWT/W337X (GBA+/-) medaka showed a decrease in GCase activity of about 50% compared to wild-type (GBA+/+) medaka (Fig. 1B). Although humans and mice lacking GCase activity die soon after birth[4,25,26], GBA-/- medaka survived for more than 3 months, enabling us to analyze the pathological progression (Fig. 1D). GBA-/- medaka showed abnormal rotating swimming movement at 2 months (S1, S2 Movie) and the abnormal appearance of a bent spine at 3 months (Fig. 1C). High levels of glucocerebroside accumulated in the brains of GBA-/- medaka (Fig. 1E), whereas the amount of galactocerebroside, an isomer of glucocerebroside, was not changed. Glucocerebroside with C18 fatty acids was the most dominant type in the brains of GBA-/- medaka (S2 Fig), an observation that is consistent with the neuronopathic GD mouse model[27].


Viable neuronopathic Gaucher disease model in Medaka (Oryzias latipes) displays axonal accumulation of alpha-synuclein.

Uemura N, Koike M, Ansai S, Kinoshita M, Ishikawa-Fujiwara T, Matsui H, Naruse K, Sakamoto N, Uchiyama Y, Todo T, Takeda S, Yamakado H, Takahashi R - PLoS Genet. (2015)

Generation of GBA nonsense mutant medaka.(A) Upper panel: DNA and amino acid sequences of GBA nonsense mutant medaka. Lower panels: Sequence data for each genotype. A = green, T = red, G = black, and C = blue. (B) Relative GCase activity in medaka brains (n = 10). (C) Abnormal posture (‘bent spine’) in GBA-/- medaka at 3 months. (D) Survival curves for each genotype. PFD, post-fertilization day. (E) Quantification of glucocerebroside and galactocerebroside in medaka brains at 3 months with SFC/MS/MS (n = 3–4). For all analyses, data are the mean ± standard error of the mean (SEM). n.s. means not significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383526&req=5

pgen.1005065.g001: Generation of GBA nonsense mutant medaka.(A) Upper panel: DNA and amino acid sequences of GBA nonsense mutant medaka. Lower panels: Sequence data for each genotype. A = green, T = red, G = black, and C = blue. (B) Relative GCase activity in medaka brains (n = 10). (C) Abnormal posture (‘bent spine’) in GBA-/- medaka at 3 months. (D) Survival curves for each genotype. PFD, post-fertilization day. (E) Quantification of glucocerebroside and galactocerebroside in medaka brains at 3 months with SFC/MS/MS (n = 3–4). For all analyses, data are the mean ± standard error of the mean (SEM). n.s. means not significant.
Mentions: We generated GBA nonsense mutant medaka to investigate the mechanisms by which GBA mutation leads to PD. To identify medaka GBA orthologs, we searched the medaka genome database (http://www.ensembl.org/Oryzias_latipes/Info/Index) with the basic local alignment search tool and found only one ortholog of human GBA. We cloned the medaka GBA with reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends and found that this gene has 11 exons encoding a protein of 522 amino acids. The amino acid sequence of medaka GBA showed 53% homology to that of human GBA (S1 Fig). Next, we screened a targeting-induced local lesions in genome (TILLING) library for medaka GBA using a high-resolution melting assay[17,24]. We identified a nonsense mutant (W337X) and generated the nonsense mutant medaka by in vitro fertilization (Fig. 1A). We examined GCase activity in the brains of GBA mutants after crossing with heterozygous mutants. GBAW337X/W337X (GBA-/-) medaka showed complete deficiency in GCase activity, and GBAWT/W337X (GBA+/-) medaka showed a decrease in GCase activity of about 50% compared to wild-type (GBA+/+) medaka (Fig. 1B). Although humans and mice lacking GCase activity die soon after birth[4,25,26], GBA-/- medaka survived for more than 3 months, enabling us to analyze the pathological progression (Fig. 1D). GBA-/- medaka showed abnormal rotating swimming movement at 2 months (S1, S2 Movie) and the abnormal appearance of a bent spine at 3 months (Fig. 1C). High levels of glucocerebroside accumulated in the brains of GBA-/- medaka (Fig. 1E), whereas the amount of galactocerebroside, an isomer of glucocerebroside, was not changed. Glucocerebroside with C18 fatty acids was the most dominant type in the brains of GBA-/- medaka (S2 Fig), an observation that is consistent with the neuronopathic GD mouse model[27].

Bottom Line: Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease.Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD).Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

ABSTRACT
Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.

No MeSH data available.


Related in: MedlinePlus