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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: 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.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.

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

Pathological analyses of GBA-/- medaka.(A) Schematic of a lateral view of the medaka brain. Each number signifies a part of the brain. 1: telencephalon, 2: optic tectum, 3: diencephalon, 4: cerebellum, 5: medulla oblongata. The brain sections used for pathological analyses in the present study are illustrated by the vertical line. (B) Hematoxylin and eosin staining. Abnormal cells observed in the periventricular gray zone of the optic tectum (arrowhead) in GBA-/- medaka at 3 months. Scale bars, 50 μm. Right panel: High-magnification image showing abnormal cells with large vacuoles (arrows). Scale bar, 5 μm. (C) Transmission electron micrographs showing abnormal macrophage-like cells. Left panel: A whole-cell image of an abnormal macrophage-like cell. Dashed lines outline a whole abnormal cell. N, Nucleus. Scale bar, 2 μm. Right panel: High-magnification image of filamentous structures in vacuoles. Scale bar, 500 nm. (D) Klüver-Barrera (KB) staining, ssDNA immunohistochemistry, APOE in situ hybridization, and GFAP immunohistochemistry in the diencephalon. Commissura posterior with decreased Luxol fast blue staining intensity (arrowhead) and ssDNA-positive dead cells (arrows) in GBA-/- medaka. APOE in situ hybridization revealed proliferating activated microglia in GBA-/- medaka. The staining intensity and area of GFAP were not changed in GBA-/- medaka. Scale bars, 50 μm. (E) Western blot analysis of GFAP and β-actin. The expression level of GFAP was not changed among genotypes. (F) Number of TH-positive neurons in the middle diencephalon, number of TH-positive neurons in the locus coeruleus, and number of TPH-positive neurons in the superior raphe at 2 and 3 months. In GBA-/- medaka, progressive neuronal loss was observed in all types of neurons (n = 4–6, *p < 0.05, ***p < 0.001). (G) Western blot analysis of TH and β-actin (n = 7, *p < 0.05, **p < 0.01). For all analyses, data are the mean ± SEM.
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pgen.1005065.g002: Pathological analyses of GBA-/- medaka.(A) Schematic of a lateral view of the medaka brain. Each number signifies a part of the brain. 1: telencephalon, 2: optic tectum, 3: diencephalon, 4: cerebellum, 5: medulla oblongata. The brain sections used for pathological analyses in the present study are illustrated by the vertical line. (B) Hematoxylin and eosin staining. Abnormal cells observed in the periventricular gray zone of the optic tectum (arrowhead) in GBA-/- medaka at 3 months. Scale bars, 50 μm. Right panel: High-magnification image showing abnormal cells with large vacuoles (arrows). Scale bar, 5 μm. (C) Transmission electron micrographs showing abnormal macrophage-like cells. Left panel: A whole-cell image of an abnormal macrophage-like cell. Dashed lines outline a whole abnormal cell. N, Nucleus. Scale bar, 2 μm. Right panel: High-magnification image of filamentous structures in vacuoles. Scale bar, 500 nm. (D) Klüver-Barrera (KB) staining, ssDNA immunohistochemistry, APOE in situ hybridization, and GFAP immunohistochemistry in the diencephalon. Commissura posterior with decreased Luxol fast blue staining intensity (arrowhead) and ssDNA-positive dead cells (arrows) in GBA-/- medaka. APOE in situ hybridization revealed proliferating activated microglia in GBA-/- medaka. The staining intensity and area of GFAP were not changed in GBA-/- medaka. Scale bars, 50 μm. (E) Western blot analysis of GFAP and β-actin. The expression level of GFAP was not changed among genotypes. (F) Number of TH-positive neurons in the middle diencephalon, number of TH-positive neurons in the locus coeruleus, and number of TPH-positive neurons in the superior raphe at 2 and 3 months. In GBA-/- medaka, progressive neuronal loss was observed in all types of neurons (n = 4–6, *p < 0.05, ***p < 0.001). (G) Western blot analysis of TH and β-actin (n = 7, *p < 0.05, **p < 0.01). For all analyses, data are the mean ± SEM.

Mentions: We performed pathological analyses of GBA-/- medaka. Patients with GD show Periodic acid-Schiff-positive Gaucher cells in affected visceral organs such as the liver, spleen, and bone marrow, whereas GBA-/- medaka showed abnormal Periodic acid-Schiff-positive cells in the spleen and kidney, but not in the liver, at 3 months (S3 Fig). Next, we examined the brains of GBA-/- medaka and found abnormal cells with large vacuoles mainly in the periventricular gray zone of the optic tectum (Fig. 2A, B). Transmission electron microscopy revealed that these cells were macrophage-like and had large vacuoles containing filamentous structures (Fig. 2C). Similar filamentous structures are observed in Gaucher cells of patients with GD and mouse models of GD[26,28,29]. The staining intensity with Luxol fast blue was decreased, and single-stranded DNA (ssDNA)-positive cells were observed in GBA-/- medaka (Fig. 2D), indicating myelin loss and cell death, respectively. In situ hybridization for apolipoprotein E (APOE), a microglial marker in teleost fish[30], revealed proliferating activated microglia in GBA-/- medaka (Fig. 2D). Teleost fish have glial fibrillary acidic protein (GFAP)-expressing radial glial cells (or ependymoglial cells) instead of astrocytes as in mammals[31]. Humans and mice with neuronopathic GD show astrogliosis in their brains[3,32], whereas neither proliferation of GFAP-positive radial glial cells nor elevated levels of GFAP were observed in GBA-/- medaka (Fig. 2D, E). To investigate the type of neuronal cells that die in GBA-/- medaka, we counted the numbers of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the middle diencephalon, which corresponds to the human substantia nigra[33], TH-positive noradrenergic neurons in the locus coeruleus, and tryptophan hydroxylase (TPH)-positive serotonergic neurons in the superior raphe at 2 and 3 months. GBA-/- medaka showed progressive cell loss of all these neurons (Fig. 2F). Consistent with these findings, the amount of TH was decreased in GBA-/- medaka (Fig. 2G). Collectively, GBA-/- medaka exhibited neuronopathic GD-like pathology including progressive and non-selective neuronal loss.


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)

Pathological analyses of GBA-/- medaka.(A) Schematic of a lateral view of the medaka brain. Each number signifies a part of the brain. 1: telencephalon, 2: optic tectum, 3: diencephalon, 4: cerebellum, 5: medulla oblongata. The brain sections used for pathological analyses in the present study are illustrated by the vertical line. (B) Hematoxylin and eosin staining. Abnormal cells observed in the periventricular gray zone of the optic tectum (arrowhead) in GBA-/- medaka at 3 months. Scale bars, 50 μm. Right panel: High-magnification image showing abnormal cells with large vacuoles (arrows). Scale bar, 5 μm. (C) Transmission electron micrographs showing abnormal macrophage-like cells. Left panel: A whole-cell image of an abnormal macrophage-like cell. Dashed lines outline a whole abnormal cell. N, Nucleus. Scale bar, 2 μm. Right panel: High-magnification image of filamentous structures in vacuoles. Scale bar, 500 nm. (D) Klüver-Barrera (KB) staining, ssDNA immunohistochemistry, APOE in situ hybridization, and GFAP immunohistochemistry in the diencephalon. Commissura posterior with decreased Luxol fast blue staining intensity (arrowhead) and ssDNA-positive dead cells (arrows) in GBA-/- medaka. APOE in situ hybridization revealed proliferating activated microglia in GBA-/- medaka. The staining intensity and area of GFAP were not changed in GBA-/- medaka. Scale bars, 50 μm. (E) Western blot analysis of GFAP and β-actin. The expression level of GFAP was not changed among genotypes. (F) Number of TH-positive neurons in the middle diencephalon, number of TH-positive neurons in the locus coeruleus, and number of TPH-positive neurons in the superior raphe at 2 and 3 months. In GBA-/- medaka, progressive neuronal loss was observed in all types of neurons (n = 4–6, *p < 0.05, ***p < 0.001). (G) Western blot analysis of TH and β-actin (n = 7, *p < 0.05, **p < 0.01). For all analyses, data are the mean ± SEM.
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pgen.1005065.g002: Pathological analyses of GBA-/- medaka.(A) Schematic of a lateral view of the medaka brain. Each number signifies a part of the brain. 1: telencephalon, 2: optic tectum, 3: diencephalon, 4: cerebellum, 5: medulla oblongata. The brain sections used for pathological analyses in the present study are illustrated by the vertical line. (B) Hematoxylin and eosin staining. Abnormal cells observed in the periventricular gray zone of the optic tectum (arrowhead) in GBA-/- medaka at 3 months. Scale bars, 50 μm. Right panel: High-magnification image showing abnormal cells with large vacuoles (arrows). Scale bar, 5 μm. (C) Transmission electron micrographs showing abnormal macrophage-like cells. Left panel: A whole-cell image of an abnormal macrophage-like cell. Dashed lines outline a whole abnormal cell. N, Nucleus. Scale bar, 2 μm. Right panel: High-magnification image of filamentous structures in vacuoles. Scale bar, 500 nm. (D) Klüver-Barrera (KB) staining, ssDNA immunohistochemistry, APOE in situ hybridization, and GFAP immunohistochemistry in the diencephalon. Commissura posterior with decreased Luxol fast blue staining intensity (arrowhead) and ssDNA-positive dead cells (arrows) in GBA-/- medaka. APOE in situ hybridization revealed proliferating activated microglia in GBA-/- medaka. The staining intensity and area of GFAP were not changed in GBA-/- medaka. Scale bars, 50 μm. (E) Western blot analysis of GFAP and β-actin. The expression level of GFAP was not changed among genotypes. (F) Number of TH-positive neurons in the middle diencephalon, number of TH-positive neurons in the locus coeruleus, and number of TPH-positive neurons in the superior raphe at 2 and 3 months. In GBA-/- medaka, progressive neuronal loss was observed in all types of neurons (n = 4–6, *p < 0.05, ***p < 0.001). (G) Western blot analysis of TH and β-actin (n = 7, *p < 0.05, **p < 0.01). For all analyses, data are the mean ± SEM.
Mentions: We performed pathological analyses of GBA-/- medaka. Patients with GD show Periodic acid-Schiff-positive Gaucher cells in affected visceral organs such as the liver, spleen, and bone marrow, whereas GBA-/- medaka showed abnormal Periodic acid-Schiff-positive cells in the spleen and kidney, but not in the liver, at 3 months (S3 Fig). Next, we examined the brains of GBA-/- medaka and found abnormal cells with large vacuoles mainly in the periventricular gray zone of the optic tectum (Fig. 2A, B). Transmission electron microscopy revealed that these cells were macrophage-like and had large vacuoles containing filamentous structures (Fig. 2C). Similar filamentous structures are observed in Gaucher cells of patients with GD and mouse models of GD[26,28,29]. The staining intensity with Luxol fast blue was decreased, and single-stranded DNA (ssDNA)-positive cells were observed in GBA-/- medaka (Fig. 2D), indicating myelin loss and cell death, respectively. In situ hybridization for apolipoprotein E (APOE), a microglial marker in teleost fish[30], revealed proliferating activated microglia in GBA-/- medaka (Fig. 2D). Teleost fish have glial fibrillary acidic protein (GFAP)-expressing radial glial cells (or ependymoglial cells) instead of astrocytes as in mammals[31]. Humans and mice with neuronopathic GD show astrogliosis in their brains[3,32], whereas neither proliferation of GFAP-positive radial glial cells nor elevated levels of GFAP were observed in GBA-/- medaka (Fig. 2D, E). To investigate the type of neuronal cells that die in GBA-/- medaka, we counted the numbers of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the middle diencephalon, which corresponds to the human substantia nigra[33], TH-positive noradrenergic neurons in the locus coeruleus, and tryptophan hydroxylase (TPH)-positive serotonergic neurons in the superior raphe at 2 and 3 months. GBA-/- medaka showed progressive cell loss of all these neurons (Fig. 2F). Consistent with these findings, the amount of TH was decreased in GBA-/- medaka (Fig. 2G). Collectively, GBA-/- medaka exhibited neuronopathic GD-like pathology including progressive and non-selective neuronal loss.

Bottom Line: 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.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.

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