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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: 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

Impairment of the autophagy-lysosome pathway in GBA-/- medaka.(A) Ubiquitin, p62, and Cathepsin D immunohistochemistry in the neuronal layer of the optic tectum at 3 months. Almost all of the cells in these figures are neurons. Ubiquitin-positive and p62-positive aggregates were observed in the perikarya of neurons in GBA-/- medaka at 3 months (arrowheads). Morphologically abnormal organelles with decreased Cathepsin D staining intensity were observed in GBA-/- medaka (arrows). Scale bars, 10 μm. (B) Transmission electron micrographs of neurons. Left panel: Neuron of a GBA+/+ medaka. Electron-dense organelles (arrows) are likely lysosomes. N, Nucleus. Scale bar, 2 μm. The inset shows a high-magnification image of electron-dense organelles. Scale bar, 500 nm. Middle panel: Neuron of a GBA-/- medaka. An aggregate containing filamentous structures (arrowhead) is continuous with an electron-dense organelle. N, Nucleus. Scale bar, 2 μm. Right panel: High-magnification image of an aggregate of filamentous structures (arrowhead) and electron-dense organelles (arrows) in a GBA-/- medaka. Scale bar, 500 nm. (C) Western blot analysis of ubiquitin, p62, and β-actin (n = 4–7, *p < 0.05, ***p < 0.001). (D) Conforcal microscope images of GBA-/- medaka. Upper panels: ubiquitin (green) and p62 (red). p62-positive aggregates colocalized with ubiquitin-positive aggregates (white arrowheads). Middle panels: LC3 (green) and p62 (red). p62-positive aggregates (yellow arrows) did not colocalize with LC3 accumulations (yellow arrowheads). Lower panels: Cathepsin D (green) and p62 (red). p62-positive aggregates did not colocalize with Cathepsin D-positive organelles. Nuclei were visualized with DAPI (blue). Scale bars, 10 μm. For all analyses, data are the mean ± SEM.
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pgen.1005065.g004: Impairment of the autophagy-lysosome pathway in GBA-/- medaka.(A) Ubiquitin, p62, and Cathepsin D immunohistochemistry in the neuronal layer of the optic tectum at 3 months. Almost all of the cells in these figures are neurons. Ubiquitin-positive and p62-positive aggregates were observed in the perikarya of neurons in GBA-/- medaka at 3 months (arrowheads). Morphologically abnormal organelles with decreased Cathepsin D staining intensity were observed in GBA-/- medaka (arrows). Scale bars, 10 μm. (B) Transmission electron micrographs of neurons. Left panel: Neuron of a GBA+/+ medaka. Electron-dense organelles (arrows) are likely lysosomes. N, Nucleus. Scale bar, 2 μm. The inset shows a high-magnification image of electron-dense organelles. Scale bar, 500 nm. Middle panel: Neuron of a GBA-/- medaka. An aggregate containing filamentous structures (arrowhead) is continuous with an electron-dense organelle. N, Nucleus. Scale bar, 2 μm. Right panel: High-magnification image of an aggregate of filamentous structures (arrowhead) and electron-dense organelles (arrows) in a GBA-/- medaka. Scale bar, 500 nm. (C) Western blot analysis of ubiquitin, p62, and β-actin (n = 4–7, *p < 0.05, ***p < 0.001). (D) Conforcal microscope images of GBA-/- medaka. Upper panels: ubiquitin (green) and p62 (red). p62-positive aggregates colocalized with ubiquitin-positive aggregates (white arrowheads). Middle panels: LC3 (green) and p62 (red). p62-positive aggregates (yellow arrows) did not colocalize with LC3 accumulations (yellow arrowheads). Lower panels: Cathepsin D (green) and p62 (red). p62-positive aggregates did not colocalize with Cathepsin D-positive organelles. Nuclei were visualized with DAPI (blue). Scale bars, 10 μm. For all analyses, data are the mean ± SEM.

Mentions: Previous studies of GD mouse models reported that p62/SQSTM1 (an autophagic substrate) accumulates in neurons and astrocytes[34], and the number of Cathepsin D-positive puncta is decreased in neurons[35]. These observations prompted us to examine the autophagy-lysosome pathway in GBA-/- medaka. Immunohistochemical analysis revealed that ubiquitin- and p62-positive aggregates were observed mainly in the perikarya of neurons (Figs. 4A, D, S8B). These aggregates were observed only in neurons and not in GFAP-positive radial glial cells or Lycopersicon Esculentum (Tomato) Lectin (LEL)-positive microglia (S8A Fig). LEL is an excellent teleost microglial marker[36]. Western blot analysis showed that the amounts of ubiquitin and p62 were increased in the brains of GBA-/- medaka (Fig. 4C). In contrast, these aggregates did not colocalize with LC3 accumulations or Cathepsin D-positive organelles (Figs. 4D, S8B). These organelles, which are putative lysosomes, showed decreased Cathepsin D staining intensity and abnormal morphology (Fig. 4A). Consistent with this, neurons that contained lysosome-like organelles filled with filamentous structures were observed with transmission electron microscopy (Fig. 4B).


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)

Impairment of the autophagy-lysosome pathway in GBA-/- medaka.(A) Ubiquitin, p62, and Cathepsin D immunohistochemistry in the neuronal layer of the optic tectum at 3 months. Almost all of the cells in these figures are neurons. Ubiquitin-positive and p62-positive aggregates were observed in the perikarya of neurons in GBA-/- medaka at 3 months (arrowheads). Morphologically abnormal organelles with decreased Cathepsin D staining intensity were observed in GBA-/- medaka (arrows). Scale bars, 10 μm. (B) Transmission electron micrographs of neurons. Left panel: Neuron of a GBA+/+ medaka. Electron-dense organelles (arrows) are likely lysosomes. N, Nucleus. Scale bar, 2 μm. The inset shows a high-magnification image of electron-dense organelles. Scale bar, 500 nm. Middle panel: Neuron of a GBA-/- medaka. An aggregate containing filamentous structures (arrowhead) is continuous with an electron-dense organelle. N, Nucleus. Scale bar, 2 μm. Right panel: High-magnification image of an aggregate of filamentous structures (arrowhead) and electron-dense organelles (arrows) in a GBA-/- medaka. Scale bar, 500 nm. (C) Western blot analysis of ubiquitin, p62, and β-actin (n = 4–7, *p < 0.05, ***p < 0.001). (D) Conforcal microscope images of GBA-/- medaka. Upper panels: ubiquitin (green) and p62 (red). p62-positive aggregates colocalized with ubiquitin-positive aggregates (white arrowheads). Middle panels: LC3 (green) and p62 (red). p62-positive aggregates (yellow arrows) did not colocalize with LC3 accumulations (yellow arrowheads). Lower panels: Cathepsin D (green) and p62 (red). p62-positive aggregates did not colocalize with Cathepsin D-positive organelles. Nuclei were visualized with DAPI (blue). Scale bars, 10 μm. For all analyses, data are the mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005065.g004: Impairment of the autophagy-lysosome pathway in GBA-/- medaka.(A) Ubiquitin, p62, and Cathepsin D immunohistochemistry in the neuronal layer of the optic tectum at 3 months. Almost all of the cells in these figures are neurons. Ubiquitin-positive and p62-positive aggregates were observed in the perikarya of neurons in GBA-/- medaka at 3 months (arrowheads). Morphologically abnormal organelles with decreased Cathepsin D staining intensity were observed in GBA-/- medaka (arrows). Scale bars, 10 μm. (B) Transmission electron micrographs of neurons. Left panel: Neuron of a GBA+/+ medaka. Electron-dense organelles (arrows) are likely lysosomes. N, Nucleus. Scale bar, 2 μm. The inset shows a high-magnification image of electron-dense organelles. Scale bar, 500 nm. Middle panel: Neuron of a GBA-/- medaka. An aggregate containing filamentous structures (arrowhead) is continuous with an electron-dense organelle. N, Nucleus. Scale bar, 2 μm. Right panel: High-magnification image of an aggregate of filamentous structures (arrowhead) and electron-dense organelles (arrows) in a GBA-/- medaka. Scale bar, 500 nm. (C) Western blot analysis of ubiquitin, p62, and β-actin (n = 4–7, *p < 0.05, ***p < 0.001). (D) Conforcal microscope images of GBA-/- medaka. Upper panels: ubiquitin (green) and p62 (red). p62-positive aggregates colocalized with ubiquitin-positive aggregates (white arrowheads). Middle panels: LC3 (green) and p62 (red). p62-positive aggregates (yellow arrows) did not colocalize with LC3 accumulations (yellow arrowheads). Lower panels: Cathepsin D (green) and p62 (red). p62-positive aggregates did not colocalize with Cathepsin D-positive organelles. Nuclei were visualized with DAPI (blue). Scale bars, 10 μm. For all analyses, data are the mean ± SEM.
Mentions: Previous studies of GD mouse models reported that p62/SQSTM1 (an autophagic substrate) accumulates in neurons and astrocytes[34], and the number of Cathepsin D-positive puncta is decreased in neurons[35]. These observations prompted us to examine the autophagy-lysosome pathway in GBA-/- medaka. Immunohistochemical analysis revealed that ubiquitin- and p62-positive aggregates were observed mainly in the perikarya of neurons (Figs. 4A, D, S8B). These aggregates were observed only in neurons and not in GFAP-positive radial glial cells or Lycopersicon Esculentum (Tomato) Lectin (LEL)-positive microglia (S8A Fig). LEL is an excellent teleost microglial marker[36]. Western blot analysis showed that the amounts of ubiquitin and p62 were increased in the brains of GBA-/- medaka (Fig. 4C). In contrast, these aggregates did not colocalize with LC3 accumulations or Cathepsin D-positive organelles (Figs. 4D, S8B). These organelles, which are putative lysosomes, showed decreased Cathepsin D staining intensity and abnormal morphology (Fig. 4A). Consistent with this, neurons that contained lysosome-like organelles filled with filamentous structures were observed with transmission electron microscopy (Fig. 4B).

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