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Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity.

Shin JY, Fang ZH, Yu ZX, Wang CE, Li SH, Li XJ - J. Cell Biol. (2005)

Bottom Line: Here, we report that mutant huntingtin accumulates in glial nuclei in HD brains and decreases the expression of glutamate transporters.Mutant htt in cultured astrocytes decreased their protection of neurons against glutamate excitotoxicity.These findings suggest that decreased glutamate uptake caused by glial mutant htt may critically contribute to neuronal excitotoxicity in HD.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Huntington disease (HD) is characterized by the preferential loss of striatal medium-sized spiny neurons (MSNs) in the brain. Because MSNs receive abundant glutamatergic input, their vulnerability to excitotoxicity may be largely influenced by the capacity of glial cells to remove extracellular glutamate. However, little is known about the role of glia in HD neuropathology. Here, we report that mutant huntingtin accumulates in glial nuclei in HD brains and decreases the expression of glutamate transporters. As a result, mutant huntingtin (htt) reduces glutamate uptake in cultured astrocytes and HD mouse brains. In a neuron-glia coculture system, wild-type glial cells protected neurons against mutant htt-mediated neurotoxicity, whereas glial cells expressing mutant htt increased neuronal vulnerability. Mutant htt in cultured astrocytes decreased their protection of neurons against glutamate excitotoxicity. These findings suggest that decreased glutamate uptake caused by glial mutant htt may critically contribute to neuronal excitotoxicity in HD.

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Expression of mutant htt in the brains of Hdh CAG(150) knock-in mice and HD patient. (A) Immunofluorescent double labeling of the striatum of heterozygous Hdh CAG(150) knock-in mice at 14–18 mo old. Arrows indicates the nuclei (blue) of GFAP (green)-positive glial cells containing EM48 labeling (red), and arrowhead indicates a neuronal nucleus, which shows more intense EM48 labeling. (B) High magnification graphs of confocal images of white matter of heterozygous Hdh CAG(150) knock-in mice. Arrows indicate glial nuclei (blue) containing EM48 labeling (red). (C) Western blotting of caudate-putamen tissues from HD and Alzheimer's disease (AD; Control) patients. The blot was probed with antiactin (bottom) and 1C2 antibody (top). (D) Light microscopic graphs of glial cells (arrows in upper panel) in white matter and neurons in the cortex (bottom) in the EM48 stained HD brain sections. (E) Immunofluorescent double labeling of white matter of HD patient brain with rabbit anti-htt (EM48) and mouse anti-GFAP. Mutant htt (red) forms aggregates (arrow) in the nucleus (blue) of an astrocytic cell that shows intense GFAP staining (green) in its processes. The control is the AD brain section. Bars, 2 μm.
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fig5: Expression of mutant htt in the brains of Hdh CAG(150) knock-in mice and HD patient. (A) Immunofluorescent double labeling of the striatum of heterozygous Hdh CAG(150) knock-in mice at 14–18 mo old. Arrows indicates the nuclei (blue) of GFAP (green)-positive glial cells containing EM48 labeling (red), and arrowhead indicates a neuronal nucleus, which shows more intense EM48 labeling. (B) High magnification graphs of confocal images of white matter of heterozygous Hdh CAG(150) knock-in mice. Arrows indicate glial nuclei (blue) containing EM48 labeling (red). (C) Western blotting of caudate-putamen tissues from HD and Alzheimer's disease (AD; Control) patients. The blot was probed with antiactin (bottom) and 1C2 antibody (top). (D) Light microscopic graphs of glial cells (arrows in upper panel) in white matter and neurons in the cortex (bottom) in the EM48 stained HD brain sections. (E) Immunofluorescent double labeling of white matter of HD patient brain with rabbit anti-htt (EM48) and mouse anti-GFAP. Mutant htt (red) forms aggregates (arrow) in the nucleus (blue) of an astrocytic cell that shows intense GFAP staining (green) in its processes. The control is the AD brain section. Bars, 2 μm.

Mentions: Examination of the striatum of Hdh CAG(150) knock-in mice at 14–18 mo of age revealed EM48 labeling in the small and dense nuclei of some GFAP-positive glial cells as compared with intense EM48 labeling in large neuronal nuclei (Fig. 5 A). The presence of mutant htt in glial nuclei was confirmed by both conventional microscopy (Fig. 5 A) and confocal imaging (Fig. 5 B) of white matter, which clearly showed that some GFAP-positive glial nuclei contained small htt aggregates. Younger Hdh CAG(150) knock-in mice (<9 mo) were not found to have obvious EM48 staining in glial nuclei (unpublished data). suggesting that the nuclear accumulation of mutant htt in glia is age dependent. A previous study reported that mutant htt is present in astrocytes of HD patient brains (Singhrao et al., 1998). We also examined postmortem brains from late stage (grade-3) HD patients and confirmed the expression of mutant htt in HD brains by Western blotting (Fig. 5 C). Despite severe degeneration in grade-3 HD brains, some GFAP-positive glial cells still remained in these brains. Small EM48-labeled aggregates (<0.3 μm) were seen in white matter and were much smaller than neuronal nuclear aggregates that often exceeded 1.5 μm and were intensively labeled by EM48 (Fig. 5 D). Immunofluorescent double labeling verified that the nuclei of GFAP-positive glial cells contained EM48 immunoreactive aggregates in HD patient brains (Fig. 5 E), which are similar in size to those in glial cells in Hdh CAG(150) knock-in mice (Fig. 5, A and B) and did not occur in the brain of Alzheimer's disease (AD) patient (Fig. 5 E). We observed that ∼12.3% of GFAP-positive cells contained htt aggregates. Given that EM48 preferentially reacts with aggregated htt and that grade-3 HD brains might have lost some glial cells or their markers, the number of glial cells expressing mutant htt, especially soluble htt, is likely to be higher.


Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity.

Shin JY, Fang ZH, Yu ZX, Wang CE, Li SH, Li XJ - J. Cell Biol. (2005)

Expression of mutant htt in the brains of Hdh CAG(150) knock-in mice and HD patient. (A) Immunofluorescent double labeling of the striatum of heterozygous Hdh CAG(150) knock-in mice at 14–18 mo old. Arrows indicates the nuclei (blue) of GFAP (green)-positive glial cells containing EM48 labeling (red), and arrowhead indicates a neuronal nucleus, which shows more intense EM48 labeling. (B) High magnification graphs of confocal images of white matter of heterozygous Hdh CAG(150) knock-in mice. Arrows indicate glial nuclei (blue) containing EM48 labeling (red). (C) Western blotting of caudate-putamen tissues from HD and Alzheimer's disease (AD; Control) patients. The blot was probed with antiactin (bottom) and 1C2 antibody (top). (D) Light microscopic graphs of glial cells (arrows in upper panel) in white matter and neurons in the cortex (bottom) in the EM48 stained HD brain sections. (E) Immunofluorescent double labeling of white matter of HD patient brain with rabbit anti-htt (EM48) and mouse anti-GFAP. Mutant htt (red) forms aggregates (arrow) in the nucleus (blue) of an astrocytic cell that shows intense GFAP staining (green) in its processes. The control is the AD brain section. Bars, 2 μm.
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fig5: Expression of mutant htt in the brains of Hdh CAG(150) knock-in mice and HD patient. (A) Immunofluorescent double labeling of the striatum of heterozygous Hdh CAG(150) knock-in mice at 14–18 mo old. Arrows indicates the nuclei (blue) of GFAP (green)-positive glial cells containing EM48 labeling (red), and arrowhead indicates a neuronal nucleus, which shows more intense EM48 labeling. (B) High magnification graphs of confocal images of white matter of heterozygous Hdh CAG(150) knock-in mice. Arrows indicate glial nuclei (blue) containing EM48 labeling (red). (C) Western blotting of caudate-putamen tissues from HD and Alzheimer's disease (AD; Control) patients. The blot was probed with antiactin (bottom) and 1C2 antibody (top). (D) Light microscopic graphs of glial cells (arrows in upper panel) in white matter and neurons in the cortex (bottom) in the EM48 stained HD brain sections. (E) Immunofluorescent double labeling of white matter of HD patient brain with rabbit anti-htt (EM48) and mouse anti-GFAP. Mutant htt (red) forms aggregates (arrow) in the nucleus (blue) of an astrocytic cell that shows intense GFAP staining (green) in its processes. The control is the AD brain section. Bars, 2 μm.
Mentions: Examination of the striatum of Hdh CAG(150) knock-in mice at 14–18 mo of age revealed EM48 labeling in the small and dense nuclei of some GFAP-positive glial cells as compared with intense EM48 labeling in large neuronal nuclei (Fig. 5 A). The presence of mutant htt in glial nuclei was confirmed by both conventional microscopy (Fig. 5 A) and confocal imaging (Fig. 5 B) of white matter, which clearly showed that some GFAP-positive glial nuclei contained small htt aggregates. Younger Hdh CAG(150) knock-in mice (<9 mo) were not found to have obvious EM48 staining in glial nuclei (unpublished data). suggesting that the nuclear accumulation of mutant htt in glia is age dependent. A previous study reported that mutant htt is present in astrocytes of HD patient brains (Singhrao et al., 1998). We also examined postmortem brains from late stage (grade-3) HD patients and confirmed the expression of mutant htt in HD brains by Western blotting (Fig. 5 C). Despite severe degeneration in grade-3 HD brains, some GFAP-positive glial cells still remained in these brains. Small EM48-labeled aggregates (<0.3 μm) were seen in white matter and were much smaller than neuronal nuclear aggregates that often exceeded 1.5 μm and were intensively labeled by EM48 (Fig. 5 D). Immunofluorescent double labeling verified that the nuclei of GFAP-positive glial cells contained EM48 immunoreactive aggregates in HD patient brains (Fig. 5 E), which are similar in size to those in glial cells in Hdh CAG(150) knock-in mice (Fig. 5, A and B) and did not occur in the brain of Alzheimer's disease (AD) patient (Fig. 5 E). We observed that ∼12.3% of GFAP-positive cells contained htt aggregates. Given that EM48 preferentially reacts with aggregated htt and that grade-3 HD brains might have lost some glial cells or their markers, the number of glial cells expressing mutant htt, especially soluble htt, is likely to be higher.

Bottom Line: Here, we report that mutant huntingtin accumulates in glial nuclei in HD brains and decreases the expression of glutamate transporters.Mutant htt in cultured astrocytes decreased their protection of neurons against glutamate excitotoxicity.These findings suggest that decreased glutamate uptake caused by glial mutant htt may critically contribute to neuronal excitotoxicity in HD.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Huntington disease (HD) is characterized by the preferential loss of striatal medium-sized spiny neurons (MSNs) in the brain. Because MSNs receive abundant glutamatergic input, their vulnerability to excitotoxicity may be largely influenced by the capacity of glial cells to remove extracellular glutamate. However, little is known about the role of glia in HD neuropathology. Here, we report that mutant huntingtin accumulates in glial nuclei in HD brains and decreases the expression of glutamate transporters. As a result, mutant huntingtin (htt) reduces glutamate uptake in cultured astrocytes and HD mouse brains. In a neuron-glia coculture system, wild-type glial cells protected neurons against mutant htt-mediated neurotoxicity, whereas glial cells expressing mutant htt increased neuronal vulnerability. Mutant htt in cultured astrocytes decreased their protection of neurons against glutamate excitotoxicity. These findings suggest that decreased glutamate uptake caused by glial mutant htt may critically contribute to neuronal excitotoxicity in HD.

Show MeSH
Related in: MedlinePlus