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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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Immunofluorescent labeling of htt-containing glia in R6/2 transgenic mouse brains. (A) Immunofluorescent labeling of brain sections containing white matter (WM) and the central region of the cerebellar cortex (Ctx) in an R6/2 mouse 8 wk old. Mouse antibody to GFAP labeled astrocytes (green) and rabbit EM48 labeled mutant htt (red). The nuclei were labeled with Hoechst dye (blue). (B) Confocal imaging of white matter showing that the nuclei of GFAP-positive glial cells contain htt aggregates (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200508072/DC1). (C) The striatal section of an R6/2 mouse at 8 wk of age was stained with antibody to GFAP and the nuclear dye Hoechst (top). EM48 immunostaining for htt and merged image are also presented (middle). Bottom panels shows merged images of striatal regions from 12- or 4-wk old R6/2 mice. Arrows indicate glial nuclei that contain EM48 staining. Small puncta represent neuropil aggregates. (D) The percentage of glial cells containing nuclear EM48 staining in the brain striatal sections of R6/2 mice at the age of 4, 8, or 12 wk. (E) The percentage of glial cells with intranuclear htt in the striatum (Str), cortex (Ctx), hippocampus (Hipp), and WM in R6/2 mice at the age of 8–10 wk. The data (mean ± SD) were obtained from three to five mice per group. **, P < 0.01 compared with the samples of 8 or 12 wk old R6/2 mice. Bars: (A) 5 μm; (B) 2.5 μm; (C)10 μm.
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fig2: Immunofluorescent labeling of htt-containing glia in R6/2 transgenic mouse brains. (A) Immunofluorescent labeling of brain sections containing white matter (WM) and the central region of the cerebellar cortex (Ctx) in an R6/2 mouse 8 wk old. Mouse antibody to GFAP labeled astrocytes (green) and rabbit EM48 labeled mutant htt (red). The nuclei were labeled with Hoechst dye (blue). (B) Confocal imaging of white matter showing that the nuclei of GFAP-positive glial cells contain htt aggregates (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200508072/DC1). (C) The striatal section of an R6/2 mouse at 8 wk of age was stained with antibody to GFAP and the nuclear dye Hoechst (top). EM48 immunostaining for htt and merged image are also presented (middle). Bottom panels shows merged images of striatal regions from 12- or 4-wk old R6/2 mice. Arrows indicate glial nuclei that contain EM48 staining. Small puncta represent neuropil aggregates. (D) The percentage of glial cells containing nuclear EM48 staining in the brain striatal sections of R6/2 mice at the age of 4, 8, or 12 wk. (E) The percentage of glial cells with intranuclear htt in the striatum (Str), cortex (Ctx), hippocampus (Hipp), and WM in R6/2 mice at the age of 8–10 wk. The data (mean ± SD) were obtained from three to five mice per group. **, P < 0.01 compared with the samples of 8 or 12 wk old R6/2 mice. Bars: (A) 5 μm; (B) 2.5 μm; (C)10 μm.

Mentions: Because the spectrum of morphological variations among glial cells makes it difficult to define astrocytes containing htt aggregates by electron microscopy, we performed immunofluorescent double labeling on thin sections (8–10 μm) of frozen brain. We first examined white matter that was enriched in astrocytes and was devoid of neurons. Astrocytes are the most abundant glial cell type in the brain and can be specifically labeled by antibody to glial fibrillary acidic protein (GFAP). Although GFAP-labeling was prominent in white matter, only a few cells displayed both strong GFAP staining and mutant htt (Fig. 2 A). This is perhaps because strong GFAP staining is only seen in reactive astrocytes that may have an increased capacity to clear mutant htt. We also performed confocal imaging analysis of white matter and confirmed that mutant htt forms small aggregates in GFAP-positive glial cells (Fig. 2 B). In addition, groups of GFAP-positive astrocytes were often found in the striatum (Fig. 2 C) and were not labeled by antibodies to other glial markers (myelin basic protein for oligodendrocytes and F4/80 for microglia) or the neuronal marker NeuN (not depicted), suggesting that they are mainly astrocytes. Glial nuclei, when stained with the Hoechst dye, are smaller and denser than neuronal nuclei. The small htt aggregates in glial nuclei as well as GFAP staining of glial processes allowed us to distinguish htt-containing glial cells from neurons that show larger and more intense EM48-labeled aggregates in their nuclei (Fig. 2, A and C). Based on these characteristics, we counted htt-containing glial cells in the HD mouse brains.


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)

Immunofluorescent labeling of htt-containing glia in R6/2 transgenic mouse brains. (A) Immunofluorescent labeling of brain sections containing white matter (WM) and the central region of the cerebellar cortex (Ctx) in an R6/2 mouse 8 wk old. Mouse antibody to GFAP labeled astrocytes (green) and rabbit EM48 labeled mutant htt (red). The nuclei were labeled with Hoechst dye (blue). (B) Confocal imaging of white matter showing that the nuclei of GFAP-positive glial cells contain htt aggregates (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200508072/DC1). (C) The striatal section of an R6/2 mouse at 8 wk of age was stained with antibody to GFAP and the nuclear dye Hoechst (top). EM48 immunostaining for htt and merged image are also presented (middle). Bottom panels shows merged images of striatal regions from 12- or 4-wk old R6/2 mice. Arrows indicate glial nuclei that contain EM48 staining. Small puncta represent neuropil aggregates. (D) The percentage of glial cells containing nuclear EM48 staining in the brain striatal sections of R6/2 mice at the age of 4, 8, or 12 wk. (E) The percentage of glial cells with intranuclear htt in the striatum (Str), cortex (Ctx), hippocampus (Hipp), and WM in R6/2 mice at the age of 8–10 wk. The data (mean ± SD) were obtained from three to five mice per group. **, P < 0.01 compared with the samples of 8 or 12 wk old R6/2 mice. Bars: (A) 5 μm; (B) 2.5 μm; (C)10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171327&req=5

fig2: Immunofluorescent labeling of htt-containing glia in R6/2 transgenic mouse brains. (A) Immunofluorescent labeling of brain sections containing white matter (WM) and the central region of the cerebellar cortex (Ctx) in an R6/2 mouse 8 wk old. Mouse antibody to GFAP labeled astrocytes (green) and rabbit EM48 labeled mutant htt (red). The nuclei were labeled with Hoechst dye (blue). (B) Confocal imaging of white matter showing that the nuclei of GFAP-positive glial cells contain htt aggregates (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200508072/DC1). (C) The striatal section of an R6/2 mouse at 8 wk of age was stained with antibody to GFAP and the nuclear dye Hoechst (top). EM48 immunostaining for htt and merged image are also presented (middle). Bottom panels shows merged images of striatal regions from 12- or 4-wk old R6/2 mice. Arrows indicate glial nuclei that contain EM48 staining. Small puncta represent neuropil aggregates. (D) The percentage of glial cells containing nuclear EM48 staining in the brain striatal sections of R6/2 mice at the age of 4, 8, or 12 wk. (E) The percentage of glial cells with intranuclear htt in the striatum (Str), cortex (Ctx), hippocampus (Hipp), and WM in R6/2 mice at the age of 8–10 wk. The data (mean ± SD) were obtained from three to five mice per group. **, P < 0.01 compared with the samples of 8 or 12 wk old R6/2 mice. Bars: (A) 5 μm; (B) 2.5 μm; (C)10 μm.
Mentions: Because the spectrum of morphological variations among glial cells makes it difficult to define astrocytes containing htt aggregates by electron microscopy, we performed immunofluorescent double labeling on thin sections (8–10 μm) of frozen brain. We first examined white matter that was enriched in astrocytes and was devoid of neurons. Astrocytes are the most abundant glial cell type in the brain and can be specifically labeled by antibody to glial fibrillary acidic protein (GFAP). Although GFAP-labeling was prominent in white matter, only a few cells displayed both strong GFAP staining and mutant htt (Fig. 2 A). This is perhaps because strong GFAP staining is only seen in reactive astrocytes that may have an increased capacity to clear mutant htt. We also performed confocal imaging analysis of white matter and confirmed that mutant htt forms small aggregates in GFAP-positive glial cells (Fig. 2 B). In addition, groups of GFAP-positive astrocytes were often found in the striatum (Fig. 2 C) and were not labeled by antibodies to other glial markers (myelin basic protein for oligodendrocytes and F4/80 for microglia) or the neuronal marker NeuN (not depicted), suggesting that they are mainly astrocytes. Glial nuclei, when stained with the Hoechst dye, are smaller and denser than neuronal nuclei. The small htt aggregates in glial nuclei as well as GFAP staining of glial processes allowed us to distinguish htt-containing glial cells from neurons that show larger and more intense EM48-labeled aggregates in their nuclei (Fig. 2, A and C). Based on these characteristics, we counted htt-containing glial cells in the HD mouse brains.

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