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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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Reduced glial protection against glutamate excitotoxicity by mutant htt in R6/2 astrocytes. (A) Cultured rat striatal neurons (15–17 DIV) were stimulated for 1 h with glutamate (0.1 mM) or NMDA (0.4 mM) in the absence (−glia) or presence (+glia) of wild-type rat cortical astrocytes. The astrocytes were removed after the stimulation. Glutamate stimulation caused neurons to degenerate and lose MAP2 staining. Wild-type astrocytes increased MAP2-positive neurons following glutamate (0.1 mM), but not NMDA (0.4 mM) stimulation, suggesting a specific protection resulting from their glutamate uptake. (B) Cultured astrocytes from littermate control (+WT glia) mouse cortex also protected against glutamate neuronal toxicity. Cultured astrocytes from R6/2 (+HD glia) mouse cortex, however, showed a decrease in protection against glutamate (0.1 mM), but not NMDA (0.4 mM), toxicity. (C) Cultured R6/2 astrocytes contained GFAP (green) in the cytoplasm and mutant htt (red) in their nuclei but had normal nuclear morphological appearance (blue). (D) The percentage of MAP2-positive striatal or cortical neurons after glutamate stimulation in the absence (No glia) or presence of wile type (+WT glia) or R6/2 (+HD glia) astrocytes. (E) The percentage of MAP2-positive striatal neurons after NMDA stimulation. The control is the number of cells without excitotoxin stimulation. The data (mean ± SEM) were obtained from four to five independent coculture experiments. *, P < 0.05; *, P < 0.01 as compared with WT astrocytes. Bars: (A and B) 20 μm; (C) 5 μm.
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fig9: Reduced glial protection against glutamate excitotoxicity by mutant htt in R6/2 astrocytes. (A) Cultured rat striatal neurons (15–17 DIV) were stimulated for 1 h with glutamate (0.1 mM) or NMDA (0.4 mM) in the absence (−glia) or presence (+glia) of wild-type rat cortical astrocytes. The astrocytes were removed after the stimulation. Glutamate stimulation caused neurons to degenerate and lose MAP2 staining. Wild-type astrocytes increased MAP2-positive neurons following glutamate (0.1 mM), but not NMDA (0.4 mM) stimulation, suggesting a specific protection resulting from their glutamate uptake. (B) Cultured astrocytes from littermate control (+WT glia) mouse cortex also protected against glutamate neuronal toxicity. Cultured astrocytes from R6/2 (+HD glia) mouse cortex, however, showed a decrease in protection against glutamate (0.1 mM), but not NMDA (0.4 mM), toxicity. (C) Cultured R6/2 astrocytes contained GFAP (green) in the cytoplasm and mutant htt (red) in their nuclei but had normal nuclear morphological appearance (blue). (D) The percentage of MAP2-positive striatal or cortical neurons after glutamate stimulation in the absence (No glia) or presence of wile type (+WT glia) or R6/2 (+HD glia) astrocytes. (E) The percentage of MAP2-positive striatal neurons after NMDA stimulation. The control is the number of cells without excitotoxin stimulation. The data (mean ± SEM) were obtained from four to five independent coculture experiments. *, P < 0.05; *, P < 0.01 as compared with WT astrocytes. Bars: (A and B) 20 μm; (C) 5 μm.

Mentions: To assess the specific protective effect of astrocytes in the removal of extracellular glutamate from the medium, we placed astrocytes, which were cultured on coverslips, on the top of neuronal cells without direct contact during 1 h stimulation with glutamate or NMDA. The astrocytes were removed after stimulation, and neurons were cultured in the absence of astrocytes for 24 h. Stimulation of 15–17 DIV striatal neurons with 0.1 mM glutamate in the absence of astrocytes led to a marked reduction in MAP2-positive neurons, whereas the presence of wild-type astrocytes significantly increased the number of MAP2-positive neurons (Fig. 9, A, B, and D). This glial protection was diminished by the glutamate transporter blockers (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200508072/DC1). NMDA (0.4 mM), whose neuronal toxicity is independent of glutamate transporters, elicited a similar reduction of MAP2-positive neurons in the absence or presence of astrocytes (Fig. 9, B and E). Also, cultured neurons were more sensitive to glutamate (0.1 mM) than to NMDA (0.4 mM; Fig. 9 A). Thus, this coculture system allowed us to specifically examine the ability of glial cells to remove extracellular glutamate and to protect neurons from glutamate excitotoxicity.


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)

Reduced glial protection against glutamate excitotoxicity by mutant htt in R6/2 astrocytes. (A) Cultured rat striatal neurons (15–17 DIV) were stimulated for 1 h with glutamate (0.1 mM) or NMDA (0.4 mM) in the absence (−glia) or presence (+glia) of wild-type rat cortical astrocytes. The astrocytes were removed after the stimulation. Glutamate stimulation caused neurons to degenerate and lose MAP2 staining. Wild-type astrocytes increased MAP2-positive neurons following glutamate (0.1 mM), but not NMDA (0.4 mM) stimulation, suggesting a specific protection resulting from their glutamate uptake. (B) Cultured astrocytes from littermate control (+WT glia) mouse cortex also protected against glutamate neuronal toxicity. Cultured astrocytes from R6/2 (+HD glia) mouse cortex, however, showed a decrease in protection against glutamate (0.1 mM), but not NMDA (0.4 mM), toxicity. (C) Cultured R6/2 astrocytes contained GFAP (green) in the cytoplasm and mutant htt (red) in their nuclei but had normal nuclear morphological appearance (blue). (D) The percentage of MAP2-positive striatal or cortical neurons after glutamate stimulation in the absence (No glia) or presence of wile type (+WT glia) or R6/2 (+HD glia) astrocytes. (E) The percentage of MAP2-positive striatal neurons after NMDA stimulation. The control is the number of cells without excitotoxin stimulation. The data (mean ± SEM) were obtained from four to five independent coculture experiments. *, P < 0.05; *, P < 0.01 as compared with WT astrocytes. Bars: (A and B) 20 μm; (C) 5 μm.
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fig9: Reduced glial protection against glutamate excitotoxicity by mutant htt in R6/2 astrocytes. (A) Cultured rat striatal neurons (15–17 DIV) were stimulated for 1 h with glutamate (0.1 mM) or NMDA (0.4 mM) in the absence (−glia) or presence (+glia) of wild-type rat cortical astrocytes. The astrocytes were removed after the stimulation. Glutamate stimulation caused neurons to degenerate and lose MAP2 staining. Wild-type astrocytes increased MAP2-positive neurons following glutamate (0.1 mM), but not NMDA (0.4 mM) stimulation, suggesting a specific protection resulting from their glutamate uptake. (B) Cultured astrocytes from littermate control (+WT glia) mouse cortex also protected against glutamate neuronal toxicity. Cultured astrocytes from R6/2 (+HD glia) mouse cortex, however, showed a decrease in protection against glutamate (0.1 mM), but not NMDA (0.4 mM), toxicity. (C) Cultured R6/2 astrocytes contained GFAP (green) in the cytoplasm and mutant htt (red) in their nuclei but had normal nuclear morphological appearance (blue). (D) The percentage of MAP2-positive striatal or cortical neurons after glutamate stimulation in the absence (No glia) or presence of wile type (+WT glia) or R6/2 (+HD glia) astrocytes. (E) The percentage of MAP2-positive striatal neurons after NMDA stimulation. The control is the number of cells without excitotoxin stimulation. The data (mean ± SEM) were obtained from four to five independent coculture experiments. *, P < 0.05; *, P < 0.01 as compared with WT astrocytes. Bars: (A and B) 20 μm; (C) 5 μm.
Mentions: To assess the specific protective effect of astrocytes in the removal of extracellular glutamate from the medium, we placed astrocytes, which were cultured on coverslips, on the top of neuronal cells without direct contact during 1 h stimulation with glutamate or NMDA. The astrocytes were removed after stimulation, and neurons were cultured in the absence of astrocytes for 24 h. Stimulation of 15–17 DIV striatal neurons with 0.1 mM glutamate in the absence of astrocytes led to a marked reduction in MAP2-positive neurons, whereas the presence of wild-type astrocytes significantly increased the number of MAP2-positive neurons (Fig. 9, A, B, and D). This glial protection was diminished by the glutamate transporter blockers (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200508072/DC1). NMDA (0.4 mM), whose neuronal toxicity is independent of glutamate transporters, elicited a similar reduction of MAP2-positive neurons in the absence or presence of astrocytes (Fig. 9, B and E). Also, cultured neurons were more sensitive to glutamate (0.1 mM) than to NMDA (0.4 mM; Fig. 9 A). Thus, this coculture system allowed us to specifically examine the ability of glial cells to remove extracellular glutamate and to protect neurons from glutamate excitotoxicity.

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