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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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Decreased expression of glutamate transporter GLT-1 and glutamate uptake in HD mouse brain. (A and B) Western blot analysis of the expression of GLT-1 in brain regions of wild-type (W1-W4) or R6/2 (HD1-HD4) mice at 4 wk (A) and 11–12 wk old (B). The same blots were probed with antibody to tubulin. The signal ratios of GLT-1 to tubulin are shown in the right. (C) RT-PCR analysis of the expression of GLT-1 and GLAST in R6/2 (HD) and wild-type (WT) mouse brain cortex. Transcripts for GLAST and actin were amplified in the same reaction (21 cycles), and the same amounts of cDNA were used for GLT-1 PCR (23 cycles). The ratios (mean ± SEM, n = 3) of GLT-1 (*, P = 0.0079, WT vs. HD) and GLAST (P = 0.11, WT vs. HD) to actin were obtained with densitometry. Panel C is the control RT-PCR without reverse transcriptase. (D) GLT-1–specific glutamate uptake in brain slices of littermate control (WT) and R6/2 (HD) mice at the age of 4–5 and 10–12 wk was obtained with DHK (1 mM) treatment. Data are presented as mean ± SEM (n = 4 each group). *, P < 0.05; **, P < 0.01 compared with WT control.
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fig3: Decreased expression of glutamate transporter GLT-1 and glutamate uptake in HD mouse brain. (A and B) Western blot analysis of the expression of GLT-1 in brain regions of wild-type (W1-W4) or R6/2 (HD1-HD4) mice at 4 wk (A) and 11–12 wk old (B). The same blots were probed with antibody to tubulin. The signal ratios of GLT-1 to tubulin are shown in the right. (C) RT-PCR analysis of the expression of GLT-1 and GLAST in R6/2 (HD) and wild-type (WT) mouse brain cortex. Transcripts for GLAST and actin were amplified in the same reaction (21 cycles), and the same amounts of cDNA were used for GLT-1 PCR (23 cycles). The ratios (mean ± SEM, n = 3) of GLT-1 (*, P = 0.0079, WT vs. HD) and GLAST (P = 0.11, WT vs. HD) to actin were obtained with densitometry. Panel C is the control RT-PCR without reverse transcriptase. (D) GLT-1–specific glutamate uptake in brain slices of littermate control (WT) and R6/2 (HD) mice at the age of 4–5 and 10–12 wk was obtained with DHK (1 mM) treatment. Data are presented as mean ± SEM (n = 4 each group). *, P < 0.05; **, P < 0.01 compared with WT control.

Mentions: Decreased expression of GLT-1 protein was observed in the HD brain by Western blotting (Fig. 3, A and B). The expression of GLT-1 seems to correlate inversely with the age-dependent nuclear accumulation of htt in glial cells, as the brain of HD mice at 11–12 wk showed a greater reduction of GLT-1 than those at 4 wk (Fig. 3, A and B). The expression level of GLAST appeared to be variable in individual mice, but was not significantly decreased in R6/2 mice as compared with littermate controls (Fig. S3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200508072/DC1). Although Hdh CAG knock-in mice did not show a significant reduction of brain GLT-1 and GLAST even at the age of 9 mo, a slight decrease of GLT-1 expression was observed in the striatum (Fig. S3, C and D). This finding supports the idea that NH2-terminal mutant htt is more toxic than full-length htt in affecting the expression of GLT-1. Consistent with previous studies (Lievens et al., 2001; Behrens et al., 2002), RT-PCR confirmed that GLT-1 transcripts were significantly reduced in HD mouse brains, while GLAST transcripts were slightly decreased (Fig. 3 C).


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)

Decreased expression of glutamate transporter GLT-1 and glutamate uptake in HD mouse brain. (A and B) Western blot analysis of the expression of GLT-1 in brain regions of wild-type (W1-W4) or R6/2 (HD1-HD4) mice at 4 wk (A) and 11–12 wk old (B). The same blots were probed with antibody to tubulin. The signal ratios of GLT-1 to tubulin are shown in the right. (C) RT-PCR analysis of the expression of GLT-1 and GLAST in R6/2 (HD) and wild-type (WT) mouse brain cortex. Transcripts for GLAST and actin were amplified in the same reaction (21 cycles), and the same amounts of cDNA were used for GLT-1 PCR (23 cycles). The ratios (mean ± SEM, n = 3) of GLT-1 (*, P = 0.0079, WT vs. HD) and GLAST (P = 0.11, WT vs. HD) to actin were obtained with densitometry. Panel C is the control RT-PCR without reverse transcriptase. (D) GLT-1–specific glutamate uptake in brain slices of littermate control (WT) and R6/2 (HD) mice at the age of 4–5 and 10–12 wk was obtained with DHK (1 mM) treatment. Data are presented as mean ± SEM (n = 4 each group). *, P < 0.05; **, P < 0.01 compared with WT control.
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fig3: Decreased expression of glutamate transporter GLT-1 and glutamate uptake in HD mouse brain. (A and B) Western blot analysis of the expression of GLT-1 in brain regions of wild-type (W1-W4) or R6/2 (HD1-HD4) mice at 4 wk (A) and 11–12 wk old (B). The same blots were probed with antibody to tubulin. The signal ratios of GLT-1 to tubulin are shown in the right. (C) RT-PCR analysis of the expression of GLT-1 and GLAST in R6/2 (HD) and wild-type (WT) mouse brain cortex. Transcripts for GLAST and actin were amplified in the same reaction (21 cycles), and the same amounts of cDNA were used for GLT-1 PCR (23 cycles). The ratios (mean ± SEM, n = 3) of GLT-1 (*, P = 0.0079, WT vs. HD) and GLAST (P = 0.11, WT vs. HD) to actin were obtained with densitometry. Panel C is the control RT-PCR without reverse transcriptase. (D) GLT-1–specific glutamate uptake in brain slices of littermate control (WT) and R6/2 (HD) mice at the age of 4–5 and 10–12 wk was obtained with DHK (1 mM) treatment. Data are presented as mean ± SEM (n = 4 each group). *, P < 0.05; **, P < 0.01 compared with WT control.
Mentions: Decreased expression of GLT-1 protein was observed in the HD brain by Western blotting (Fig. 3, A and B). The expression of GLT-1 seems to correlate inversely with the age-dependent nuclear accumulation of htt in glial cells, as the brain of HD mice at 11–12 wk showed a greater reduction of GLT-1 than those at 4 wk (Fig. 3, A and B). The expression level of GLAST appeared to be variable in individual mice, but was not significantly decreased in R6/2 mice as compared with littermate controls (Fig. S3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200508072/DC1). Although Hdh CAG knock-in mice did not show a significant reduction of brain GLT-1 and GLAST even at the age of 9 mo, a slight decrease of GLT-1 expression was observed in the striatum (Fig. S3, C and D). This finding supports the idea that NH2-terminal mutant htt is more toxic than full-length htt in affecting the expression of GLT-1. Consistent with previous studies (Lievens et al., 2001; Behrens et al., 2002), RT-PCR confirmed that GLT-1 transcripts were significantly reduced in HD mouse brains, while GLAST transcripts were slightly decreased (Fig. 3 C).

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