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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 GLT-1 in glial cells expressing mutant htt. (A and B) Western blots of cultured astrocytes from R6/2 (HD) and littermate control (WT) mice. Astrocytes that had been cultured 4–6 wk and treated with (+) or without (–) 0.25 mM dBcAMP were examined by Western blotting with antibodies to GFAP and GLT-1. The same blots were also probed with an antibody to tubulin. Two blots (a, b) containing different cell samples are presented. (C) Densitometric analysis of signals of immunoreactive bands. The ratios (mean ± SEM, n = 3–4) of GLAST (*, P = 0.045, WT vs. HD), GLT-1 (*, P = 0.013; **, P < 0.001 WT vs. HD), and GFAP to tubulin. (D) Western blot analysis of the expression of htt-23Q and htt-130Q in adenovirus-infected glial cells. Bracket indicates the stacking gel in which aggregated htt-130Q is present. GLT-1 expression is reduced in htt-130Q astrocytes cocultured with cortical or striatal neurons. (E) [3H]Glutamate uptake assays of astrocytes infected with htt-23Q or htt-130Q. Note that the glutamate uptake in htt-130Q–infected cells is lower than in htt-23Q infected cells. The data are presented as mean ± SEM (*, P < 0.05; **, P < 0.01). The Vmax of [3H]glutamate uptake in htt-23Q and htt-130Q infected cells was 11.1 ± 0.84 and 7.3 ± 1.21 nmol/min/mg protein, respectively. There was no significant difference in the apparent glutamate Kms (37.7 ± 3.24 and 38.6 ± 6.3 for htt-23Q and htt-130Q, respectively).
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fig7: Decreased expression of GLT-1 in glial cells expressing mutant htt. (A and B) Western blots of cultured astrocytes from R6/2 (HD) and littermate control (WT) mice. Astrocytes that had been cultured 4–6 wk and treated with (+) or without (–) 0.25 mM dBcAMP were examined by Western blotting with antibodies to GFAP and GLT-1. The same blots were also probed with an antibody to tubulin. Two blots (a, b) containing different cell samples are presented. (C) Densitometric analysis of signals of immunoreactive bands. The ratios (mean ± SEM, n = 3–4) of GLAST (*, P = 0.045, WT vs. HD), GLT-1 (*, P = 0.013; **, P < 0.001 WT vs. HD), and GFAP to tubulin. (D) Western blot analysis of the expression of htt-23Q and htt-130Q in adenovirus-infected glial cells. Bracket indicates the stacking gel in which aggregated htt-130Q is present. GLT-1 expression is reduced in htt-130Q astrocytes cocultured with cortical or striatal neurons. (E) [3H]Glutamate uptake assays of astrocytes infected with htt-23Q or htt-130Q. Note that the glutamate uptake in htt-130Q–infected cells is lower than in htt-23Q infected cells. The data are presented as mean ± SEM (*, P < 0.05; **, P < 0.01). The Vmax of [3H]glutamate uptake in htt-23Q and htt-130Q infected cells was 11.1 ± 0.84 and 7.3 ± 1.21 nmol/min/mg protein, respectively. There was no significant difference in the apparent glutamate Kms (37.7 ± 3.24 and 38.6 ± 6.3 for htt-23Q and htt-130Q, respectively).

Mentions: Next, we examined whether mutant htt in astrocytes directly affects the expression of glutamate transporters. Western blots revealed that the basal level of GLT-1 was lower in cultured astrocytes from R6/2 mouse brains than those from littermate controls (Fig. 7 A), whereas the basal level of GFAP did not differ. We then treated the astrocytes with dibutyryl cyclic adenosine monophosphate (dBcAMP), which can significantly increase the expression of GLT-1 by regulating its transcription (Eng et al., 1997). The difference in the expression of GLT-1, but not GFAP, between control and R6/2 astrocytes was enhanced by dBcAMP treatment (Fig. 7, A and B), suggesting that mutant htt negatively affects gene transcription of GLT-1. The expression of GLAST in HD glial cells and its up-regulation by dBcAMP were also reduced as compared with wild-type cells (Fig. 7, A and B), but this reduction was not as great as the change in GLT-1. Because GLAST expression is not significantly decreased in HD brains (Lievens et al., 2001; Behrens et al., 2002), in vivo neuron–glia interactions may attenuate the inhibitory effect of mutant htt on the protein expression of GLAST in the brain.


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 GLT-1 in glial cells expressing mutant htt. (A and B) Western blots of cultured astrocytes from R6/2 (HD) and littermate control (WT) mice. Astrocytes that had been cultured 4–6 wk and treated with (+) or without (–) 0.25 mM dBcAMP were examined by Western blotting with antibodies to GFAP and GLT-1. The same blots were also probed with an antibody to tubulin. Two blots (a, b) containing different cell samples are presented. (C) Densitometric analysis of signals of immunoreactive bands. The ratios (mean ± SEM, n = 3–4) of GLAST (*, P = 0.045, WT vs. HD), GLT-1 (*, P = 0.013; **, P < 0.001 WT vs. HD), and GFAP to tubulin. (D) Western blot analysis of the expression of htt-23Q and htt-130Q in adenovirus-infected glial cells. Bracket indicates the stacking gel in which aggregated htt-130Q is present. GLT-1 expression is reduced in htt-130Q astrocytes cocultured with cortical or striatal neurons. (E) [3H]Glutamate uptake assays of astrocytes infected with htt-23Q or htt-130Q. Note that the glutamate uptake in htt-130Q–infected cells is lower than in htt-23Q infected cells. The data are presented as mean ± SEM (*, P < 0.05; **, P < 0.01). The Vmax of [3H]glutamate uptake in htt-23Q and htt-130Q infected cells was 11.1 ± 0.84 and 7.3 ± 1.21 nmol/min/mg protein, respectively. There was no significant difference in the apparent glutamate Kms (37.7 ± 3.24 and 38.6 ± 6.3 for htt-23Q and htt-130Q, respectively).
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Related In: Results  -  Collection

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fig7: Decreased expression of GLT-1 in glial cells expressing mutant htt. (A and B) Western blots of cultured astrocytes from R6/2 (HD) and littermate control (WT) mice. Astrocytes that had been cultured 4–6 wk and treated with (+) or without (–) 0.25 mM dBcAMP were examined by Western blotting with antibodies to GFAP and GLT-1. The same blots were also probed with an antibody to tubulin. Two blots (a, b) containing different cell samples are presented. (C) Densitometric analysis of signals of immunoreactive bands. The ratios (mean ± SEM, n = 3–4) of GLAST (*, P = 0.045, WT vs. HD), GLT-1 (*, P = 0.013; **, P < 0.001 WT vs. HD), and GFAP to tubulin. (D) Western blot analysis of the expression of htt-23Q and htt-130Q in adenovirus-infected glial cells. Bracket indicates the stacking gel in which aggregated htt-130Q is present. GLT-1 expression is reduced in htt-130Q astrocytes cocultured with cortical or striatal neurons. (E) [3H]Glutamate uptake assays of astrocytes infected with htt-23Q or htt-130Q. Note that the glutamate uptake in htt-130Q–infected cells is lower than in htt-23Q infected cells. The data are presented as mean ± SEM (*, P < 0.05; **, P < 0.01). The Vmax of [3H]glutamate uptake in htt-23Q and htt-130Q infected cells was 11.1 ± 0.84 and 7.3 ± 1.21 nmol/min/mg protein, respectively. There was no significant difference in the apparent glutamate Kms (37.7 ± 3.24 and 38.6 ± 6.3 for htt-23Q and htt-130Q, respectively).
Mentions: Next, we examined whether mutant htt in astrocytes directly affects the expression of glutamate transporters. Western blots revealed that the basal level of GLT-1 was lower in cultured astrocytes from R6/2 mouse brains than those from littermate controls (Fig. 7 A), whereas the basal level of GFAP did not differ. We then treated the astrocytes with dibutyryl cyclic adenosine monophosphate (dBcAMP), which can significantly increase the expression of GLT-1 by regulating its transcription (Eng et al., 1997). The difference in the expression of GLT-1, but not GFAP, between control and R6/2 astrocytes was enhanced by dBcAMP treatment (Fig. 7, A and B), suggesting that mutant htt negatively affects gene transcription of GLT-1. The expression of GLAST in HD glial cells and its up-regulation by dBcAMP were also reduced as compared with wild-type cells (Fig. 7, A and B), but this reduction was not as great as the change in GLT-1. Because GLAST expression is not significantly decreased in HD brains (Lievens et al., 2001; Behrens et al., 2002), in vivo neuron–glia interactions may attenuate the inhibitory effect of mutant htt on the protein expression of GLAST in the brain.

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