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Arundic acid attenuates retinal ganglion cell death by increasing glutamate/aspartate transporter expression in a model of normal tension glaucoma.

Yanagisawa M, Aida T, Takeda T, Namekata K, Harada T, Shinagawa R, Tanaka K - Cell Death Dis (2015)

Bottom Line: To test this hypothesis, we examined the effect of arundic acid on GLAST expression and glutamate uptake.We found that arundic acid induces GLAST expression in vitro and in vivo.Thus, discovering compounds that can enhance EAAT1 expression and activity may be a novel strategy for therapeutic treatment of glaucoma.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

ABSTRACT
Glaucoma is the second leading cause of blindness worldwide and is characterized by gradual visual impairment owing to progressive loss of retinal ganglion cells (RGCs) and their axons. Glutamate excitotoxicity has been implicated as a mechanism of RGC death in glaucoma. Consistent with this claim, we previously reported that glutamate/aspartate transporter (GLAST)-deficient mice show optic nerve degeneration that is similar to that observed in glaucoma. Therefore, drugs that upregulate GLAST may be useful for neuroprotection in glaucoma. Although many compounds are known to increase the expression of another glial glutamate transporter, EAAT2/GLT1, few compounds are shown to increase GLAST expression. Arundic acid is a glial modulating agent that ameliorates delayed ischemic brain damage by attenuating increases in extracellular glutamate. We hypothesized that arundic acid neuroprotection involves upregulation of GLAST. To test this hypothesis, we examined the effect of arundic acid on GLAST expression and glutamate uptake. We found that arundic acid induces GLAST expression in vitro and in vivo. In addition, arundic acid treatment prevented RGC death by upregulating GLAST in heterozygous (GLAST(+/-)) mice. Furthermore, arundic acid stimulates the human GLAST ortholog, EAAT1, expression in human neuroglioblastoma cells. Thus, discovering compounds that can enhance EAAT1 expression and activity may be a novel strategy for therapeutic treatment of glaucoma.

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Arundic acid increases GLAST expression and transport activity in the mouse retina. (a) Effects of arundic acid treatment on GLAST, GLT1 and EAAC1 mRNA levels in the retina of GLAST+/− mice. Arundic acid (10 mg/kg, given daily from P22 to P35) increased the GLAST mRNA level (N=6), whereas the mRNA levels of GLT1 (N=6) and EAAC1 (N=6) are unaffected. *P<0.05 as determined by a Student's t-test. (b) Arundic acid (10 mg/kg) increases GLAST protein expression in the retina of GLAST+/− mice relative to vehicle-treated control mice (N=6). A representative western blot of GLAST protein expression is shown; the quantified data represent the mean±S.E.M. *P<0.05 as determined by a Student's t-test. (c) Effect of arundic acid on glutamate uptake activity in the retina of GLAST mutant mice. Relative glutamate uptake velocity was quantified from six independent experiments performed in duplicate for each data point. Data represent the mean±S.E.M. *P<0.05, **P<0.01 as determined by one-way ANOVA with Tukey–Kramer's post hoc analysis
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fig2: Arundic acid increases GLAST expression and transport activity in the mouse retina. (a) Effects of arundic acid treatment on GLAST, GLT1 and EAAC1 mRNA levels in the retina of GLAST+/− mice. Arundic acid (10 mg/kg, given daily from P22 to P35) increased the GLAST mRNA level (N=6), whereas the mRNA levels of GLT1 (N=6) and EAAC1 (N=6) are unaffected. *P<0.05 as determined by a Student's t-test. (b) Arundic acid (10 mg/kg) increases GLAST protein expression in the retina of GLAST+/− mice relative to vehicle-treated control mice (N=6). A representative western blot of GLAST protein expression is shown; the quantified data represent the mean±S.E.M. *P<0.05 as determined by a Student's t-test. (c) Effect of arundic acid on glutamate uptake activity in the retina of GLAST mutant mice. Relative glutamate uptake velocity was quantified from six independent experiments performed in duplicate for each data point. Data represent the mean±S.E.M. *P<0.05, **P<0.01 as determined by one-way ANOVA with Tukey–Kramer's post hoc analysis

Mentions: To determine whether the effects of arundic acid are similar in vivo, we examined glutamate transporter expression in isolated mouse retinas treated with arundic acid. Three glutamate transporters are expressed around the synapses of RGCs in the plexiform layer, GLAST, GLT1 and excitatory amino-acid carrier 1 (EAAC1).21 We administered arundic acid or vehicle to GLAST+/− mice during postnatal day (P) 22 to P35, and evaluated the glutamate transporter expression levels by qPCR. Arundic acid treatment increased GLAST mRNA expression in the retinas of GLAST+/− mice, but did not alter GLT1 or EAAC1 mRNA levels (Figure 2a). Next, we asked whether arundic acid is capable of increasing GLAST protein expression in the retinas of GLAST+/− mice. Using western blot analysis, we found that 14-day arundic acid treatment significantly increased endogenous GLAST protein expression (Figure 2b). To test whether the increases in GLAST mRNA and protein expression are accompanied by enhanced glutamate transport activity, we conducted l-[3,4-3H]-glutamate uptake assays in isolated retinas from GLAST+/− and GLAST−/− mice. Fourteen-day arundic acid treatment led to a 1.23-fold increase in glutamate uptake in the retinas of GLAST+/− mice, compared with those treated with vehicle alone. We also observed that arundic acid increased GLAST protein expression and glutamate uptake activity in the retinas of wild-type mice (Supplementary Figures 2a and b). By contrast, arundic acid treatment did not affect glutamate uptake activity in the retinas of GLAST−/− mice (Figure 2c). These results suggest that arundic acid treatment increases retinal glutamate uptake activity by selectively increasing GLAST expression in vivo.


Arundic acid attenuates retinal ganglion cell death by increasing glutamate/aspartate transporter expression in a model of normal tension glaucoma.

Yanagisawa M, Aida T, Takeda T, Namekata K, Harada T, Shinagawa R, Tanaka K - Cell Death Dis (2015)

Arundic acid increases GLAST expression and transport activity in the mouse retina. (a) Effects of arundic acid treatment on GLAST, GLT1 and EAAC1 mRNA levels in the retina of GLAST+/− mice. Arundic acid (10 mg/kg, given daily from P22 to P35) increased the GLAST mRNA level (N=6), whereas the mRNA levels of GLT1 (N=6) and EAAC1 (N=6) are unaffected. *P<0.05 as determined by a Student's t-test. (b) Arundic acid (10 mg/kg) increases GLAST protein expression in the retina of GLAST+/− mice relative to vehicle-treated control mice (N=6). A representative western blot of GLAST protein expression is shown; the quantified data represent the mean±S.E.M. *P<0.05 as determined by a Student's t-test. (c) Effect of arundic acid on glutamate uptake activity in the retina of GLAST mutant mice. Relative glutamate uptake velocity was quantified from six independent experiments performed in duplicate for each data point. Data represent the mean±S.E.M. *P<0.05, **P<0.01 as determined by one-way ANOVA with Tukey–Kramer's post hoc analysis
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig2: Arundic acid increases GLAST expression and transport activity in the mouse retina. (a) Effects of arundic acid treatment on GLAST, GLT1 and EAAC1 mRNA levels in the retina of GLAST+/− mice. Arundic acid (10 mg/kg, given daily from P22 to P35) increased the GLAST mRNA level (N=6), whereas the mRNA levels of GLT1 (N=6) and EAAC1 (N=6) are unaffected. *P<0.05 as determined by a Student's t-test. (b) Arundic acid (10 mg/kg) increases GLAST protein expression in the retina of GLAST+/− mice relative to vehicle-treated control mice (N=6). A representative western blot of GLAST protein expression is shown; the quantified data represent the mean±S.E.M. *P<0.05 as determined by a Student's t-test. (c) Effect of arundic acid on glutamate uptake activity in the retina of GLAST mutant mice. Relative glutamate uptake velocity was quantified from six independent experiments performed in duplicate for each data point. Data represent the mean±S.E.M. *P<0.05, **P<0.01 as determined by one-way ANOVA with Tukey–Kramer's post hoc analysis
Mentions: To determine whether the effects of arundic acid are similar in vivo, we examined glutamate transporter expression in isolated mouse retinas treated with arundic acid. Three glutamate transporters are expressed around the synapses of RGCs in the plexiform layer, GLAST, GLT1 and excitatory amino-acid carrier 1 (EAAC1).21 We administered arundic acid or vehicle to GLAST+/− mice during postnatal day (P) 22 to P35, and evaluated the glutamate transporter expression levels by qPCR. Arundic acid treatment increased GLAST mRNA expression in the retinas of GLAST+/− mice, but did not alter GLT1 or EAAC1 mRNA levels (Figure 2a). Next, we asked whether arundic acid is capable of increasing GLAST protein expression in the retinas of GLAST+/− mice. Using western blot analysis, we found that 14-day arundic acid treatment significantly increased endogenous GLAST protein expression (Figure 2b). To test whether the increases in GLAST mRNA and protein expression are accompanied by enhanced glutamate transport activity, we conducted l-[3,4-3H]-glutamate uptake assays in isolated retinas from GLAST+/− and GLAST−/− mice. Fourteen-day arundic acid treatment led to a 1.23-fold increase in glutamate uptake in the retinas of GLAST+/− mice, compared with those treated with vehicle alone. We also observed that arundic acid increased GLAST protein expression and glutamate uptake activity in the retinas of wild-type mice (Supplementary Figures 2a and b). By contrast, arundic acid treatment did not affect glutamate uptake activity in the retinas of GLAST−/− mice (Figure 2c). These results suggest that arundic acid treatment increases retinal glutamate uptake activity by selectively increasing GLAST expression in vivo.

Bottom Line: To test this hypothesis, we examined the effect of arundic acid on GLAST expression and glutamate uptake.We found that arundic acid induces GLAST expression in vitro and in vivo.Thus, discovering compounds that can enhance EAAT1 expression and activity may be a novel strategy for therapeutic treatment of glaucoma.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

ABSTRACT
Glaucoma is the second leading cause of blindness worldwide and is characterized by gradual visual impairment owing to progressive loss of retinal ganglion cells (RGCs) and their axons. Glutamate excitotoxicity has been implicated as a mechanism of RGC death in glaucoma. Consistent with this claim, we previously reported that glutamate/aspartate transporter (GLAST)-deficient mice show optic nerve degeneration that is similar to that observed in glaucoma. Therefore, drugs that upregulate GLAST may be useful for neuroprotection in glaucoma. Although many compounds are known to increase the expression of another glial glutamate transporter, EAAT2/GLT1, few compounds are shown to increase GLAST expression. Arundic acid is a glial modulating agent that ameliorates delayed ischemic brain damage by attenuating increases in extracellular glutamate. We hypothesized that arundic acid neuroprotection involves upregulation of GLAST. To test this hypothesis, we examined the effect of arundic acid on GLAST expression and glutamate uptake. We found that arundic acid induces GLAST expression in vitro and in vivo. In addition, arundic acid treatment prevented RGC death by upregulating GLAST in heterozygous (GLAST(+/-)) mice. Furthermore, arundic acid stimulates the human GLAST ortholog, EAAT1, expression in human neuroglioblastoma cells. Thus, discovering compounds that can enhance EAAT1 expression and activity may be a novel strategy for therapeutic treatment of glaucoma.

Show MeSH
Related in: MedlinePlus