Volume 6 Issue 8 - November 28, 2008
A Possible Therapeutic Strategy for CNS Repair:To blockade calcium regulation of glutamate aspartate transporter regulation in astrocytes
Yu-Peng Liu1, Chung-Shi Yang2, Shun-Fen Tzeng1,*

1Department of Life Sciences, National Cheng Kung University, Tainan City, Taiwan
2Center for Nanomedicine Research, National Health Research Institutes, Zhunan, Taiwan

Journal of Neurochemistry. 2008 Apr;105(1):137-150.

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Astrocytes, the predominant glial population in the CNS, regulate neuronal maturation, neuronal metabolic/neurotransmitter homeostasis and synaptic plasticity in developing and adult CNS.  During excitatory synaptic transmission in the healthy CNS, glutamate is briefly released at a high concentration into the extracellular space, and normal physiological levels are maintained by re-uptake of glutamate through neuronal or glial glutamate transporters (Fig. 1). However, extracellular glutamate overload occurs after CNS traumatic injury and causes excessive Ca2+ influx into neurons through overactivation of neuronal ionotropic glutamate receptors, leading to neuronal excitotoxicity. Additionally, numerous glutamate-induced CNS neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), epilepsy, ischemia and traumatic brain injury, have been reported to associate with dysfunction of astrocytic glutamate transporters.  Therefore, the function of glutamate transporters, especially astrocytic glutamate transporters especially the astrocytic glutamate transporters (glutamate aspartate transporter; GLAST/EAAT-1) and glutamate transporter-1 (GLT-1/EATT-2) is of great importance for protecting neurons from glutamate-induced excitotoxicity. 
Figure 1. Glutamate transporters in astrocytes are responsible for synaptic glutamate homeostasis.

Cadmium (Cd) is a toxic heavy metal that is widely used in paints and in batteries. Cd is also found in foods, water, and tobacco leaves, and easily accumulates in many human organs, such as kidney, liver, lung, testis, bone, and the blood system. A number of studies have indicated that Cd enables to enter into the CNS via change of the blood-brain barrier (BBB) permeability or through the olfactory pathway. Cd entry into the CNS is thought to associate with severe neurodegenerative disorders, such as ALS and Alzheimer disease. The clinical study on the ALS patient in the nickel-Cd battery factory have revealed that Cd impairs BBB and enhances glutamate-induced excitotoxicity in the brain, implying that the dysfunction of astrocytic glutamate transporters may occur in Cd-contaminated microenvironment. In the present study, we showed that Cd at concentrations below lethal doses inhibited the GLAST expression in astrocytes that suppressed the astrocytic glutamate uptake activity. This provides evidence indicating the important role of Cd-induced Ca2+ influx in regulating the astrocytic GLAST expression.


Astrocytic GLT-1 and GLAST downrgulated by cadmium.
30-50% inhibition of astrocytic glutamate uptake was induced by exposure to CdCl2 at 5, 7 and 10 μM (Fig. 2A). Moreover, quantitative real time RT-PCR (Q-PCR) analysis indicated that GLAST mRNA expression was reduced by a 24 h-treatment with CdCl2 (Fig. 2B). Moreover, this inhibition in astrocytic GLAST mRNA expression was time-dependent. In addition, DHK, a selective inhibitor for GLT-1, was used to examine whether GLT-1 has a role in the glutamate uptake of Cd-treated astrocytes. Our result showed that DHK addition had no effect on the astrocytic glutamate uptake or the Cd-induced inhibition of glutamate uptake. The amount of intracellular glutamate is also mediated by cystine/glutamate exchanger so called system Xc- which drives the import of L-cystine into cells along with the efflux of L-glutamate to extracellular space. Therefore, to rule out the possibility that Cd may regulate system Xc- to reduce intracellular glutamate, we applied its inhibitor LQ to the culture. LQ did not change Cd-induced inhibition of astrocytic glutamate uptake ability. These findings support our suggestion that the decline of GLAST mRNA levels by Cd is a main factor for causing a reduction in the astrocytic glutamate uptake ability.

Transfection of astrocytes with human GLAST promoter construct showed that the hGLAST promoter activity in astrocytes was significantly reduced by CdCl2 (Fig. 2C). Was the reduction of GLAST mRNA levels due to the instability of GLAST mRNA in Cd-treated astrocytes? To answer this issue, the astrocytes were treated withCdCl2, followed by the application of actinomycin D to block gene transcription. Q-PCR analysis indicated that there was a decreased level of GLAST mRNA in Cd-treated astrocytes and control astrocytes along with a time-dependent pattern (Fig. 2D). However, Cd-treated astrocytes showed no accelerated rate of GLAST mRNA decay when compared to that observed in control astrocytes. Thus, our observations suggest that the decline of GLAST mRNA levels in Cd-treated astrocytes was resulted from the downregulation of GLAST transcription.
Figure 2.  Inhibition of astrocytic glutamate uptake and GLAST gene expression by CdCl2.   (A). Significant reduction of astrocytic glutamate uptake was observed after exposure to CdCl2. *p < 0.05; #p < 0.05 compared with the culture exposed to 5 μM CdCl2. (B). Astrocyte cultures were treated for 24 h by CdCl2 at the different concentrations. The cultures were then subjected to RNA extraction and Q-PCR analysis for GLAST mRNA levels. * p<0.05. (C). Astrocytes were transiently transfected by plasmid DNA containing human GLAST promoter for 24 h, and were then exposed for another 24 h to CdCl2 at the different concentrations. The cell lysate was subjected to the promoter activity assay. * p<0.05. (D). Astrocytes were exposed to10 μM of CdCl2 for 6 h, and followed by treatment with actinomycin D to inhibit gene transcription for different time periods. The inset indicates no difference in the rate of GLAST mRNA decay between control and Cd-treated groups. 

Dramatic increase in intracellular Ca2+ levels by cadmium.
Cd has been reported to increase intracellular Ca2+ levels, and thereby inhibit GABA-activated ion currents in snail neurons. To test the hypothesis that Cd might inhibit the transcription of astrocytic glutamate transporters through the Ca2+-dependent signaling pathway, the change of intracellular Ca2+ levels in astrocytes was examined after the addition of CdCl2. The application of CdCl2 (5 μM and 10 μM) to astrocytic cultures dramatically increased intracellular Ca2+ levels within 20 seconds (Fig. 3A). The results were further confirmed by the observation that 10 μM of CdCl2 failed to induce an increase in [Ca2+]i, when astrocytes were pretreated with the intracellular Ca2+ chelator, BAPTA-AM for 30 min (Fig. 3A). To further verify whether Cd-induced intracellular Ca2+ rise was due to Ca2+ influx, 10 μM of CdCl2 in Ca2+-free buffer was applied into the astrocytic cultures. As expected, an intracellular Ca2+ rise was not observed (Fig 3A), demonstrating that Cd causes an increase in [Ca2+]i via the induction of dramatic Ca2+ influx.

Ca2+ signaling involved in the inhibition of astrocytic glutamate uptake and GLAST mRNA expression.
The observations described as above prompted us to investigate the Ca2+ effect on the glutamate uptake activity of Cd-treated astrocytes. A set of experiments was then performed using extracellular Ca2+ chelator, EGTA, and intracellular Ca2+ chelator BAPTA-AM.  Cd-induced inhibition in astrocytic glutamate uptake activity was completely blocked when pre-exposure of astrocytes to the Ca2+ chelator, EGTA or BAPTA-AM for 5 min and 2 h, respectively (Fig. 3B). These findings indicate that the signaling pathway triggered by Cd-induced [Ca2+]i rise restrains the astrocytic glutamate uptake function. 

To determine the role of Cd-induced Ca2+ signaling in GLAST mRNA expression, astrocytes were pre-exposed to EGTA and BAPTA-AM. Pretreatment with EGTA completely abolished Cd effect on GLAST mRNA expression, and BAPTA-AM also substantially restored GLAST mRNA expression in Cd-treated astrcoytes. The effect of the two Ca2+ chelators on GLAST mRNA expression in Cd-treated astrocytes was confirmed by the observations that either EGTA or BAPTA-AM remarkably suppressed Cd-induced inhibition of the GLAST promoter activity (Fig. 3C). We also found that exposure of astrocytes to Cd increases the binding of AP-1 (activator protein-1) and transcription factor CRE binding protein B (CREB) to their DNA binding motifs derived from human GLAST promoter. This increase can be abolished by pretreatment with EGTA and BAPTA-AM. 
Figure 3.  Exposure to CdCl2 increases Ca2+ influx into astrocytes, and then induce the inhibition of GLAST gene expression. (A). A series of Ca2+ images indicate that intracellular Ca2+ levels in astrocytes were increased by CdCl2(5 μM and 10 μM), but decreased by 10 μM CdCl2 in the presence of BAPTA-AM. The time-course tracing of the relative fluorescence intensity F/F0 indicated that intracellular Ca2+ levels were increased at 20 or 30 seconds after the addition of CdCl2 (5 μM or 10 μM). (B). Astrocytes were pre-treated by distinct Ca2+ chelators. The cells were then exposed to CdCl2 (10 μM) for 24 h. GLAST mRNA expression was determined by Q-PCR. (C). Astrocytes were transiently transfected by plasmid DNA containing human GLAST promoter for 24 h, incubated in the medium containing EGTA and BAPTA-AM, and then followed by treatment with 10 μM CdCl2 for another 24 h. The cell lysate was subjected to the promoter activity assay. Noted that the inhibitors of several protein kinases had no effect on Cd-induced inhibition of GLAST gene expression. *p<0.05.

In conclusion, multiple Ca2+ signaling pathways are critically important in the inhibition of astrocytic glutamate uptake activity via the reduction of GLAST transcription, which may be regulated by Ca2+-activated AP-1/CREB and/or unknown repressors. Of the most significant is our study that the rise of intracellular Ca2+ in astrocytes during injury to the CNS may result in the elimination of astrocytic glutamate uptake function, which in turn may enhances secondary tissue damage. Therefore, the blockage of Ca2+ influx into astrocytes could be an effective CNS therapeutic strategy via the maintenance of astrocytic glutamate uptake activity.
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