c Impairment of astrocytic glutamate transporter (GLT-1; EAAT2) function is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD) and manganism, the latter being induced by chronic exposure to high levels of manganese (Mn). Mn decreases EAAT2 promoter activity and mRNA and protein levels, but the molecular mechanism of Mn-induced EAAT2 repression at the transcriptional level has yet to be elucidated. We reveal that transcription factor Yin Yang 1 (YY1) is critical in repressing EAAT2 and mediates the effects of negative regulators, such as Mn and tumor necrosis factor alpha (TNF-␣), on EAAT2. YY1 overexpression in astrocytes reduced EAAT2 promoter activity, while YY1 knockdown or mutation of the YY1 consensus site of the EAAT2 promoter increased its promoter activity and attenuated the Mn-induced repression of EAAT2. Mn increased YY1 promoter activity and mRNA and protein levels via NF-B activation. This led to increased YY1 binding to the EAAT2 promoter region. Epigenetically, histone deacetylase (HDAC) classes I and II served as corepressors of YY1, and, accordingly, HDAC inhibitors increased EAAT2 promoter activity and reversed the Mn-induced repression of EAAT2 promoter activity. Taken together, our findings suggest that YY1, with HDACs as corepressors, is a critical negative transcriptional regulator of EAAT2 and mediates Mn-induced EAAT2 repression.

Raloxifene (RX), a selective estrogen receptor modulator (SERM), exerts neuroprotection in multiple clinical and experimental settings. Astrocytic glutamate transporters GLT-1 (EAAT2) and GLAST (EAAT1) are the main glutamate transporters in the central nervous system, taking up most of excess glutamate from the synaptic cleft to prevent excitotoxic neuronal death. Since drugs targeting astrocytic glutamate transporters to enhance their expression and function represent potential therapeutics for neurodegenerative disorders associated with excitotoxicity, we tested if RX modulates the expression and function of GLT-1 and GLAST in rat primary astrocytes. The results showed that RX significantly increased glutamate uptake and expression of GLT-1 mRNA and protein levels. RX enhanced GLT-1 expression by the activation of multiple signaling pathways including ERK, EGFR and CREB mediated by estrogen receptors (ERs) ER-α, ER-β and GPR30. At the transcriptional level, NF-κB played a critical role in RX-induced GLT-1 expression as RX increased NF-κB reporter activity and induced binding of NF-κB p65 and p50 to the GLT-1 promoter. RX attenuated the reduction of GLT-1 expression and glutamate uptake induced by manganese (Mn) whose chronic high levels of exposure cause manganism. RX also upregulated GLAST by increasing its promoter activity and protein levels via the NF-κB pathway and ERs. Our findings provide new insight into the mechanism of RX-induced enhancement of GLT-1 and GLAST expression, as well as the attenuation of Mn-reduced expression of these transporters. These findings will be highly valuable for developing therapeutics of neurodegenerative diseases associated with impaired astrocytic glutamate transporters.

Background: Tamoxifen (TX), a selective estrogen receptor modulator, enhances glutamate transporter (GLT-1) expression in astrocytes. Results: TX up-regulated GLT-1 expression via the CREB and NF-B pathways. Conclusion: TX enhanced GLT-1 expression at the transcriptional level. Significance: Understanding the mechanisms of TX action on GLT-1 will contribute to the development of neuroprotectants against excitotoxicity.

Background:The mechanism for transcriptional regulation of EAAT1 remains to be elucidated. Results: EGF-activated NF-B is a positive regulator of EAAT1, whereas manganese-activated YY1, with HDACs acting as co-repressors, is a negative regulator. Conclusion: NF-B and YY1 are two critical transcriptional regulators of EAAT1. Significance: Identifying the molecular targets of EAAT1 regulation is crucial to develop therapeutics against neurological disorders associated with impairment of EAAT1.

Cellular ionic homeostasis, fundamentally K þ homeostasis, has been implicated as a critical regulator of apoptosis. The intracellular K þ efflux on apoptotic insult and suppression of apoptosis by high concentration of extracellular K þ or after inhibition of this efflux by K þ channel blockers have established the crucial role of K þ in turning on the apoptotic machinery. Several contrasting observations have reported the antiapoptotic effect of intracellular K þ concentration to be the result of inhibition of cytochrome c release from mitochondria, but the exact inhibitory mechanism remains obscure. However, here we show the blockage of K þ efflux during apoptosis did not affect cytochrome c release from the mitochondria, still completely inhibited the formation of the apoptosome comprising Apaf-1, cytochrome c, caspase-9 and other accessories. As a consequence of this event, procaspase-9, -3, -8 and other death-related proteins were not processed. Furthermore, physiological concentrations of K þ also inhibited the processing of procaspase-3 by purified caspase-8 or -9, the nucleosomal DNA fragmentation by purified DFF40/CAD and the nuclear fragmentation to varying extents. Altogether, these findings suggest that the efflux of K þ is prerequisite not only for the formation of the apoptosome but also for the downstream apoptotic signaltransduction pathways.