Previous work has shown that the 68-kDa immediate-early protein of herpes simplex virus type 1 (HSV-1), also known as ICP22, is involved in the control of viral gene expression, although the precise mechanism remains to be elucidated. In order to study the function(s) of this protein, we constructed expression vectors containing the coding sequence of the ICP22 gene placed under the control of the SV40 or HCMV promoter. After cell transfection, ICP22 synthesis was studied by immunoblotting, using a specific antiserum. In transient expression experiments in COS cells in which the ICP22 vector was under the control of the SV40 promoter, we found that ICP22 was able to inhibit chloramphenicol acetyltransferase (CAT) expression under the control of either the alpha 22 (IE4) promoter or other immediate-early promoters, such as alpha 4 (IE3), alpha 0 (IE1), and alpha 27 (IE2). CAT expression under the control of the alpha 4 (IE3) promoter was inhibited in these cells by expression of ICP22 under the control of the HCMV promoter; it was also inhibited in RAT-1 cells by ICP22 expressed under the control of the SV40 or HCMV promoter. In contrast, CAT expression directed by the SV40 or HCMV promoters was only weakly or not inhibited by the ICP22 vectors. We also constructed an expression vector for UL13, a gene whose product is implicated in the phosphorylation of ICP22. Although CAT expression under the control of the alpha 4 (IE3) promoter was also negatively regulated by the UL13 gene product, the effects of the ICP22 (directed by the SV40 or HCMV promoter) and UL13 vectors were not synergistic; furthermore, at a particular molar ratio of the two vectors, inhibition of CAT activity was partially reversed. The results in the present work suggest that ICP22 can negatively regulate the expression of immediate-early viral genes and that its phosphorylation by UL13 protein kinase might be involved in the modulation of its function.

Astrocytes play a key role by catabolizing glutamate from extracellular space into glutamine and tricarboxylic acid components. We previously produced an astrocytic cell line that constitutively expressed glutamic acid decarboxylase (GAD67), which converts glutamate into GABA to increase the capacity of astrocytes to metabolize glutamate. In this study, GAD-expressing astrocytes in the presence of glutamate were shown to have increased energy metabolism, as determined by a moderate increase of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, by an increased ATP level, and by enhanced lactate release. These changes were due to GAD transgene expression because transient expression of a GAD antisense plasmid resulted in partial suppression of the ATP level increase. These astrocytes had an increased survival in response to glucose deprivation in the presence of glutamate compared with the parental astrocytes, and they were also able to enhance survival of a neuronal-like cell line (PC12) under glucose deprivation. This protection may be partially due to the increased lactate release by GAD-expressing astrocytes because PC12 cell survival was enhanced by lactate and pyruvate under glucose deprivation. These results suggest that the establishment of GAD expression in astrocytes enhancing glutamate catabolism could be an interesting strategy to increase neuronal survival under hypoglycemia conditions. Key Words: Glutamic acid decarboxylase transgene -Neuroprotection-Energetic metabolism-PC12 cells. J. Neurochem. 75, 56 -64 (2000).Astrocytes are responsible for extracellular homeostasis and secrete neurotrophic factors (Blakemore and Franklin, 1991;La Gamma et al., 1993). They remove glutamate from the extracellular medium by means of specific transporters (Rothstein et al., 1994) and can metabolize glutamate into glutamine using glutamine synthetase (Westergaard et al., 1995). The glutamate that enters astrocytes can also be metabolized in the tricarboxylic acid (TCA) cycle, providing energy (Sonnewald et al., 1997) and playing an important role, especially during the absence of carbohydrates (Bakken et al., 1998). Moreover, when the extracellular glutamate concentration is increased, glutamic acid decarboxylase (GAD) is activated, and GABA release is enhanced in GABAergic neurons (Weiss, 1988;Harris and Miller, 1989). This GABA release is increased in the presence of astrocytes (Westergaard et al., 1992).To increase glutamate metabolism in astrocytes, we previously transduced an astrocyte cell line with a retrovirus expressing the GAD transgene, directed by the glial fibrillary acid protein promoter, chosen because glial fibrillary acidic protein is one of the genes most commonly activated during focal injury (Mucke et al., 1991). The astrocytic clones were able to express functional GAD and release GABA in the presence of extracellular glutamate. The GAD transgene seems to be more efficient at catabolizing glutamate than the other glutamate catabolic pathways (Sacchettoni et al., 199...

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