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Histone acetylation is implicated in the regulation of herpes simplex virus type 1 (HSV-1) latency. However, the role of histone acetylation in HSV-1 reactivation is less clear. In this study, the well established model system, quiescently-infected, neuronally-differentiated PC12 (QIF-PC12) cells, was used to address the participation of histone acetylation in HSV-1 reactivation. In this model, sodium butyrate and trichostatin A (TSA), two histone deacetylase inhibitors, stimulated production of infectious HSV-1 progeny from a quiescent state. To identify viral genes responsive to TSA, we analyzed representative α, β, and γ viral genes using quantitative real-time polymerase chain reaction. Only the latency-associated transcript (LAT) accumulated in response to TSA treatment, under culture conditions that restricted virus replication and spread. This led us to evaluate the importance of LAT expression on TSA-induced reactivation. In QIF-PC12 cells, the LAT deletion mutant virus dLAT2903 reactivated equivalently with its wild type parental strain (McKrae) after TSA treatment, as well as forskolin and heat stress treatment. Both viruses also reactivated equivalently from latently infected trigeminal ganglia explants from rabbits. In contrast, there was a marked reduction in the recovery of dLAT2903, as compared to wild type virus, from the eyes of latently infected rabbits following epinephrine iontophoresis. These combined in vitro, ex vivo and in vivo data suggest that LAT is not required for reactivation from latently infected neuronal cells per se, but may enhance processes that allow for the arrival of virus at, or close to, the site of original inoculation (i.e., recrudescence).

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A diverse body of evidence indicates a role for the lipid biomediator lysophosphatidic acid (LPA) in the CNS. This study identifies and characterizes the induction of neuronal death by LPA. Treatment of cultured hippocampal neurons from embryonic rat brains with 50~eM LPA resulted in neuronal necrosis, as determined morphologically and by the release of lactate dehydrogenase. A concentration of LPA as low as 10~iMled to the release of lactate dehydrogenase. In contrast, treatment of neurons with 0.1 or 1.0 j.tM LPA resulted in apoptosis, as determined by chromatin condensation. In addition, neuronal death induced by 1 ,aM LPA was characterized as apoptotic on the basis of terminal dUTP nick end-labeling (TUNEL) staining, externalization of phosphatidylserme, and protection against chromatin condensation, TU N EL staining, and phosphatidylserine externalization by treatment with N-benzyloxycarbonyl-Val-Ala-Aspfluoromethyl ketone, a broad-spectrum inhibitor of caspases, i.e., members of the interleukin-1/3 converting enzyme family. Studies with antagonists of ionotropic glutamate receptors did not indicate a significant role for these receptors in apoptosis induced by 1 1iM LPA. LPA (1 1iM) also induced a decrease in mitochondrial membrane potential. Moreover, pretreatment of neurons with cyclosporin A protected against the LPA-induced decrease in mitochondrial membrane potential and neuronal apoptosis. Thus, LPA, at pathophysiological levels, can induce neuronal apoptosis and could thereby participate in neurodegenerative disorders. Key Words: Lysophosphatidic acid-Apoptosis-Neurons-Mitochondrial membrane potential -Cyclosporin A.

The brain is a rich source of the lipid biomediator lysophosphatidic acid, and lysophosphatidic acid levels can significantly increase following brain trauma. Responses of primary rat brain astrocytes to this novel lipid are defined in the current study. Treatment of cells with lysophosphatidic acid resulted in a time‐ and dose‐dependent inhibition of glutamate uptake. Inhibition of glutamate uptake was specific because the related phospholipids, phosphatidic acid, lysophosphatidylcholine, and lysophosphatidylglycerol, did not inhibit this uptake under comparable conditions, i.e., treatment with 10 µM lipid for 30 min. Lysophosphatidic acid treatment of cells resulted in an increase in lipid peroxidation, as measured by the thiobarbituric acid assay. This increase in content of thiobarbituric acid‐reactive substances was largely inhibited by treatment with dithiothreitol or propyl gallate; however, such treatment did not affect the lysophosphatidic acid‐induced inhibition of glutamate uptake. Lysophosphatidic acid also inhibited glucose uptake with a dose‐response curve that paralleled the inhibition of glutamate uptake. By impairing uptake of glutamate by astrocytes, lysophosphatidic acid may exacerbate excitotoxic processes in various neurodegenerative conditions.

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Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA‐induced events in astrocytes were defined. As little as 1 nM LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca2+]i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca2+]i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca2+]i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca2+]i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA‐induced increase in [Ca2+]i was inhibited by a protein kinase C inhibitor, Ro 31‐8220, and a calcium‐dependent protein kinase C inhibitor, Gö 6976. The increase in [Ca2+]i was important for the LPA‐induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA‐stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA‐induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin‐sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca2+]i, lipid peroxidation, and DNA synthesis.