Borna disease virus (BDV) frequently persists in the brain of infected animals.To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system. Borna disease virus (BDV) is a neurotropic, enveloped virus with a nonsegmented negative-strand RNA genome (2). It naturally infects the central nervous system (CNS) of a broad range of mammalian species (14), where it efficiently establishes persistence in neuronal and nonneuronal cells (4). We recently reported the recovery of a recombinant BDV from cDNA (7, 13) that expresses the green fluorescent protein (GFP) from an additional transcription unit integrated near the 5Ј end of its genome (12). The first-generation GFP vector originating from tissue culture-adapted BDV strain He/80 (13) was severely attenuated in the CNS of adult rats (12) and unable to productively infect mice (our unpublished data). Although mice are resistant to infections with primary BDV isolates, the virus can be adapted to gain replication competence in the CNS of these animals (6, 9). After serial passage of recombinant BDV strain He/80 in the brains of MRL mice, we identified five point mutations that confer replication competence in mice (1). Three of these mutations cause amino acid changes, two in the polymerase L and one in the phosphoprotein P. The other two mutations are silent and located within putative regulatory sequences flanking the initiation codon of the X gene. In a previous study we showed that incorporation of the two adaptive mutations into the L gene (L RD ) improves the growth properties of the GFP-expressing virus in the CNS of rats (12), but the virus remained severely growth retarded in mice (our unpublished data). The mutation in the P gene was previously shown to reduce the sensitivity of the viral polymerase complex for the inhibitory activity of X (1). A recombinant virus carrying this P mutation in combination with the two mutations in L (BDV-P K L RD ) showed strongly enhanced replication speed in mice, whereas the silent mutations within the X gene were found to reduce replication speed and pathogenicity of BDV-P K L RD in MRL mice (1) and rats (our unpublished data). In the present study we incorporated all five adaptive mutations into the BDV genome in order to generate a fully mouse-adapted GFP-expressing BDV vector, designated mBDV-GFP. We found that mBDV-GFP productively infected the central and the peripheral nervous systems of C57BL/6 mice and expressed easily detectable amounts of GFP during the e...

Oxygen deprivation during ischemic or hemorrhagic stroke results in ATP depletion, loss of ion homeostasis, membrane depolarization, and excitotoxicity. Pharmacologic restoration of cellular energy supply may offer a promising concept to reduce hypoxic cell injury. In this study, we investigated whether carbimazole, a thionamide used to treat hyperthyroidism, reduces neuronal cell damage in oxygen-deprived human SK-N-SH cells or primary cortical neurons. Our results revealed that carbimazole induces an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) that was associated with a marked inhibition of global protein synthesis. Translational inhibition resulted in significant bioenergetic savings, preserving intracellular ATP content in oxygen-deprived neuronal cells and diminishing hypoxic cellular damage. Phosphorylation of eEF2 was mediated by AMP-activated protein kinase and eEF2 kinase. Carbimazole also induced a moderate calcium influx and a transient cAMP increase. To test whether translational inhibition generally diminishes hypoxic cell damage when ATP availability is limiting, the translational repressors cycloheximide and anisomycin were used. Cycloheximide and anisomycin also preserved ATP content in hypoxic SK-N-SH cells and significantly reduced hypoxic neuronal cell damage. Taken together, these data support a causal relation between the pharmacologic inhibition of global protein synthesis and efficient protection of neurons from ischemic damage by preservation of high-energy metabolites in oxygen-deprived cells. Furthermore, our results indicate that carbimazole or other translational inhibitors may be interesting candidates for the development of new organprotective compounds. Their chemical structure may be used for computer-assisted drug design or screening of compounds to find new agents with the potential to diminish neuronal damage under ATP-limited conditions.

Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited.