The growth compromised HSV-2 mutant DeltaRR prevents kainic acid-induced apoptosis and loss of function in organotypic hippocampal cultures.
We have previously shown that the HSV-2 anti-apoptotic protein ICP10PK is delivered by the replication incompetent virus mutant DeltaRR and prevents kainic acid (KA)-induced epileptiform seizures and neuronal cell loss in the mouse and rat models of temporal lobe epilepsy. The present studies used DeltaRR and the ICP10PK deleted virus mutant DeltaPK to examine the mechanism of neuroprotection. DeltaRR-infected neuronal cells expressed a chimeric protein in which ICP10PK is fused in frame to LacZ (p175) while retaining ICP10PK kinase activity. DeltaPK-infected neuronal cells expressed a mutant ICP10 protein that is deleted in the PK domain and is kinase negative (p95). p175 and p95 were expressed in CA3 (86+/-3%) and CA1 (69+/-7%) cells from DeltaRR or DeltaPK-infected organotypic hippocampal cultures (OHC) and 80-85% of the ICP10 positive cells co-stained with antibody to beta(III) Tubulin (neuronal marker). DeltaRR, but not DeltaPK, inhibited KA-induced cell death and caspase-3 activation in CA3 neurons, an inhibition seen whether DeltaRR was delivered 2 days before or 2 days after KA administration (95% neuroprotection). Neuroprotection was associated with ERK and Akt activation and was abrogated by simultaneous treatment with the MEK (U0126) and PI3-K (LY294002) inhibitors. DeltaRR-mediated neuroprotection was associated with increased expression of the anti-apoptotic protein Bag-1 and decreased expression of the pro-apoptotic protein Bad. The surviving neurons retained normal synaptic function potentially related to increased expression of the transcription factor CREB. The data indicate that DeltaRR is a promising platform for neuroprotection from excitotoxic injury.
Gober
Michael D
MD
Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore, MD 21201-1559, USA.
Laing
Jennifer M
JM
Thompson
Scott M
SM
Aurelian
Laure
L
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D002470:Cell Survival / Q000502:physiology; D002522:Chlorocebus aethiops; D019610:Cytoprotection / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics*; D018258:Herpesvirus 2, Human / Q000235:genetics*; D006624:Hippocampus / Q000254:growth & development / Q000378:metabolism / Q000821:virology; D006801:Humans; D007608:Kainic Acid / Q000633:toxicity; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D050506:Mutant Chimeric Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000821:virology*; D009498:Neurotoxins / Q000633:toxicity; D009924:Organ Culture Techniques; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012264:Ribonucleotide Reductases / Q000235:genetics*; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured; D014709:Vero Cells
D050506:Mutant Chimeric Proteins; D009498:Neurotoxins; C100671:ICP10 protein, herpes simplex virus type 2; D012264:Ribonucleotide Reductases; D017346:Protein Serine-Threonine Kinases; D007608:Kainic Acid