Ž . Molecular Brain Research 42 1996 350–353 Short communication A bcl-2 expressing viral vector protects cortical neurons from excitotoxicity even when administered several hours after the toxic insult William W.-G. Jia a,b, ) , Yihong Wang c , Dong Qiang c , Frank Tufaro d , Rodney Remington c , Max Cynader c a Department of Surgery and Ophthalmology, UniÕersity of British Columbia, 2550 Willow Street, VancouÕer, BC V5Z 3N9 Canada b Department of Medical Oncology, British Columbia Cancer Research Centre, VancouÕer, Canada c Department of Ophthalmology, UniÕersity of British Columbia, VancouÕer, Canada d Department of Microbiology and Immunology, UniÕersity of British Culumbia, VancouÕer, Canada Accepted 13 August 1996 Abstract The product of the bcl-2 oncogene has been shown to play an important role in apoptosis and programmed cell death. In this study, a herpes simplex virus type-1 vector was constructed to carry the human bcl-2 gene. The possible role of bcl-2 in protecting neurons from excitoxicity was investigated by using the viral vector to deliver the gene into neuronal cultures before or after the cells were exposed to glutamate under conditions in which 50–80% of neurons died. Infection with the bcl-2 expressing vector 24 h prior to glutamate treatment effectively prevented the cell death that normally follows this treatment. Moreover, infection with the vector as late as 8 h after the glutamate insult still resulted in substantial neuroprotective effects. These results have potential implications for new therapies in stroke or ischemic neuropathies. Keywords: Apoptosis; Glutamate; Cortical neuron; Bcl-2; Herpes simplex virus It has been widely reported that there is delayed neu- ronal death in the central nervous system after an ischemic w x insult 13 and much recent evidence supports the hypothe- w x sis that this type of cell death is apoptotic 3,8,10,13 and is related to elevated glutamate release from depolarized w x w x neurons 5,12 . Bcl-2 is a 26 kDa protein 14 that regu- lates cell survival and inhibits cell death induced by a w x variety of insults 7,9,15 . It has been shown that sympa- thetic neurons injected with the product of the bcl-2 gene are protected from apoptosis that normally results from wx growth factor deprivation 6 . Transferring the bcl-2 gene to neurons before glutamate treatment or ischemia has also w x been reported to confer neuroprotection 1,11 . We now report that expression of Bcl-2 from a herpes simplex virus vector can protect infected neurons from apoptosis induced by glutamate toxicity even when administered several hours after the glutamate insult. A herpes amplicon vector, avBcl-2, containing an HSV- 1 origin of replication and packaging site was constructed ) Ž . Corresponding author. Fax: q1 604 875-4663. to contain a human bcl-2 gene driven by a promoter from the immediate early gene of the human cytomegalovirus. Infectious viral stocks were generated with a replication defective helper virus. The bcl-2 gene was constructed down stream to a promoter from the immediate early gene of human cytomegalovirus. A mixed gliarneuron culture system was utilized to mimic the in vivo situation. Glial cells derived from rat cortex were first seeded in culture dishes for 1 week followed by layering of neurons from Ž prenatal rat cerebral cortex. Preliminary experiments data . not shown using neuron and glial specific markers showed that 95% of cells in the upper layers of these cultures were neurons. Neuronal cultures were infected with the avBcl-2 Ž . at various multiplicities of infection MOI . Viral stocks yielding over 60% positive staining in neuronal cells were used for the experiments. The majority of infected cells were neurons with only a few glial cells showing im- Ž . munopositive reactions to the Bcl-2 antibody Fig. 1 . Infection with helper virus only did not result in any Bcl-2 immunoreactivity, indicating that the viral infection itself did not induce expression of endogenous Bcl-2 in this culture system. No Bcl-2 expression was found in mock- infected cultures. 0169-328Xr96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. Ž . PII S0169-328X 96 00223-9