Calcium ion metabolism, excitotoxicity, and neuronal survival following calbindin D28k gene transfer via Herpes simplex virus amplicon vector

There is a pressing need for development of effective interventions which can prevent or ameliorate the calamitous effects of neuronal death resulting from neurologic insults or diseases. Since viral gene transfer vectors might serve to alter neuronal physiology prior to or immediately following a neurologic insult, the experiments reported here represent the first detailed study of the in vitro and in vivo neurotoxicity of Herpes simplex virus (HSV) amplicon vectors. This work demonstrates that viral vector toxicity can be influenced by the genetic makeup of the vector backbone, that one vector can be toxic to certain parts of the brain but not to others, that toxicity associated with vector infection may be due to components of the cells in which the vectors are generated; and that viral toxicity may be prevented by NMDA antagonists or by ultraviolet irradiation of the vectors prior to infection. A fuller understanding of the biology of such vectors is therefore imperative.; Excitatory amino acid (EAA) overstimulation of neurons causes a marked rise in cytoplasmic calcium ion concentration {dollar}rm(lbrack Casp{lcub}2+{rcub}rbracksb{lcub}i{rcub}){dollar} which is followed by neuron death from hours to days later. If the rise in {dollar}rmlbrack Casp{lcub}2+{rcub}rbracksb{lcub}i{rcub}{dollar} is prevented by placing Ca{dollar}sp{lcub}2+{rcub}{dollar} chelators in the cytosol of the stimulated cells, EAA neurotoxicity can be ameliorated. I tested whether calbindin D28k functions as an intracellular calcium ion buffer by constructing HSV amplicon vectors to deliver rat calbindin cDNA to hippocampal neurons in vitro. Neurons were infected with vectors delivering calbindin or a negative control, or mock-infected. Infected cells were made aglycemic after 12 or 24 hours of hypoglycemia, or were challenged with various glutamatergic insults or sodium cyanide, during fura-2 calcium ratiometric imaging. In response to hypoglycemic and glutamatergic challenges, neurons overexpressing calbindin mobilized less Ca{dollar}sp{lcub}2+{rcub}{dollar} as compared to controls. The calbindin vector decreased neuron death due to hypoglycemia, but not aglycemia, and due to glutamatergic but not hypoxic insults. These results are examined in the context of the potential of vectors like this for neuronal gene therapy.