EXPRESSION OF THE CYCLIC-AMP-DEPENDENT PROTEIN-KINASE (PKA) CATALYTICSUBUNIT FROM A HERPES-SIMPLEX VIRUS VECTOR EXTENDS THE SURVIVAL OF RAT SYMPATHETIC NEURONS IN THE ABSENCE OF NGF

Superior cervical ganglion neurons from neonatal rats are dependent on nerve growth factor for their survival both in vivo and in vitro. In culture this requirement can be largely replaced by cAMP or its analog ues. Since activation of protein kinase A by cAMP is likely to be the pathway by which it exerts its survival-promoting effect, we have test ed the feasibility of using herpes simplex virus (HSV) as a vector for expressing survival-promoting genes in neurons by cloning the catalyt ic subunit of the cAMP-dependent protein kinase (PKAcat) with a metall othionein gene promoter into the HSV thymidine kinase gene by homologo us recombination. About 95% of the neurons became infected using 2.5 p .f.u. per cell. When this construct was used to express PKAcat in supe rior cervical ganglion neurons, in the presence of nerve growth factor (NGF) increases of 1.9- to 2.4-fold in PKA activity were found 8-10 h after infection; levels remained elevated (1.4- to 2.1-fold) up to 18 h, returning to basal by 24 h. After infection in the absence of NGF, cumulative activity over 24 h was similar to 3.5-fold lower in the fi rst 24 h. Although the level of the inhibitory regulatory subunit type I was raised by 18 h, this is unlikely to completely explain the tran sient activity of PKAcat. When neurons were induced to express maximum PKAcat levels in the presence of NGF and then deprived of NGF, surviv al was extended by up to 2 days, demonstrating a direct role for PKA i n promoting survival. By this time, some neurite degeneration was begi nning which appeared to be partly due to toxic effects of the virus. H owever, replenishment with NGF supported further survival, showing tha t at this time the neurons were still viable. Similar rates of surviva l were obtained using a tsK-based PKAcat vector, but no significant su rvival was obtained with parental HSV or tsK virus strains. These data demonstrate the feasibility, and highlight some of the problems, of u sing HSV-based vectors as tools for expressing functional survival pro teins in sympathetic neurons.