Expression of the cyclic AMP-dependent protein kinase (PKA) catalytic subunit 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 analogues. Since activation of protein kinase A by cAMP is likely to be the pathway by which it exerts its survival-promoting effect, we have tested the feasibility of using herpes simplex virus (HSV) as a vector for expressing survival-promoting genes in neurons by cloning the catalytic subunit of the cAMP-dependent protein kinase (PKAcat) with a metallothionein gene promoter into the HSV thymidine kinase gene by homologous 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 superior 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 approximately 3.5-fold lower in the first 24 h. Although the level of the inhibitory regulatory subunit type I was raised by 18 h, this is unlikely to completely explain the transient activity of PKAcat. When neurons were induced to express maximum PKAcat levels in the presence of NGF and then deprived of NGF, survival was extended by up to 2 days, demonstrating a direct role for PKA in promoting survival. By this time, some neurite degeneration was beginning which appeared to be partly due to toxic effects of the virus. However, replenishment with NGF supported further survival, showing that at this time the neurons were still viable. Similar rates of survival were obtained using a tsK-based PKAcat vector, but no significant survival was obtained with parental HSV or tsK virus strains. These data demonstrate the feasibility, and highlight some of the problems, of using HSV-based vectors as tools for expressing functional survival proteins in sympathetic neurons.
Buckmaster
E A
EA
Department of Human Anatomy, University of Oxford, UK.
Tolkovsky
A M
AM
D000345:Affinity Labels; D000818:Animals; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D005822:Genetic Vectors / Q000502:physiology*; D007150:Immunohistochemistry; D008715:Methionine / Q000378:metabolism; D009414:Nerve Growth Factors / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D009474:Neurons / Q000166:cytology*; D051381:Rats; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism*; D013564:Sympathetic Nervous System / Q000166:cytology*; D014158:Transcription, Genetic
D000345:Affinity Labels; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D008715:Methionine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases