Effect of preproTRH antisense on thyrotropin-releasing hormone synthesis and viability of cultured rat diencephalic neurons

To investigate a possible neurotropic role for thyrotropin-releasing hormon e (TRH) in the central nervous system, we used recombinant antisense TRH ad enovirus (TRHav) to "knock out" TRH in cultured 17-d fetal rat diencephalon . The morphology along with beta -galactosidase (beta -gal) enzyme histoche mistry (X-gal staining) and TRH content (femtomoles/well) were used to meas ure the effect of antisense TRH virus. Control adenovirus mediated beta -ga l transfection efficiency was nearly 85%, as shown by positive X-gal staini ng, and was without effect on cell morphology, TRH content, or the normal r esponse to glucocorticoid (dexamethasone) exposure with enhanced TRH expres sion. A significant 90% decline in TRH content as well as changes in neuron al morphology (shrunken cell bodies and short dendrites) were observed afte r 14 but not 7 d following TRHav treatment. The addition of synthetic TRH p eptide at 2.5 muM along with TRHav, but not dexamethasone, partly prevented the morphologic changes. No morphologic changes were seen in wild-type AtT 20 cells, a pituitary cell line that does not produce TRH. To investigate w hether neuronal death from loss of proTRH was owing to apoptosis, neuronal DNA change by means of fluorescent dye H-33342 staining, TUNEL staining, an d DNA laddering analysis was examined. Eighty to 90% positive H-33342 and T UNEL staining as well as a 180- to 200-bp DNA fragment on DNA laddering ana lysis were found as compared to control. These results indicate that proTRH gene expression prevents neuronal apoptosis and may play a role in neurona l development and function.