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 hormone (TRH) in the central nervous system, we used recombinant antisense TRH adenovirus (TRHav) to "knock out" TRH in cultured 17-d fetal rat diencephalon. The morphology along with beta-galactosidase (beta-gal) enzyme histochemistry (X-gal staining) and TRH content (femtomoles/well) were used to measure the effect of antisense TRH virus. Control adenovirus mediated beta-gal transfection efficiency was nearly 85%, as shown by positive X-gal staining, and was without effect on cell morphology, TRH content, or the normal response to glucocorticoid (dexamethasone) exposure with enhanced TRH expression. A significant 90% decline in TRH content as well as changes in neuronal morphology (shrunken cell bodies and short dendrites) were observed after 14 but not 7 d following TRHav treatment. The addition of synthetic TRH peptide at 2.5 microM along with TRHav, but not dexamethasone, partly prevented the morphologic changes. No morphologic changes were seen in wild-type AtT20 cells, a pituitary cell line that does not produce TRH. To investigate whether neuronal death from loss of proTRH was owing to apoptosis, neuronal DNA change by means of fluorescent dye H-33342 staining, TUNEL staining, and DNA laddering analysis was examined. Eighty to 90% positive H-33342 and TUNEL staining as well as a 180- to 200-bp DNA fragment on DNA laddering analysis were found as compared to control. These results indicate that proTRH gene expression prevents neuronal apoptosis and may play a role in neuronal development and function.
Luo
L G
LG
Division of Endocrinology, Rhode Island Hospital, Brown Medical School, Providence 02903, USA.
Lee
S L
SL
Lechan
R M
RM
Jackson
I M
IM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D002470:Cell Survival; D002478:Cells, Cultured; D053938:DNA Fragmentation; D016373:DNA, Antisense / Q000494:pharmacology*; D004027:Diencephalon / Q000166:cytology*; D004622:Embryo, Mammalian; D007031:Hypothalamus / Q000166:cytology; D020287:In Situ Nick-End Labeling; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics*; D051381:Rats; D013973:Thyrotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D016373:DNA, Antisense; D011498:Protein Precursors; D013973:Thyrotropin-Releasing Hormone; D001616:beta-Galactosidase