The expression and function of one subtype of serotonin (5-HT) receptors, the 5-HT2C receptor, is regulated by RNA editing and alternative splicing of its encoded pre-mRNA. In postmortem prefrontal cortical tissue from depressed suicide victims, the editing pattern of 5-HT2C pre-mRNA differs significantly from the editing pattern of control brains that could potentially result in a decreased efficiency of 5-HT2C receptor-G protein interactions. The overall objectives of the proposed research plan are to varify further a possible relationship between depression and an altered editing pattern of prefrontal cortical 5-HT2C pre-mRNA, and to define the possible functional consequences of this altered editing pattern and to test possible underlying mechanisms.
Three specific aims are proposed to attain these objectives.
In specific aim 1, the investigator proposes an extended replication of the differential 5-HT2C editing finding piloting this work. The 5-HT2C receptor editing pattern would be compared in brains of groups of male depressed suicide victims and depressed non-suicide subjects to assess the relative contribution of depression versus suicide. Editing patterns of 5-HT2C receptor mRNAs would be identified by nucleotide sequencing of cDNAs generated by RT-PCR. Proposed specific aim 2 would assess the functional significance of altered 5 HT2C receptor mRNA editing using stably transfected lines of NIH3T3 cells that express the three major altered 5-HT2C receptor isoforms associated with depression, as well as the more rare nonedited and fully edited isoforms. Functional assays would determine the comparative ability of the edited and nonedited isoforms to couple to G protein and activate the phospholipase C second messenger system. The effects of RNA editing on the guanyl nucleotide sensitivity of 5-HT2C receptor-G protein interactions, and the interaction of different receptor isoforms would also be preliminarily assessed. Related functional experiments would compare GTP(S binding to membranes of prefrontal cortical tissues of depressed suicide victims and depressed non-suicide subjects to determine the effect of 5-HT2C editing alterations associated with depression on basal and agonist-promoted activation of G proteins by 5-HT2C receptors.
Specific aim 3 would compare the effects of mechanistically distinct antidepressants, and the 5-HT2A/2C receptor antagonist ketanserin, on the neocortical 5-HT2C receptor RNA editing pattern, in wild-type mice. These studies would attempt to assess whether the observed alteration of 5-HT2C RNA editing in depression reflects medication effects.