Molecular Dissection of Melatonin Synthesis In Vivo

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DESCRIPTION: (provided by applicant) Melatonin is a nocturnal hormone rhythmically synthesized and released from the pineal gland due to the actions of four enzymes: tryptophan hydroxylase (TPFI), aromatic amino acid decarboxylase (AAADC), serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Although current evidence suggests that in rats cAMP signaling mediates both transcriptional and post-transcriptional control of melatonin formation, little is known about the in vivo targets of cAMP in the pineal. We hypothesize that cAMP regulates melatonin synthesis principally by PKA activation, which phosphorylates key proteins involved in the biosynthesis of melatonin; phosphorylation of these proteins results in a combination of increased transcription and decreased degradation of these biosynthetic enzymes. We plan to investigate the role of the cAMP signaling pathway in pineal circadian rhythms in the intact animal by using an integrated molecular and physiological approach. Aim 1 will establish the importance of the cAMP-dependent protein kinase (PKA) and CREB in transcriptional activation of NAT and melatonin formation in vivo. We will determine the effects of pharmacological inhibitors and activators of PKA catalysis in vivo by microdialysis and examine their influence on NAT transcriptional activation and repression during the day and night. We will also deliver recombinant adenoviral vectors expressing A-CREB or constitutively active PKA into the intact pineals to examine their effects on NAT mRNA and melatonin production. Aim 2 will evaluate the role of FKA in NAT protein stability in vivo. We will utilize a phospho-NAT specific antibody to study the functional significance of PKA-mediated NAT phosphorylation; we will characterize NAT from a mutant strain of rat we discovered that has lower NAT protein level due to a point mutation in a PKA phosphorylation site; lastly, we will use in vivo viral vector delivery and in vivo on-line microdialysis to study the stability and function of NAT mutants. Aim 3 will characterize the role of cAMP in the generation of our newly discovered tri-phasic circadian serotonin release in the pineal in vivo by again utilizing pharmacologic and molecular manipulations of cAMP signaling combined with in vivo physiological measurements of serotonin rhythms. These experiments will further our understanding of the in vivo signal transduction mechanisms that control transcriptional and post-transcriptional activation of melatonin and serotonin formation, which may play a role in the pathogenesis of sleep, psychiatric, and neurological disorders.