The unique regulatory mechanisms guiding pituitary adenylate cyclase activating polypeptide (PACAP) expression reflect the functional diversity of PACAP in the nervous system. PACAP peptides not only have essential roles in basic neuronal communication and signaling, but also have neurotrophic properties that promote neuronal survival, mitosis, proliferation and differentiation. Using sympathetic neurons of the rat superior cervical ganglion (SCG), we showed that PACAP is dramatically induced only in models of neuronal injury and increased activity. Depolarization of sympathetic neurons, a well established model of activity-dependent modulation, stimulates cellular PACAP levels. Axotomy of neurons, a model of neuronal injury, augments PACAP gene expression, which may have functional roles in the regeneration responses to injury. The mechanisms underlying these striking inductions hinge on the expression of both long and novel short proPACAP transcripts, which we have shown to be generated by alternative 3' endonucleolytic cleavage and polyadenylation, and alternative 5' noncoding region exon splicing. Sequence elements in the 3' and 5' untranslated regions (UTR) of mature transcripts are critical for mRNA stability and translatability; shorter transcripts are usually more stable, and 5' noncoding region sequences definitively modulate translational efficiency. Hence, despite identical precursor protein coding regions, differences in the structures of the noncoding regions of alternative proPACAP transcript variants will have significant impact on mRNA stability and translatability, and, ultimately, on the amount of PACAP synthesized. Accordingly, these studies will test the hypotheses that (1) depolarization of sympathetic neurons induces expression of alternative 3' and 5' UTR proPACAP mRNA variants, (2) modulation of alternative PACAP transcript variant expression controls PACAP production by regulating transcript stability and translatability, (3) alternative proPACAP transcript variant expression is a key mechanism regulating PACAP induction during neuronal injury and repair, (4) increased neuronal PACAP contributes to the downstream induction of other neuropeptides, neuronal survival and neurite outgrowth. The proposed studies are important for understanding the molecular mechanisms and functional roles of PACAP in neuron regeneration and repair responses to injury. The results of these analyses will be immediately relevant not only with respect to neurotrophic functions modulating neuronal survival, mitosis, proliferation and differentiation, but also the transmitter roles of PACAP in neuronal communication.
