Identifying Novel Roles for Peptidergic Signaling in Mice

Despite accumulating evidence demonstrating the essential roles played by neuropeptides, it has proven challenging to use this information to develop therapeutic strategies. Peptidergic signaling can involve juxtacrine, paracrine, endocrine and neuronal signaling, making it difficult to define physiologically important pathways. One of the final steps in the biosynthesis of many neuropeptides requires a single enzyme, peptidylglycine α-amidating monooxygenase (PAM), and lack of amidation renders most of these peptides biologically inert. PAM, an ancient integral membrane enzyme that traverses the biosynthetic and endocytic pathways, also affects cytoskeletal organization and gene expression. While mice, zebrafish and flies lacking Pam ( Pam KO/KO ) are not viable, we reasoned that cell-type specific elimination of Pam expression would generate mice that could be screened for physiologically important and tissue-specific deficits. Pam cKO/cKO mice, with loxP sites flanking the 2 exons deleted in the global Pam KO/KO mouse, were indistinguishable from wildtype mice. Eliminating Pam expression in excitatory forebrain neurons reduced anxiety-like behavior, increased locomotor responsiveness to cocaine and improved thermoregulation in the cold. A number of amidated peptides play essential roles in each of these behaviors. Although atrial natriuretic peptide (ANP) is not amidated, Pam expression in the atrium exceeds levels in any other tissue. Eliminating Pam expression in cardiomyocytes increased anxiety-like behavior and improved thermoregulation. Atrial and serum levels of ANP fell sharply Pam Myh6-cKO/cKO in mice and RNASeq analysis identified changes in gene expression in pathways related to cardiac function. Use of this screening platform should facilitate the development of new therapeutic approaches targeted to peptidergic pathways. SIGNIFICANCE Peptidergic signaling, which plays key roles in the many pathways that control thermoregulation, salt and water balance, metabolism, anxiety, pain perception and sexual reproduction, is essential for the maintenance of homeostasis. Despite the fact that peptides generally signal through G protein coupled receptors, it has proven difficult to use knowledge about peptide synthesis, storage and secretion to develop effective therapeutics. Our goal was to develop an in vivo bioassay system that would reveal physiologically meaningful deficits associated with disturbed peptidergic signaling. We did so by developing a system in which an enzyme essential for the production of many bioactive peptides could be eliminated in a tissue-specific manner.