Identifying roles for peptidergic signaling in mice

Abstract

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 (PamKO/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. Conditional PamcKO/cKO mice, with loxP sites flanking the 2 exons deleted in the global PamKO/KO mouse, were indistinguishable from wild-type 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 in PAM myosin heavy chain 6 conditional knockout mice, and RNA sequencing analysis identified changes in gene expression in pathways related to cardiac function. Use of this screening platform should facilitate the development of therapeutic approaches targeted to peptidergic pathways.

Cell‐type specific knockout of peptidylglycine α‐amidating monooxygenase reveals specific behavioral roles in excitatory forebrain neurons and cardiomyocytes

“…As expected, PAM expression in the central nervous system of Pam Myh6‐cKO/cKO mice was unaltered 14 . Nevertheless, as observed in Pam +/− mice, Pam Myh6‐cKO/cKO mice exhibited increased anxiety‐like behavior 14 . The near‐total loss of atrial secretory granules and low serum levels of atrial natriuretic peptide (ANP) in Pam Myh6‐cKO/cKO mice suggest that anxiety‐like behavior is altered through a very different pathway 14,15 .…”

“…Using mice expressing Cre‐recombinase under control of the myosin heavy chain six promoter, PAM expression was eliminated in cardiomyocytes. As expected, PAM expression in the central nervous system of Pam Myh6‐cKO/cKO mice was unaltered 14 . Nevertheless, as observed in Pam +/− mice, Pam Myh6‐cKO/cKO mice exhibited increased anxiety‐like behavior 14 .…”

“…The distinctly different behavioral effects of deleting Pam only in excitatory neurons vs. reducing Pam levels in both excitatory and inhibitory neurons indicates an important role for amidated peptides 16‐24 . Previous examination of the cortex and hippocampus in Pam Emx1‐cKO/cKO mice revealed elimination of PAM expression in excitatory neurons, with no effect on Pam in GABAergic neurons 14 . In this study, expression of PAM and GAD in the basolateral and lateral amygdala of Pam Emx1‐cKO/cKO mice was evaluated and tests of social interaction were conducted.…”

“…Neuropeptides play a key role in modulating excitatory/inhibitory balance 12,13 . With the availability of viable mice with both alleles of Pam floxed, the biochemical, physiological and behavioral consequences of completely ablating PAM expression in excitatory forebrain neurons using mice expressing Cre‐recombinase under control of the Emx1 promoter was examined 14,15 . Unlike Pam +/− mice, Pam Emx1‐cKO/cKO mice exhibited decreased anxiety‐like behavior and increased cold tolerance 14 .…”

Physiological signaling in the absence of amidated peptides

“…ANP is known to decrease plasma sodium and thus works to maintain normal blood pressure. However, Powers et al (3) find that while PAM deletion in cardiac myocytes decreases ANP levels, it does not result in the predicted increases in blood pressure in mice subjected to a high-salt diet. This result is surprising, since global knockout of ANP in mice results in hypertension, even in mice on a normal salt diet (20).…”