We have investigated the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway(RSP). Using our CPE knockout (KO) mouse, we showed that 50% of newly synthesized POMC in primary cultures of the pituitary anterior lobe cells was degraded and suggests that in the absence of efficient sorting to the granules of the RSP due to the lack of CPE, POMC was targeted for degradation. However, some of the remaining POMC was sorted into the RSP. A candidate for a compensatory sorting receptor is Secretogranin III (SgIII), which has been shown to bind POMC. SgIII, is found in neuroendocrine cells and is involved in trafficking of chromogranin A (CgA) to the RSP. We used RNA interference (siRNA) to knock down SgIII and CPE expression in AtT20 cells, we demonstrated increased POMC secretion via the constitutive secretory pathway in both cases. Increased constitutive secretion of CgA was only observed in the SgIII knockdown cells. In double CPE-SgIII knock down cells, increased constitutive secretion of POMC was observed and stimulated secretion of ACTH was perturbed. These results demonstrate that CPE is involved in the trafficking of POMC to the RSP; and that SgIII may play a compensatory role for CPE in the sorting of POMC to the RSP in addition to a more general role in the RSP trafficking process. We recently found that transmembrane CPE is not only associated with large dense core vesicles (LDCVs), but also with synaptic vesicles (SVs) in mouse hypothalamus and synaptic-like microvesicles in PC12 cells. High K+ stimulated release of glutamate from hypothalamic neurons was diminished in CPE-KO mice. Electron microscopy revealed that the number of SVs located in the pre-active zone (within 200nm of the plasma membrane at the active zone) of synapses was significantly decreased in hypothalamic neurons of CPE-KO mice compared with wild-type mice. Total internal reflective fluorescence (TIRF) microscopy using PC12 cells as a model showed that overexpression of the CPE cytoplasmic tail reduced the steady-state level of synaptophysin-containing synaptic-like microvesicles accumulated in the area within 200 nm from the sub-plasma membrane (TIRF zone). Our findings show that the CPE cytoplasmic tail, which interacts with gamma adduccin and actin, is a new mediator for the localization of SVs in the actin-rich pre-active zone in hypothalamic neurons and the TIRF zone of PC12 cells. Our recent studies in pituitary AtT-20 cells have provided evidence for an autocrine mechanism for up-regulating LDCV biogenesis to replenish LDCVs following stimulated exocytosis of these vesicles. The autocrine signal was identified as serpinin, a novel 26 amino acid CgA-derived peptide cleaved from the C-terminal of CgA. Serpinin is released in an activity-dependent manner from LDCVs. Secreted serpinin was found to activate adenyl cyclase to increase cAMP levels, and protein kinase A in the cell. This then led to the translocation of the transcription factor sp1 from the cytoplasm into the nucleus and an increase in transcription of a protease inhibitor, protease nexin 1 (PN-1), which then inhibited granule protein degradation in the Golgi complex. The stabilization of those proteins increased their levels in the Golgi, resulting in significantly enhanced LDCV formation. We have also identified a N-terminal modified form of serpinin, pyroglutamate-serpinin (pGlu-serpinin) in pituitary AtT-20 cells and heart tissue. pGlu-serpinin was found to have neuroprotective activity against oxidative stress in AtT-20 cells and low K+-induced apoptosis in rat cortical neurons. In collaboration with Dr. Bruno Tota (University of Calabria), pGlu-serpinin was found to have positive inotropic activity in cardiac function, with no change in blood pressure and heart rate. pGlu-serpinin acts through a 1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway. pGlu-serpinin and other CgA derived cardioactive peptides emerge as novel -adrenergic inotropic and lusitropic modulators, Together, they can play a key role in how the myocardium orchestrates its complex response to sympathochromaffin stimulation. CPE plays a significant role in obesity, and recently the gene has been coined an obesity susceptibility gene. We showed that extremely obese CPE-KO mice have low bone mineral density and concluded that that the lack of processing of pro-CART to mature CART, a peptide that promotes bone formation, is likely responsible for the poor bone density in these mice. Additionally in collaboration with Dr. Lecka-Czernik (Univ. of Toledo), we found that CPE is enriched in a rat messenchymal stem cell line from bone marrow and thus CPE may be involved in regulating bone formation at another level. This possibility is currently being explored. CPE-KO mice have deficiencies in their nervous system function, including learning and memory. We showed that in 6-14 week old CPE-KO mice, dendritic pruning was poor in cortical and hippocampal neurons which would affect synaptogenesis. Additionally electrophysiological measurements showed a defect in the generation of long term potentiation (LTP) in hippocampal slices of these mice. This defect is attributed to the loss of neurons in the CA3 region of the hippocampus of CPE KO animals observed at 4 weeks of age and older. These neurons, which are normally enriched in CPE, were normal at 3 weeks of age just before the animals were weaned. When weaning was delayed a week, this degeneration was not observed till postnatal week 5 in the CPE KO mice. We have now shown that the degeneration is attributed to the stress of weaning which included maternal separation, ear tagging and tail clipping for genotyping. We also examined the effect of restrained stress on CPE expression in hippocampal neurons. When mice were subjected to acute restrained stress for 1h and then sacrificed 0, 1-24h post stress, they showed an immediate and transient decrease of CPE-mRNA expression in the hippocampus. In contrast after mild chronic stress (1h/day for 7days), which increases glucocorticoid secretion, the mice showed an increase in CPE mRNA and protein in the hippocampus, and no neuronal degeneration was evident. Furthermore, when hippocampal neurons were treated with synthetic glucocorticoid, dexamethasone, there was a significant increase in CPE mRNA and protein in the cells. These observations suggest that the increase in CPE may mediate neuronal survival during stress and lack of CPE results in neuronal degeneration. To this end, we have overexpressed or applied recombinant CPE exogenously in rat hippocampal neurons in culture and shown increased survival and neuroprotective effect of CPE on these neurons when subjected to oxidative stress with hydrogen peroxide treatment or glutamate cytotoxicity. The expression of CPE was examined in mouse embryos to determine if it could play a role in early embryonic development. We found that WT CPE and CPE-delta N mRNA was expressed as early as day E5.5 and increased each day, peaking at E8.5, falling slightly at E9.5 prior to expression of the endocrine system. CPE mRNA expression decreased sharply at E 10.5 -11.5 to below E5.5 levels and then increased sharply at E12.5 in parallel with the development of the endocrine system and continued to increase to adulthood. However, CPE-deltaN mRNA increased maximally at E10.5 followed by a precipitous decrease at E11.5-12.5, and then a small increase till PN1. In contrast to CPE, CPE-deltaN is absent in the adult hippocampus. In situ hybridization studies indicate that CPE and CPEdeltaN mRNA are expressed primarily in the fore brain and somites in mouse embryos. We have begun to study the role of CPE and CPE-delta N during embryonic development of the nervous system using neurospheres to study proliferation and differentiation.