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. Transport of hormone and BDNF vesicles to the plasma membrane for activity-dependent secretion is critical for endocrine function and synaptic plasticity. We showed that the cytoplasmic tail of a transmembrane form of CPE in hormone or BDNF-containing dense core secretory vesicles plays an important role in their transport to the release site in pituitary cells and hippocampus, respectively. Overexpression of the CPE cytoplasmic tail in the cytoplasm to compete with the endogenous tail diminished localization of endogenous POMC, BDNF and fluorescence-tagged CPE in the processes of endocrine AtT20 cells;and hippocampal neurons;as well as movement of BDNF- and POMC/CPE- vesicles to the processes in these cells. S-tagged CPE tail pulled down microtubule-based motors, dynactin (p150), dynein and KIF1A/KIF3A from AtT20 cell and brain cytosol, indicating their involvement in vesicle transport. Finally, overexpression of the CPE tail inhibited the regulated secretion of ACTH from AtT20 cells. We also showed that the CPE tail interacted with C-terminus of -adducin, a component of the cytoskeleton that binds and stabilizes F-actin. Overexpression of the C-terminal 38 amino acid of -adducin inhibited the exit of POMC vesicles at the trans-Golgi network. Thus these studies demonstrate that the vesicular CPE cytoplasmic tail plays a novel mechanistic role in facilitating the exit of POMC/ACTH vesicles from the TGN via -adducin/actin interaction;and anchoring POMC and BDNF vesicles to the microtubule-based motor system for transport along the processes to the plasma membrane for activity-dependent secretion in endocrine cells and neurons. We recently found that transmembrane CPE is not only associated with large dense core vesicles, 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 of synapses was significantly decreased in hypothalamic neurons of CPE-KO mice compared with WT mice. Total internal reflective fluorescence (TIRF) microscopy using PC12 cells 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, mediates the localization of SVs in the actin-rich pre-active zone in hypothalamic neurons and the TIRF zone of PC12 cells. We identified a novel 26 amino acid CgA-derived peptide from the C-terminal of CgA that regulates granule biogenesis in an autocrine way. Serpinin, released in an activity-dependent manner from LDCVs, can activate adenyl cyclase to increase cAMP levels, and protein kinase A in the cell leading to expression of a protease nexin 1 (PN1) by the transcription factor sp1Since PN1 inhibits granule protein degradation in the Golgi complex, their stabilization increases 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. Thus pGlu-serpinin is a potential drug for treating heart failure with no side effects seen with other positive inotropic drugs on the market. 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. However, recently, 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 we are currently investigating other possible roles of CPE in bone formation. CPE-KO mice have central nervous system deficiencies, 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 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. Our results demonstrate that the degeneration is correlated with the stress of weaning and maternal separation and that CPE may support neuronal survival. 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), the mice showed an increase in CPE mRNA and protein in the hippocampus, and no neuronal degeneration was evident. This is consistent with a neuroprotective role of CPE. To further investigate this role, we overexpressed CPE in rat hippocampal neurons in culture and found increased survival of these neurons. Additionally when we subjected these CPE overexpressing neurons to oxidative stress with hydrogen peroxide treatment, they were protected against apoptosis. Furthermore, when hippocampal neurons were treated with synthetic glucocorticoid, dexamethasone, there was a significant increase in CPE mRNA and protein in the cells. These findings taken together support our hypothesis that stress-induced secretion of glucocorticoid up-regulates CPE expression in hippocampal neurons to protect them from degeneration in vivo.

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Xiao, Lan; Yang, Xuyu; Sharma, Vinay Kumar et al. (2018) Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-?l variants in embryonic mouse brain. FASEB J :fj201800359R
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