We continue to investigate the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway (RSP). Both proteins have been shown to be capable of trafficking POMC into the RSP, a mechanism that may reflect multiple components working together to accomplish the very important task of an endocrine cell, that being the controlled regulated secretion of bioactive peptide hormones. Using RNA interference to knock down SgIII and CPE, we showed that both proteins affect the normal secretion of POMC in AtT20 cells, i.e. POMC was secreted at an elevated rate through the constitutive secretory pathway when either CPE or SgIII are reduced. When both are knocked-down, the affect is augmented, suggesting that POMC trafficking is dependent on both proteins for efficient trafficking to the RSP for subsequent processing to ACTH. With Dr. Josh Park, Uni. of Toledo, we showed that snapin connects a microtubule motor complex consisting of kinesin-2, cytoplasmic dynein, and dynactin to the cytoplasmic tail of CPE on POMC vesicles to mediate their transport in anterior pituitary AtT-20 cells. Snapin directly binds to CPE cytoplasmic tail and interacts with microtubule motors. Overexpression of snapin reduced process-localization, processivity and velocity of movement of ACTH/POMC vesicles, similar to overexpression of CPE C-terminal tail. Knockdown of snapin decreased stimulated ACTH secretion. Moreover, A kinase anchor protein 150 (AKAP150), a scaffold for protein kinase A and calcineurin, associate with snapin-microtubule motor complex to facilitate the process-localization of ACTH/POMC vesicles. Thus, our study uncovered a new molecular complex that mediates and possibly regulates post-Golgi transport of ACTH/POMC vesicles to the process terminals of AtT20 cells. With Dr. Bruno Tota (Uni. of Calabria), we have investigated the effect of pGlu-serpinin, a CgA-derived peptide, on cardio-protection. Using normotensive (WKY) and hypertensive (SHR) rats as models; we showed that pGlu-serpinin mimicked pre-conditioning and post-conditioning-induced cardioprotection. In both WKY and SHR rats, pGlu-serpinin improved left ventricle function recovery after ischemia. Moreover, it reduced ischemic induced contracture state and decreased infarct size. In pGlu-serpinin mediated post-conditioning pharmacological cardiac protection, the mechanism involved the activation of the reperfusion injury salvage kinase (RISK) pathway. Our recent work showing the negative regulation of Wnt-3a by CPE in collaboration with Dr. Rina Rosin-Arbersfeld spurred the investigation of neurite outgrowth in PC12 cells and cortical neurons by Wnt-3a and Wnt-5a and the role CPE/NF-alpha1 may play. We have shown that CPE/NF-alpha1 can modulate the NGF-induced neurite outgrowth by reducing B-catenin in the canonical Wnt-3a pathway as well as Rho, an effector of the Wnt-3a non-canonical pathway. Interestingly, we showed that Wnt-5a can complex with CPE and induce neurite outgrowth which can be enhanced by NGF. Recently we have investigated NF-alpha1 in preventing restraint stress-induced depression. Prolonged (6h/d for 21 days), but not short-term (1h/d for 7d) restraint stress reduced fibroblast growth factor 2 (FGF2) in the hippocampus, leading to depressive-like behavior in mice. We found that mice after short-term restraint stress increased hippocampal NF-alpha1, FGF2 and doublecortin, a marker for immature neurons, suggesting increased neurogenesis. Indeed we showed that in cultured hippocampal neurons, exogenous NF-alpha1 could increase FGF2 expression. Moreover, NF-alpha1-KO mice exhibited severely reduced hippocampal FGF2 levels and immature neuron numbers in the subgranular zone. These mice displayed depressive-like behavior that was rescued by FGF2 administration. Thus, NF-alpha1 prevents stress-induced depression by up-regulating hippocampal FGF2 expression which leads to enhanced neurogenesis and anti-depressant activity. Analysis of the CPE promoter identified potential PPARgamma binding sites that could induce its expression. We showed that rosiglitazone, a PPARgamma agonist and anti-diabetic drug with additional anti-depression activities, could induce the expression of CPE/NF-alpha1 in Neuro2a cells and hippocampal neurons. The induced NF-alpha1 protected the cells against oxidative stress induced by hydrogen peroxide through up-regulation of BCL-2, a pro-survival mitochondrial protein. The expression of NF-alpha1 and a splice variant isoform, CPE-deltaN was examined in mouse embryos to determine if it could play a role in development and neuroprotection of embryos. We found that WT CPE and CPE-deltaN 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 decrease at E11.5-12.5, and then a small increase till PN1. In contrast to NF-alpha1, CPE-deltaN is virtually absent in the adult brain. In situ hybridization studies indicate that WT CPE and CPEdeltaN mRNA are expressed primarily in the fore brain and somites in mouse embryos. We showed that CPE-deltaN plays a role in protecting embryonic cortical and hippocampal neurons from glutamate neurotoxicity and oxidative stress through up-regulating FGF2 expression which then increased the pro-survival protein, BCl2. We also studied the role of NF-alpha1 during embryonic development of the nervous system using neurospheres to study proliferation and differentiation. Exogenous addition of NF-alpha1 to E13.5 neocortex-derived neurospheres, which contains stem cells and neuroprogenitors, resulted in reduced proliferation of the neurospheres without causing cell death. NF-alpha1 down-regulated the wnt-pathway in the neurospheres leading to reduced levels of beta-catenin which is known to enhance proliferation. Differentiation studies using neurospheres from 7d cultures that were dissociated into single cells and cultured for an additional 5d showed an increase in astrocytes in the presence of NF-alpha1, without altering the percentage of neuronal and oligodendrocyte populations. Interestingly, dissociated cells from neurospheres derived from NF-alpha1-KO mouse embryos showed decreased astrocytes and increased neurons. Our results suggest a novel role of NF-alpha1 as an extracellular signal to differentiate neural stem cells into astrocytes. We identified a mutant CPE from the cortex of an Alzheimer Disease (AD) patient which we call CPE-QQ. When expressed in Neuro2a cells it was not secreted but degraded by the proteosome. Immunocytochemical studies showed CPE-QQ co-stained with Calnexin, an ER marker and overexpression in hippocampal neurons increased levels of ER stress marker CHOP, decreased levels of pro-survival protein, BCL-2, and increased neuronal cell death. This indicates that CPE-QQ induces cell death through ER stress and down regulation of BCL-2. Transgenic mice overexpressing CPE-QQ exhibited memory deficits as tested by the Morris water maze but their spatial learning ability was unimpaired. These mutant mice showed less neurites in the CA3 region and the dentate gyrus of the hippocampus and the medial prefrontal cortex, indicative of neurodegeneration. Moreover they showed diminished neurogenesis in the subgranular zone and hyperphosphorylation of tau at ser395, a hallmark of AD. These studies have substantiated a neuroprotective role of CPE/NF&#61537;-1 in humans and identified a new gene, CPE/NF-alpha1, with a mutation that can cause neurodegeneration linked to AD and other dementia.