We have investigated 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. Together with Dr. Josh Park, Uni. of Toledo, studied the role of the CPE-cytoplasmic tail in trafficking of secretory vesicles to the plasma membrane for secretion. 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 post-Golgi transport of ACTH/POMC vesicles to the process terminals of AtT20 cells for secretion. 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. These studies have been extended to lower vertebrates showing that pGlu-serpinin depresses myocardial performance in teleost and amphibian hearts, thus supporting an evolutionary role of serpinins in the sympatho-adrenergic control of the vertebrate heart. Studies in collaboration with Dr. Rina Rosin-Arbersfeld (TelAviv Univ,) showed negative regulation of the Wnt-3a pathway by CPE in HEK293 cells. Extracellular CPE binds to Wnt-3a-Frizzled complex and causes down-regulation of the wnt signaling by disrupting the signalosome route. In addition we found that CPE co-localizes and co-secretes with CPE in HEK293 cells. Overexpression of CPE or a non-enzymatic mutant CPE causes aggregation of CPE with Wnt3a in the ER, leading to decreased secretion of Wnt3a and decrease in wnt signaling. Thus CPE levels regulates the wnt-signaling pathway in cells, intracellularly and extracellularly, independent of its enzymatic activity. Physiologically, we showed in PC12cells and cortical neurons that CPE also known as NF-alpha1, can modulate NGF-induced neurite outgrowth by negatively regulating beta-catenin in the canonical Wnt-3a pathway, as well as Rho, an effector of the Wnt-3a non-canonical pathway. We also showed that Wnt-5a can complex with CPE and induce neurite outgrowth which can be enhanced by NGF. 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. NF-alpha1 is therefore a potential therapeutic target. To this end, we found that rosiglitazone, a PPARgamma agonist and anti-diabetic drug with additional anti-depression activities, induced the expression of CPE/NF-alpha1 and doublecortin expression when fed to mice. This indicates that PPAR-gamma agonists can be potentially useful as anti-depressant drugs. We have identified a CPE mutation in the cortex of an Alzheimer Disease (AD) patient which results in a CPE mutant protein with a string of nine amino acids added in the sequence, and we call this protein, CPE-QQ. When expressed in Neuro2a cells it was not secreted but degraded by the proteosomes. 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. Moreover, these mice also showed depressive-like behavior by the forced swim test. 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-1 in humans and identified a new gene, CPE/NF-alpha1, with a mutation that can cause neurodegeneration. 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. Furthermore, in vivo studies show that NF-alpha1 KO mice had 49% fewer GFAP+ astrocytes in the neocortex compared to WT-mice at postnatal day1, the time of astrocytogenesis. Our results indicate that NF-alpha1 plays a critical and novel role as an extracellular signal to differentiate neural stem cells into astrocytes for normal neurodevelopment.
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