Neuropeptides and Carboxypeptidase E/ Neurotrophic Factor-1 in Neural and Cognitive Functions

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

We have studied the role of the CPE-cytoplasmic tail in trafficking of secretory vesicles to the plasma membrane for secretion. In collaboration with Dr. Josh Park, University of Toledo, we showed that snapin binds directly to the short cytoplasmic tail of CPE on POMC vesicles and to dynactin which in turn binds to microtubule motors consisting of kinesin-2, or cytoplasmic dynein to mediate their transport wwin anterior pituitary AtT-20 cells. 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 (Univ. of Calabria), we investigated the effect of pGlu-serpinin, a CgA-derived peptide, on cardio-protection. pGlu-serpinin mimicked pre-conditioning and post-conditioning-induced cardioprotection in both WKY and SHR rats, as well as improved left ventricle function recovery after ischemia. In pGlu-serpinin mediated post-conditioning pharmacological cardiac protection, the mechanism involved the activation of the reperfusion injury salvage kinase (RISK) pathway. pGlu-serpinin also depressed myocardial performance in teleost and amphibian hearts, thus supporting an evolutionary role of serpinins in sympatho-adrenergic control of the vertebrate heart. Currently, the major focus of our research is on the novel, neurotrophic functions of CPE/NF-alpha1. A human with a null mutation of CPE has been reported to have severe learning disability besides obesity, diabetes and infertility due to lack of CPE. We have also identified a CPE mutation in an Alzheimer Disease (AD) patient which results in a CPE mutant protein with an additional nine amino acids that we named CPE-QQ. When expressed in Neuro2a cells, CPE-QQ was not secreted but degraded by proteosomes. Immunocytochemical studies showed CPE-QQ localized to the endoplasmic reticulum (ER) and overexpression in hippocampal neurons increased ER stress and decreased levels of pro-survival protein, BCL-2, resulting in increased neuronal cell death. Transgenic mice overexpressing CPE-QQ exhibited memory deficits in the Morris water maze test but their spatial learning ability was unimpaired. Moreover, these mice showed depressive-like behavior by the forced swim test. These mutant mice had fewer neurites in the hippocampal CA3 region and the dentate gyrus, and the medial prefrontal cortex, indicative of neurodegeneration. They showed diminished neurogenesis in the subgranular zone and hyperphosphorylation of tau at ser395, a hallmark of AD. These studies indicate that this human mutation in CPE/NF-alpha1 is neurotoxic, and lead to neurodegeneration and cognitive decline. In addition we have found another human mutation, W235R in CPE/NF-alpha1, that caused ER stress and neuronal cell death. Our in vitro studies showed that CPE/NF-alpha1 acts extracellularly as a neurotrophic factor, independent of its enzymatic activity to protect hippocampal neurons against oxidative stress via activation of the ERK- or AKT- pathways to up-regulate BCL-2 expression. The neuroprotective effect of CPE/NF-alpha1 was demonstrated in vivo using a transgenic knock-in mouse model expressing a non-enzymatic form of CPE/NF-alpha1, CPE-E342Q, but no WT form. Knock-out mice lacking CPE/NF-alpha1showed complete degeneration of the hippocampal CA3 region and cognitive dysfunction after social (maternal separation) and physical stress (ear tagging and tail clipping) associated the weaning paradigm. However, the CPE-E342Q mice, although lacking WT-CPE showed no degeneration or cognitive dysfunction after the weaning paradigm, similar to WT mice, indicating that CPE/NF-alpha1could prevent stress-induced hippocampal neurodegeneration and cognitive dysfunction, independent of its enzymatic activity. We also investigated CPE/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. Mice after short-term restraint stress increased hippocampal NF-alpha1, FGF2 and doublecortin, a marker for immature neurons, suggesting increased neurogenesis. NF-alpha1 added to cultured hippocampal neurons, increased 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, CPE/NF-alpha1 prevents stress-induced depression by up-regulating hippocampal FGF2 expression which leads to enhanced neurogenesis and anti-depressant activity. We found that rosiglitazone, a PPARgamma agonist and anti-diabetic drug which has anti-depression activities, induced the expression of CPE/NF-alpha1 and doublecortin expression when fed to mice. Thus PPAR-gamma agonists can be potentially useful anti-depressant drugs. We also studied the role of CPE/NF-alpha1 in embryonic development of the nervous system. We showed that addition of CPE/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. These CPE/NF-alpha1 treated neurospheres showed down-regulation of the wnt pathway protein, beta-catenin, which is known to promote proliferation. Differentiation studies using neurospheres in culture that were dissociated into single cells 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 day 1, the time of astrocytogenesis. Thus, NF-alpha1 plays a critical and novel role as an extracellular signal to differentiate neural stem cells into astrocytes for normal neurodevelopment. Recently, we have cloned three CPE mRNAs in embryonic mouse brain. They were 2.1, 1.9 and 1.73 kb in size, encoding 53kD, 47kD and 40kD proteins representing wild-type(WT) and two N-terminal truncated isoforms (CPE-deltaN), respectively. Interestingly, all these isoforms showed a surge of expression at embryonic E10.5 and at postnatal day 1. While CPE-WT continued to be expressed in adult mouse brain, the CPE-deltaN forms were not. Furthermore, we showed that overexpression of 40kD CPE-deltaN in a mouse hippocampal neuronal cell line and primary cortical neurons up-regulated the expression of Insulin-like growth factor binding protein 2, death associated protein 1 and ephrinA1 which mediate cell proliferation, programmed cell death and neuronal migration, respectively, during neurodevelopment. Our findings emphasize the importance of the CPE-NF-alpha1 in regulating embryonic neurodevelopment and mutations of the gene could lead to various neurological deficits.