We have studied the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway (RSP). Both proteins were 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 effect was 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, Univ. of Toledo, we studied the role of the CPE-cytoplasmic tail in trafficking of secretory vesicles to the plasma membrane for secretion. We showed that snapin binds directly to the cytoplasmic tail of CPE on POMC vesicles and connects it to a microtubule motor complex consisting of kinesin-2, cytoplasmic dynein, and dynactin to mediate their transport via microtubules in 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 our major focus is on discovering novel non-enzymatic functions of CPE/NF-alpha1 as a neurotrophic factor. 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 levels of ER stress marker CHOP, decreased levels of pro-survival protein, BCL-2, and 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 substantiate a neuroprotective role of CPE/NF-alpha1 in humans and identified CPE/NF-alpha1, as a new gene that can cause neurodegeneration when mutated. 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. 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 studied the role of CPE/NF-alpha1 in embryonic development of the nervous system. The wnt-pathway is highly involved in development. In collaboration with Dr. Rina Rosin-Arbersfeld (Tel Aviv Univ.) we 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 wnt signaling by disrupting the signalosome route. We further 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 gene in neurodevelopment and mutations could lead to various neurological deficits.

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43
Fiscal Year
2018
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U.S. National Inst/Child Hlth/Human Dev
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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
Imbrogno, S; Mazza, R; Pugliese, C et al. (2017) The Chromogranin A-derived sympathomimetic serpinin depresses myocardial performance in teleost and amphibian hearts. Gen Comp Endocrinol 240:1-9
Cong, Lin; Cheng, Yong; Cawley, Niamh X et al. (2017) A Novel Single Nucleotide T980C Polymorphism in the Human Carboxypeptidase E Gene Results in Loss of Neuroprotective Function. PLoS One 12:e0170169
Selvaraj, Prabhuanand; Xiao, Lan; Lee, Cheol et al. (2017) Neurotrophic Factor-?1: A Key Wnt-?-Catenin Dependent Anti-Proliferation Factor and ERK-Sox9 Activated Inducer of Embryonic Neural Stem Cell Differentiation to Astrocytes in Neurodevelopment. Stem Cells 35:557-571
Qin, X-Y; Wu, H-T; Cao, C et al. (2017) A meta-analysis of peripheral blood nerve growth factor levels in patients with schizophrenia. Mol Psychiatry :
Cawley, Niamh X; Rathod, Trushar; Young, Sigrid et al. (2016) Carboxypeptidase E and Secretogranin III Coordinately Facilitate Efficient Sorting of Proopiomelanocortin to the Regulated Secretory Pathway in AtT20 Cells. Mol Endocrinol 30:37-47
Qin, X-Y; Cao, C; Cawley, N X et al. (2016) Decreased peripheral brain-derived neurotrophic factor levels in Alzheimer's disease: a meta-analysis study (N=7277). Mol Psychiatry :
Cheng, Yong; Loh, Y Peng; Birch, Nigel P (2016) Neuroserpin Attenuates H2O2-Induced Oxidative Stress in Hippocampal Neurons via AKT and BCL-2 Signaling Pathways. J Mol Neurosci :
Cheng, Yong; Li, Zhaojin; Kardami, Elissavet et al. (2016) Neuroprotective effects of LMW and HMW FGF2 against amyloid beta toxicity in primary cultured hippocampal neurons. Neurosci Lett 632:109-13
Qin, Xiao-Yan; Feng, Jin-Chao; Cao, Chang et al. (2016) Association of Peripheral Blood Levels of Brain-Derived Neurotrophic Factor With Autism Spectrum Disorder in Children: A Systematic Review and Meta-analysis. JAMA Pediatr 170:1079-1086

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