Neuropeptides and Carboxypeptidase E/ Neurotrophic Factor-alpha1 in Neural and Cognitive Functions

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

We studied the role of the CPE-cytoplasmic tail in trafficking of secretory vesicles to the plasma membrane for secretion. With Dr.J.Park, Univ.Toledo/NIA, we showed that the cytoplasmic tail of transmembrane CPE on POMC vesicles binds directly to snapin, which in turn binds to microtubule motors, kinesin-2 and kinesin-3 to mediate their anterograde transport. Anterior pituitary AtT-20 cells, overexpressing snapin showed reduced process-localization and velocity of movement of ACTH/POMC vesicles, similar to CPE cytoplasmic tail overexpression. Knockdown of snapin decreased stimulated ACTH secretion.Upon protein kinase A (PKA) activation by forskolin, the interactions of kinesin-2 and kinesin-3 with CPE and ACTH vesicle levels at the terminus of At20 cells were significantly increased. Our study has uncovered a new molecular complex consisting of the CPE cytoplasmic tail-snapin-kinesin 2 and 3 that mediates post-Golgi transport of ACTH/POMC vesicles to the process terminals of AtT20 cells for secretion in a PKA-dependent manner. Currently, our major focus is on the neurotrophic functions of CPE/NF-alpha1. Humans with null and mis-sense CPE mutations have been reported to have severe learning disability in early childhood, obesity, diabetes and infertility, due to lack of CPE. We have identified a CPE mutation in an Alzheimer Disease (AD) patient which results in a CPE mutant protein with an additional nine amino acids, 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 neuronal cell death. Transgenic mice overexpressing CPE-QQ exhibited memory deficits in the Morris water maze test, but their spatial learning ability was unimpaired. These mice showed depressive-like behavior by the forced swim test. They had fewer neurites in the hippocampus and medial prefrontal cortex, indicative of neurodegeneration. They showed diminished neurogenesis in the sub-granular zone and hyperphosphorylation of tau, a hallmark of AD. Thus, this human mutation in CPE/NF-alpha1 is neurotoxic, leading to neurodegeneration and cognitive decline. Our in vitro studies showed that CPE/NF-alpha1 acts extracellularly, 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. CPE knock-out mice lacking CPE/NF-alpha1 showed complete degeneration of hippocampal CA3 neurons and cognitive dysfunction after social (maternal separation) and physical stress (ear tagging and tail clipping) associated with the weaning paradigm, despite having WT levels of other growth factors such as BDNF, GDNF, NGF and NT3. Treatment of WT mice with ANA12, an inhibitor of TrkB, the BDNF receptor did not result in neurodegeneration of the CA3 region after weaning stress. Most importantly, CPE-E342Q mice, although lacking WT-CPE and had endocrinological deficits, showed no hippocampal degeneration or cognitive dysfunction after the weaning paradigm, indicating that CPE/NF-alpha1 is critical, but not BDNF, in preventing stress-induced hippocampal cell death, independent of its enzymatic activity. To further understand the mechanism of the trophic action of CPE/NF-alpha1, we searched for a membrane receptor for this factor. Screening a human G-protein coupled receptor (GPCR) library, we found a serotonin receptor, HTR1E with no known function that interacted with CPE/NF-alpha1. This interaction was confirmed by co-immunoprecipitation and pulldown assays. Binding studies revealed a Kd=13.82nM. Molecular dynamics studies indicated that CPE/NF-alpha1 interacts with HTR1E via 3 salt-bridges stabilized by several hydrogen bonds, and is independent of the serotonin binding pocket. Immunohistochemistry revealed co-localization of HTR1E and CPE/NF-alpha1 on the surface of hippocampal neurons. Signal transduction studies showed that HTR1E-CPE/NF-alpha1 interaction activated the Erk1/2-CREB pathway via recruitment of beta-arrestin. This in turn activated the BCL2 pro-survival pathway. We showed that HTR1E-CPE/NF-alpha1 interaction mediated neuroprotection of human primary neurons against H2O2 -induced cytotoxicity and glutamate-induced neurotoxicity. These findings indicate that CPE/NF-alpha1 interacts with HTR1E to promote neuronal survival. We have examined the role of 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, CPE/NF-alpha1-KO mice exhibited severely reduced hippocampal FGF2 levels and immature neuron numbers in the sub-granular 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. Interestingly, we found that rosiglitazone, a PPAR-gamma agonist and anti-diabetic drug which has anti-depression and neuroprotective activities, induced CPE/NF-alpha1 and doublecortin expression when fed to mice. Thus PPAR-gamma agonists can be potentially useful as anti-depressant and neuroprotective drugs. We have investigated the role of CPE/NF-alpha1 in embryonic development of the nervous system. 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-beta catenin pathway known to promote proliferation. Differentiation studies using neurospheres 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, 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 role in differentiating neural stem cells into astrocytes for normal neurodevelopment. With Dr. Lecka-Czernik, Univ. Toledo, we showed that murine skeletal stem cells, treated with CPE or CPE-E342Q protein enhanced Erk phosphorylation and up-regulated expression of two wnt pathway markers, Cxn43 and Axin2 and genes associated with fatty acid metabolism and energy dissipation. These cells exhibited transient accumulation of small lipid droplets, increased oxidative phosphorylation and cellular dependence on fatty acids as fuel for energy production. Thus, CPE-NF-alpha1 besides acting as a trophin in brain, plays a trophic role in regulating bone mass and energy metabolism, independent of its enzymatic activity. Given CPE-NF-alpha1's trophic role in preventing stress-induced neuronal cell death, our future work will explore the use of CPE-NF-alpha1 as a therapeutic agent for treating neurodegenerative diseases.