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Neurosecretory Proteins in Neuroprotection, Neurodevelopment, and Cancer

• Y. Peng Loh, PhD, Head, Section on Cellular Neurobiology
• Niamh X. Cawley, PhD, Staff Scientist
• Hong Lou, MD, Senior Research Assistant
• Xuyu Yang, PhD, Research Fellow
• Yong Cheng, PhD, Postdoctoral Fellow
• Prabhuanand Selvaraj, PhD, Postdoctoral Fellow
• Leila Toulabi, PhD, Postdoctoral Fellow
• Lin Cong, MD, Special Volunteer
• Vida Falahatian, MD, Special Volunteer
• Zhaojin Li, MS, Special Volunteer
• Alicja Woronowicz, MD, PhD, Special Volunteer
• Wei Yao, PhD, Special Volunteer

We study the cell biology of neuroendocrine cells and the function of neuropeptides and the neurotrophic factor carboxypeptidase E (CPE)/Neurotrophic Factor-α1 (NF-α1) in health and disease. Our focus is three-fold, to: (1) investigate the mechanisms of biogenesis and intracellular trafficking of dense-core secretory granules containing neuropeptides and their processing enzymes; (2) investigate the role of serpinin, a novel chromogranin A–derived peptide discovered in our lab, in neural and cardiac function; and (3) determine the non-enzymatic neurotrophic role of CPE/NF-α1 in neuronal function and cancer. Our work led to the discovery of novel molecular mechanisms of protein trafficking to the regulated secretory pathway (RSP) and identified players and mechanisms that control secretory granule biogenesis and transport in neuroendocrine cells. Recently, we found a new role for CPE/NF-α1 as a trophic factor that mediates neuroprotection, neurodevelopment, and anti-depression. We also identified a splice variant of CPE/NF-α1 (CPE-DN) that drives metastasis in various cancer types. Using cell lines, primary cell cultures, mouse models, and human tumor specimens and sera, our studies have deepened the understanding of neurodegenerative diseases, memory, learning, depression, cardiac function, obesity, and metastasis in cancer.

Mechanism of sorting, transport, and regulated secretion of neuroproteins

The intracellular sorting of pro-neuropeptides and neurotrophins to the RSP is essential for processing, storage, and release of active proteins and peptides in the neuroendocrine cell. We investigated the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), proinsulin, and brain-derived neurotrophic factor (BDNF) to the RSP. Our studies showed that these pro-proteins undergo homotypic oligomerization as they traverse the cell from the site of synthesis in the endoplasmic reticulum (ER) to the trans-Golgi network (TGN). In the TGN, the pro-proteins are sorted into dense-core granules of the RSP for processing by prohormone convertases and CPE and then secreted. We showed that the sorting of prohormones to the RSP occurs by a receptor-mediated mechanism. Site-direct mutagenesis studies identified a 3-D consensus sorting motif consisting of two acidic residues found in POMC, proinsulin, and BDNF. We identified the transmembrane form of CPE as an RSP sorting receptor that is specific for the sorting signal of these proproteins.

We also investigated the role of secretogranin III (SgIII) as a surrogate sorting receptor for membrane CPE in targeting POMC to the RSP. Using RNA interference (siRNA) to knock down SgIII or CPE expression in pituitary AtT20 cells, we demonstrated in both cases that POMC secretion via the constitutive secretory pathway was elevated. In double CPE-SgIII knock-down cells, elevated constitutive secretion of POMC and stimulated secretion of ACTH were perturbed. Thus, CPE mediates trafficking of POMC to the RSP, and SgIII may play a compensatory role for CPE in POMC sorting to the RSP.

Transport of vesicles containing hormone or BDNF to the plasma membrane for activity-dependent secretion is critical for endocrine function and synaptic plasticity. We showed that the cytoplasmic tail of a transmembrane form of CPE in hormone- or BDNF–containing dense-core secretory vesicles plays an important role in their transport to the vesicles' release site. In hippocampal neurons and pituitary cells, overexpression of the CPE tail inhibited the movement of BDNF– and POMC/CPE–containing vesicles to the processes, respectively. The transmembrane CPE tails on the POMC/ACTH and BDNF vesicles anchors these organelles, which interact with the microtubule-based motors dynactin and KIF1A/KIF3A to effect anterograde vesicle movement to the plasma membrane for activity-dependent secretion in endocrine cells and neurons.

Serpinin, a chromogranin A–derived peptide, regulates secretory granule biogenesis, cell survival, cardiac function, and angiogenesis.

Our previous studies in pituitary AtT-20 cells provided evidence that an autocrine mechanism up-regulates large dense-core vesicle (LDCV) biogenesis to replenish LDCVs following stimulated exocytosis of the vesicles. We identified the autocrine signal as serpinin, a novel 26 amino-acid, chromogranin A (CgA)–derived peptide cleaved from the C-terminus of CgA. Serpinin is released in an activity-dependent manner from LDCVs and activates adenyl cyclase to raise cAMP levels and protein kinase A in the cell. This leads to translocation of the transcription factor Sp1 from the cytoplasm into the nucleus and enhanced transcription of a protease inhibitor, protease nexin 1 (PN-1), which then inhibits granule protein degradation in the Golgi complex, stabilizing and raising granule protein levels in the Golgi and enhancing LDCV formation. We also identified modified forms of serpinin, pyroglutamyl-serpinin (pGlu-serpinin) and a C-terminally extended form, serpinin-RRG, in the secretion medium of AtT20 cells and in rat heart tissue. pGlu-serpinin is synthesized and stored in secretory granules and secreted in an activity-dependent manner from AtT20 cells. We observed pGlu-serpinin immunostaining in nerve terminals of neurites in mouse brain, olfactory bulb, and retina, suggesting a role as a neurotransmitter or neuromodulator. Additionally, pGlu-serpinin exhibited neuroprotective activity against oxidative stress in AtT20 cells and against low K⁺–induced apoptosis in rat cortical neurons. In collaboration with Bruno Tota, we found that pGlu-serpinin has positive inotropic activity in cardiac function, with no change in blood pressure and heart rate. pGlu-serpinin acts through a β1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway in the heart. pGlu-serpinin and other CgA–derived cardio-active peptides emerge as novel β-adrenergic inotropic and lusitropic modulators. Together, they can play a key role in the myocardium’s orchestration of its complex response to sympatho-chromaffin stimulation. Additionally, we recently found that pGlu serpinin is a powerful cardioprotectant after ischemia.

Role of CPE/NF-α1 in neuroprotection and anti-depression during stress

We generated a CPE/NF-α1 knock-out (KO) mouse to study the function of CPE in vivo. This KO mouse exhibited obesity, infertility, and diabetes as well as learning and memory deficits and depressive-like behavior (Reference 1). Interestingly, a null mutation in the gene encoding CPE/NF-α1 was recently identified in a female who has clinical features such as obesity, type 2 diabetes, learning disabilities, and hypogonadotrophic hypogonadism, similar to the Cpe-KO mouse, indicating the importance of CPE. Using the Cpe-KO mice as a model to study the nervous system deficiencies and Morris water maze and object-preference tests, we showed defects in learning and memory and, in the forced swim test, depressive-like behavior. Analysis of the brain of 6- to 14-week-old Cpe–KO mice revealed poor dendritic pruning in cortical and hippocampal neurons, which could affect synaptogenesis. Electrophysiological measurements showed a defect in the generation of long-term potentiation in hippocampal slices. A major cause of the defects is the loss of neurons in the CA3 region of the hippocampus. Hippocampal neurons in CA3 region are enriched in CPE and were normal at three weeks of age just before weaning, indicating that the defect was not developmental. The degeneration is likely caused by epileptic-like neuronal firing, releasing large amounts of glutamate during weaning stress. Hence CPE/NF-α1 is important for the survival of the CA3 neurons. We then showed that CPE/NF-α1, either overexpressed or applied externally to cultured hippocampal or cortical neurons, protected these neurons from apoptosis induced by oxidative stress with hydrogen peroxide. Moreover, a non-enzymatically active form of CPE/NF-α1 had the same effect, indicating that its action is independent of enzymatic activity. We propose that CPE/NF-α1 acts extracellularly as a signaling molecule by binding to a receptor to mediate neuroprotection. To this end, we demonstrated that ¹²⁵I CPE/NF-α1 bound to HT22 cells, an immortalized hippocampal neuronal cell line, in a saturable manner, and that the binding was specifically displaced by non-iodinated CPE/NF-α1 but not by bovine serum albumin, suggesting the existence of a receptor.

The mechanism of action of CPE/NF-α1 in neuroprotection involves the activation of the ERK1/2 and the Akt signaling pathways, which then leads to enhanced transcription/translation of a pro-survival mitochondrial protein, Bcl2, inhibition of caspase 3 activation, and promotion of neuronal survival (Reference 2). Furthermore, this CPE/NF-α1–mediated neuroprotection pathway is activated by rosiglitazone, a PPARg ligand, which binds to PPARg binding sites in the CPE promoter. Examination of this pathway during stress in vivo revealed that, after mild chronic restraint stress (CRS) for 1h/day for seven days, mice showed significantly elevated levels of CPE/NF-α1 mRNA and protein, as well as of the anti-apoptosis protein Bcl2, in the hippocampus. In situ hybridization studies indicated especially elevated CPE/NF-α1 mRNA levels in the CA3 region and no gross neuronal cell death after mild CRS. Furthermore, primary hippocampal neurons in culture showed elevated CPE/NF-α1 and Bcl2 expression and a decline in Bax, a pro-apoptotic protein, after treatment with the synthetic glucocorticoid dexamethasone. This up-regulation was mediated by glucocorticoid binding to glucocorticoid-regulatory element (GRE) sites on the promoter of the Cpe gene. Thus, during mild CRS, when glucocorticoid is released, CPE/NF-α1 and Bcl2 expression are coordinately up-regulated to mediate neuroprotection of hippocampal neurons. The importance of CPE as a neuroprotective agent was demonstrated by the absence of an increase in Bcl2 in the hippocampus of Cpe-KO mice, leading to the degeneration of the CA3 neurons.

The relevance of CPE/NF-α1 in neuroprotection in humans was indicated by our studies on a mutation of the CPE gene found in an Alzheimer patient (Reference 3). Our search in GeneBank EST database identified a sequence entry from cortex of an Alzheimer’s disease (AD) patient that had three adenosine inserts in the CPE gene, thereby introducing nine amino acids, including two glutamines, into the mutant protein, herein called CPE-QQ. Expression of CPE-QQ in Neuro2a cells indicates that it is not secreted. Co-expression of wild-type (WT) and CPE-QQ in Neuro2a cells resulted in degradation of both forms of the protein and reduction in secretion of WT CPE. Immunocytochemical studies show that CPE-QQ stains in the perinuclear region of the cells and co-stained with Calnexin, an ER marker, consistent with localization of the mutant in the ER. Moreover, many cells appear rounded, indicating unhealthy cells that might be undergoing ER stress, unlike the cells expressing WT, which show staining in the cell body and neurites. Overexpression of CPE-QQ in rat primary hippocampal neurons resulted in increased levels of the 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 expression. We then generated transgenic mice overexpressing CPE-QQ and showed that, at 50 weeks but not at 11 weeks, the animals exhibited memory deficits compared with WT mice, but that their spatial learning ability was unimpaired. The CPE-QQ mice were neither obese nor diabetic. However, they showed significantly fewer neurites in the CA3 region, the dentate gyrus of the hippocampus, and the medial prefrontal cortex, indicative of neurodegeneration. Moreover, they exhibited decreased neurogenesis in the subgranular zone and hyperphosphorylation of tau at ser³⁹⁵, a hallmark of AD. The studies substantiated a neuroprotective role of CPE/NFα-1 in humans and identified a mutation in the CPE gene that can cause neurodegeneration linked to AD and perhaps other forms of dementia.

Stress also induces depression. Huda Akil’s group (University of Michigan) reported that fibroblast growth factor 2 (FGF2) is an anti-depressant. We found that prolonged (6h/day for 21 days) restraint stress reduced CPE/NFα-1 and FGF2 in the hippocampus of mice and induced depressive-like behavior. However, after short-term restraint stress, mice did not show depressive-like behavior despite elevated corticosterone levels indicative of stress. Moreover, hippocampal CPE/NFα-1, FGF2, and doublecortin, a marker for neurogenesis, were elevated in these mice, suggesting that the anti-depressive effects of CPE/NF-α1 are mediated through increased neurogenesis. Indeed, we found that exogenously applied CPE/NF-α1 could up-regulate FGF2 mRNA and protein expression in cultured hippocampal neurons, indicating that CPE/NF-α1 regulates FGF2 expression. CPE/NF-α1-KO mice exhibited severely reduced hippocampal FGF2 levels and immature neuron numbers in the subgranular zone. The mice displayed depressive-like behavior, which was rescued by FGF2 administration. Thus, CPE/NF-α1 prevents stress-induced depression by up-regulating hippocampal FGF2 expression, which leads to enhanced neurogenesis and anti-depressive activity (Reference 4). Furthermore, we found that rosiglitazone, an anti-diabetic drug, can trigger this pathway (Reference 4). Roziglitazone has previously been shown to be effective in treating diabetic patients with bi-polar disorders.

CPE/NFα-1 inhibits proliferation and induces differentiation of embryonic stem cells.

CPE/NFα-1mRNA is expressed in mouse embryos as early as day E5.5 and increases sharply at E12.5, in parallel with the development of the endocrine system, and continues to increase into adulthood. In situ hybridization studies indicate that CPE/NF-α1 is expressed primarily in the forebrain in mouse embryos, suggesting a role of CPE/NF-α1 in neurodevelopment. We therefore began studying neural stem cell proliferation. Exogenous recombinant CPE/NF-α1 was added to E13.5 neocortex-derived neurospheres, which contain stem cells and neuroprogenitors. CPE/NF-α1 treatment reduced the number and size of the neurospheres formed, suggesting inhibition of proliferation and maintenance of the ‘stemness’ of the stem cells in the neurospheres. CPE/NF-α1 down-regulated the wnt pathway in the neurospheres, leading to reduced levels of β-catenin, a protein known to enhance proliferation, suggesting that CPE/NF-α1’s inhibitory effect on proliferation is mediated by negatively regulating the wnt pathway. We carried out differentiation studies using neurospheres from seven-day cultures that were dissociated into single cells and cultured for an additional five days. We observed an increase in astrocytes in the presence of CPE/NF-α1 without alteration in the percentage of neuronal and oligodendrocyte populations. Interestingly, dissociated cells from neurospheres derived from Cpe/Nf-α1-KO mouse embryos showed fewer astrocytes but more neurons. In vivo, Cpe/Nf-α1-KO mouse cortex (at P1, the time of astrocytogenesis) showed 49% fewer astrocyte numbers than in WT animals, confirming the ex vivo data. Our results suggest a novel role for CPE/NF-α1 as an extracellular signal to inhibit proliferation and induce differentiation of neural stem cells into astrocytes, thus playing an important role in neurodevelopment (Reference 5).

Neurite outgrowth is key to the formation of synapses and the neural network during development. We found that CPE/NF-α1 prevented Wnt-3a inhibition of nerve growth factor (NGF)–stimulated neurite outgrowth in PC12 cells and cortical neurons. Moreover, CPE/NF-α1 augmented Wnt-5a–mediated neurite outgrowth. Thus, the interplay between NGF preventing neurite outgrowth, which is inhibited by Wnt-3a, and augmenting neurite outgrowth, which is mediated by Wnt-5a and CPE/NF-α1, could play an important role in regulating these positive and negative cues, which are critical for neurodevelopment.

Carboxypeptidase E/CPE-deltaN in tumorigenesis

Our studies indicate an important role of the CPE gene in mediating tumor growth, survival, and metastasis. We identified and cloned a novel splice isoform CPE (CPE-deltaN) mRNA, which encodes a 40kD N-terminal truncated protein that is elevated in metastatic hepatocellular, colon, breast, head, and neck carcinoma cell lines. CPE-deltaN is translocated from the cytoplasm to the nucleus of metastatic cancer cells. Overexpression of CPE-deltaN in hepatocellular carcinoma (HCC) cells promoted their proliferation and migration. In nude mice, siRNA knockdown of CPE-DN expression in highly metastatic HCC cells inhibited their growth and metastasis. CPE-deltaN promoted migration by up-regulating expression of the metastasis gene Nedd9, through interaction with histone deacetylase (HDAC) 1 or 2 (Reference 1). The enhanced invasive phenotype of HCC cells stably transfected with CPE-deltaN was suppressed when Nedd9 was silenced by siRNA. Thus, CPE-deltaN plays a role in tumor metastasis. Wild-type (WT) CPE, on the other hand, plays a different role in tumor progression. In some cancer cell lines, e.g., glioma cell lines, which express and secrete WT CPE, it has been shown to enhance proliferation. However, in other cancer cells, e.g., the pancreatic cancer cell line Panc-1, which does not express WT CPE protein, there was little effect of transfected WT CPE on proliferation. In vivo, the interplay between CPE-deltaN and WT CPE could influence the metastatic potential of tumors.

A prospective study was carried out to evaluate the role of CPE/CPE-deltaN mRNA as a biomarker for predicting recurrence in 120 HCC patients from the Liver Network patients in Taiwan. The study focused on Stage I and II patients, given that these patients generally have better prognosis, but whose tumor recurrence rate is still high. Using the same methodology as we had published previously, we determined the Tumor/Normal (T/N) ratio of CPE/CPE-deltaN mRNA. The follow-up time ranged from 9 to 106 months. Our results demonstrated that the recurrence-free survival of HCC patients was significantly associated with CPE expression level (T/N greater than 2) for both stage I and II patients (Reference 6). The CPE/CPE mRNA expression level in HCC could therefore be a useful clinical biomarker for predicting tumor recurrence in HCC patients who are in an early pathology stage and able to receive curative resection.

Using circulating exosomes in humans, we are also developing a blood assay to determine the CPE/CPE-deltaN biomarker levels in cancer versus normal controls. We found elevated levels of CPE/CPE-deltaN mRNA in secreted exosomes from different highly metastatic cancer cells. Even though exosome numbers themselves were not correlated with the metastatic potential of the cells, the CPE/CPE deltaN mRNA content inside the exosomes were. Thus, such a blood assay could offer a non-invasive method for the diagnosis and assessment of treatment efficacy of cancer patients.

Publications

1. Cawley NX, Wetsel WC, Murthy SR, Park JJ, Pacak K, Loh YP. New roles of carboxypeptidase E in endocrine and neural function and cancer. Endocr Rev 2012;33:216-253.
2. Cheng Y, Cawley NX, Loh YP. Carboxypeptidase E/NF-alpha1: a new neurotrophic factor against oxidative stress-induced apoptotic cell death mediated by ERK and PI3-K/AKT pathways. PLoS One 2013;8:e71578.
3. Cheng Y, Cawley NX, Yanik T, Murthy SRK, Liu C, Kasikci F, Abebe D, Loh YP. A human carboxypeptidase E gene mutation in an Alzheimer’s disease patient causes neurodegeneration, memory deficits and depression. Translat Psychiatry 2016;6(12):e973.
4. Cheng Y, Rodriguiz RM, Murthy SRK, Senatorov V, Thouennon E, Cawley NX, Aryal D, Ahn S, Lecka-Czernik B, Wetsel WC, Loh YP. Neurotrophic factor-alpha1 prevents stress-induced depression through enhancement of neurogenesis and is activated by rosiglitazone. Mol Psychiatry 2015;20:744-754.
5. Selvaraj P, Xiao L, Lee C, Murthy SRK, Cawley NX, Lane M, Merchenthaler I, Ahn S, Loh YP. Neurotrophic factor-alpha1: a key Wnt-beta-catenin dependent anti-proliferation factor and ERK-Sox9 activated inducer of embryonic neural stem cell differentiation to astrocytes in neurodevelopment. Stem Cells 2016;Epub ahead of print.
6. Huang S-H, Wu H-D, Chen Y-T, Murthy SR, Chiu YT, Chang Y, Chang IC, Yang X, Loh YP. Carboxypeptidase E is a prediction marker for tumor recurrence in early stage hepatocellular carcinoma. Tumour Biol 2016;37:9745-9753.

Collaborators

• Soyhun Ahn, PhD, Unit on Developmental Neurogenetics, NICHD, Bethesda, MD
• Shiu-Feng Huang, MD, PhD, National Health Research Institutes, Zhunan, Taiwan
• Jennifer Jones, MD, PhD, Vaccine Branch, NCI
• Jacqueline Jonklaas, MD, Georgetown University Medical Center, Washington, DC
• Beata Lecka-Czernik, PhD, University of Toledo, Toledo, OH
• Istvan Merchenthaler, PhD, University of Maryland, Baltimore, MD
• Saravana Murthy, PhD, Scanogen, Inc., Baltimore, MD
• Karel Pacak, MD, PhD, Section on Medical Neuroendocrinology, NICHD, Bethesda, MD
• Joshua J. Park, PhD, University of Toledo, Toledo, OH
• Rina Rosin-Arbesfeld, PhD, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
• Bruno Tota, MD, Università della Calabria, Cosenza, Italy
• Y-Ching Wang, PhD, National Cheng Kung University, Tainin, Taiwan
• William Wetsel, PhD, Duke University, Durham, NC
• Tulin Yanik, PhD, Middle East Technical University, Ankara, Turkey

Contact

For more information, email lohp@mail.nih.gov or visit http://scn.nichd.nih.gov.

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