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
- Erwan Thouennon, 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
- Nikoletta Lendvai, MD, Predoctoral Student
- Jane Huang, BS, Postbaccalaureate Fellow
We study the cell biology of neuroendocrine cells and the function of neuropeptides and the neurotrophic factor 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 carboxypeptidase E (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 (CPE-deltaN) 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 diseases related to neurodegeneration, memory, learning, depression, cardiac function, obesity, and metastasis in cancer.
Mechanism of sorting and vesicle transport of pro-neuropeptides, neurotrophins, and their processing enzymes to the regulated secretory pathway for secretion
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 investigated the role of membrane CPE and secretogranin III (SgII) as sorting receptors for 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 primary pituitary and AtT20 cells, overexpression of the CPE tail inhibited the movement of BDNF– and POMC/CPE–containing vesicles to the processes, respectively. The CPE tail interacts with the microtubule-based motors dynactin and KIF1A/KIF3A to effect anterograde vesicle movement to the plasma membrane for secretion. CPE anchors POMC/ACTH and BDNF vesicles to the microtubule-based motor system for transport along the processes to the plasma membrane for activity-dependent secretion in endocrine cells and neurons.
Role of CPE/NF-α1 in neuroprotection, stress, and neurodevelopment
We showed that CPE knockout (KO) mice exhibit nervous system deficiencies. Morris water maze and object-preference tests indicate defects in learning and memory, and forced swim tests indicate depression. In 6- to 14-week-old CPE–KO mice, dendritic pruning was poor in cortical and hippocampal neurons, which could affect synaptogenesis. Electrophysiological measurements revealed a defect in the generation of long-term potentiation in hippocampal slices of the mice. A major cause for the defect was the loss of neurons in the CA3 region of the hippocampus of CPE–KO animals observed at four weeks of age and older when the animals are weaned. Hippocampal neurons in CA3 region are enriched in CPE and were normal at 3 weeks of age just before weaning. When weaning was delayed for a week, the degeneration was not observed. When given carbamezapine i.p. at two weeks of age, degeneration was prevented. The results suggest that the degeneration is correlated with possible epileptic-like neuronal firing during the stress of weaning and that CPE is important for the survival of CA3 neurons during that period. We then showed that, when CPE was applied externally to cultured hippocampal or cortical neurons, they were protected from apoptosis after inducing oxidative stress with hydrogen peroxide, indicating that CPE acts as an extracellular signaling molecule in neuroprotection. Its action involved activation of the ERK1/2 and the Akt signaling pathways, which then caused phosphorylation and translocation of the transcription factor Sp1 from the cytosol to the nucleus. This then led to enhanced transcription/translation of BCL2, a pro-survival mitochondrial protein, inhibition of caspase 3 activation and promotion of neuronal survival (Reference 1). Furthermore, we showed that this CPE–mediated neuroprotection pathway can be activated by rosiglitazone, a PPARg ligand, because the CPE promoter contains a PPARg binding site (Reference 2). Thus, CPE is a novel neuroprotective trophic factor, which we renamed Neurotrophic factor-alpha1 (NF-α1). We then demonstrated that CPE/NF-α1 has a neuroprotective role in vivo. During and after mild chronic restraint stress (CRS) for 1h/day for seven days, CPE/NF-α1 mRNA and protein levels, as well as those of the anti-apoptotic factor Bcl2, were significantly elevated in the hippocampus. In situ hybridization studies indicated especially elevated CPE/NF-α1 mRNA expression in the CA3 region and no gross neuronal cell death after mild CRS. Studies on primary hippocampal neurons in culture demonstrated elevated CPE and Bcl2 expression and a decline in Bax, a pro-apoptotic protein, after treatment with the synthetic glucocorticoid dexamethasone; the regulation was mediated by glucocorticoid binding to glucocorticoid-regulatory element (GRE) sites on the promoter of the cpe gene. The findings indicate that, 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 the increase in Bcl2 in the hippocampus of CPE–KO mice, leading to the degeneration of the CA3 neurons.
We also investigated the role of NF-α1 in preventing restraint stress–induced depression. Prolonged (6h/day for 21 days), but not short-term (1h/day for 7days), restraint stress reduced fibroblast growth factor 2 (FGF2) in the hippocampus, leading to depressive-like behavior in mice. We found that, after short-term restraint stress in mice, hippocampal NF-α1, FGF2, and doublecortin, a marker for immature neurons, rose, suggesting increased neurogenesis. Indeed, we showed that, in cultured hippocampal neurons, exogenous NF-α1 could raise FGF2 expression. After prolonged restraint stress, mice showed reduced NF-α1 and FGF2 levels. Moreover, 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, NF-α1 prevents stress-induced depression by up-regulating hippocampal FGF2 expression, which leads to enhanced neurogenesis and anti-depressant activity (Reference 3). Furthermore, we found that rosiglitazone, an anti-diabetic drug, can trigger this pathway (Reference 3) and has previously been shown to be effective in treating diabetic patients with bi-polar disorders.
Recently, we found that NF-α1 plays a role during embryonic development. NF-α1mRNA was expressed in mouse embryos as early as day E5.5, rising each day, peaking at E8.5, and falling slightly at E9.5. NF-α1 mRNA expression then declined sharply at E10.5–11.5 to below E5.5 levels and then rose sharply at E12.5, in parallel with the development of the endocrine system, and continued to increase into adulthood. In situ hybridization studies indicate that NF-α1 is expressed primarily in the forebrain and dorsal root ganglia in mouse embryos. To study neural stem cell proliferation, exogenous recombinant NF-α1 was added to E13.5 neocortex–derived neurospheres, which contain stem cells and neuroprogenitors. 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. 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 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 NF-α1 without alteration in the percentage of neuronal and oligodendrocyte populations. Interestingly, dissociated cells from neurospheres derived from NF-α1–KO mouse embryos showed fewer astrocytes but more neurons. In vivo, NF-α1–KO mouse cortex (E16.5) showed lower astrocyte numbers than in WT animals, confirming the ex vivo data. Our results suggest a novel role of NF-α1 as an extracellular signal to differentiate neural stem cells into astrocytes.
We also studied the role of NF-α1 in neurite outgrowth, which is key to the formation of synapses and the neural network during development. We found that NF-α1 prevented Wnt-3a inhibition of NGF–stimulated neurite outgrowth in PC12 cells and cortical neurons. Moreover, NF-α1 augmented Wnt-5a–mediated neurite outgrowth. Thus, by interplaying with NGF to prevent neurite outgrowth inhibited by Wnt-3a and augmenting neurite outgrowth mediated by Wnt-5a, NF-α1, could play an important role in regulating these positive and negative cues that are critical for neurodevelopment (Reference 4).
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 enhanced LDCV formation. We also identified modified forms of serpinin, pyroglutamyl-serpinin (pGlu-serpinin) and a C-terminally extended form, serpinin-RRG, in 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.
Carboxypeptidase E/CPE-deltaN in tumorigenesis and as a biomarker for predicting future metastasis
Our studies indicate an important role of the CPE gene in mediating tumor growth, survival, and metastasis. Recently, we described a novel splice isoform of CPE (CPE-deltaN) 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. siRNA knockdown of CPE-deltaN expression in highly metastatic HCC cells inhibited their growth and metastasis in nude mice. CPE-deltaN promoted migration by up-regulating expression of the metastasis gene Nedd9, through interaction with histone deacetylase (HDAC) 1 or 2. The enhanced invasive phenotype of HCC cells stably transfected with CPE-deltaN was suppressed when Nedd9 was silenced by siRNA. Microarray studies of HCC cells overexpressing CPE-deltaN showed elevated expression of 27 genes associated with metastasis, including Nedd9, claudin 2 (cldn2), matrix metallopeptidase 1 (mmp1), and inositol 1,4,5-trisphosphate 3-kinase A (itpka), while 30 genes associated with tumor suppressor function, such as insulin-like growth factor binding protein 5 and 3 (igfbp5 and igfbp3) were down-regulated.
CPE and CPE-deltaN, each play distinctive roles in tumor progression. In some cancer cell lines, overexpression of wild-type (WT) CPE has been demonstrated to enhance proliferation, e.g., glioma cell lines; however, in other cell lines, e.g., PANC-1, there was no effect of WT CPE on proliferation. Interestingly, wild-type full-length CPE secreted by neuroendocrine tumors negatively regulates the canonical wnt pathway and likely mediates the anti-metastatic effects observed when tumor cells are treated with WT CPE. In vivo, the interplay between CPE-deltaN and WT CPE could influence the metastatic potential of tumors.
In clinical studies, we showed that CPE/CPE-deltaN is a good prognostic biomarker for HCC and lung adenocarcinoma. Previously, we carried out a retrospective study on hepatocellular carcinoma patients and showed that CPE-deltaN is a powerful prognostic biomarker for predicting future recurrence (Reference 5). We have now carried out a prospective study to further evaluate the role of CPE-deltaN mRNA as a biomarker for predicting recurrence in 120 HCC patients from the Liver Network patients in Taiwan. We focused on Stage I and II patients, given that these patients generally have better prognosis, but the 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-DN mRNA. The follow up time ranged from 9.0 months 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 identical to that found in our previous retrospective study. CPE mRNA expression level in HCC can therefore be a useful biomarker clinically for predicting tumor recurrence in HCC patients who are in the early pathology stage and able to receive curative resection.
In collaboration with Y-Ching Wang, we carried out a prospective study to evaluate CPE-deltaN as a biomarker for predicting recurrence and death in patients with lung adenocarcinoma, the major sub-type of non-small cell lung carcinoma, which accounts for 85% of lung cancer. Eighty six patients were recruited and followed up for up to seven years post-resection of the tumor to determine recurrence and death. Kaplan-Meier survival analysis showed that patients with high CPE-deltaN copy numbers had a shorter time of disease-free survival and shorter time to death. In subgroup analyses, the association of disease-free survival time with CPE-deltaN copy number was particularly strong among stage I and II lung cancer patients. Thus CPE-deltaN mRNA is a potentially powerful biomarker for predicting recurrence and death at all histo-pathological stages in lung adenocarcinoma patients and is especially useful in identifying patients at high risk of recurrence at early stages I and II of the disease, which will facilitate treatment strategies after surgery.
We also successfully developed a blood assay using circulating exosomes to determine the CPE/CPE-deltaN biomarker levels and showed significant differences between pheochromocytoma and normal control patients. This assay will be very useful for screening patients for cancer.
- Cheng Y, Cawley NX, Loh YP. Carboxypeptidase E/NF-a1: a new neurotrophic factor against oxidative stress-induced apoptotic cell death mediated by ERK and PI3-K/AKT pathways. PLoS One 2013; 8:e71578.
- Thouennon E, Cheng Y, Falahatian V, Cawley NX, Loh YP. Rosiglitazone-activated PPARg induces neurotrophic factor-a1 transcription contributing to neuroprotection. J Neurochem 2015; 134:463-470.
- 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-a1 prevents stress-induced depression through enhancement of neurogenesis and is activated by rosiglitazone. Mol Psychiatry 2015; 20:744-754.
- Selvaraj P, Huang JSW, Chen A, Skalka N, Rosin-Abesfeld R, Loh YP. Neurotrophic factor-a1 modulates NGF-induced neurite outgrowth through interaction with Wnt-3a and Wnt-5a in PC12 cells and cortical neurons. Mol Cell Neurosci 2015; 11:222-233.
- Lee TK, Murthy SRK, Cawley NX, Dhanvantari S, Hewitt SM, Lou H, Lau T, Ma S, Huynh T, Wesley RA, Ng IO, Pacak K, Poon RT, Loh YP. An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers. J Clin Invest 2011; 121:880-892.
- Soyhun Ahn, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
- Shiu-Feng Huang, MD, PhD, National Health Research Institutes, Zhunan, Taiwan
- 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, Program in Reproductive and Adult Endocrinology, 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
- William Wetsel, PhD, Duke University, Durham, NC
- Y-Ching Wang, PhD, National Cheng Kung University, Tainin, Taiwan
- Tulin Yanik, PhD, Middle East Technical University, Ankara, Turkey