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Secretory Protein Trafficking, Neuroprotection, and Cancer

Y. Peng Loh, PhD, Head, Section on Cellular Neurobiology
Niamh X. Cawley, PhD, Staff Scientist
Hong Lou, MD, Senior Research Assistant
Yong Cheng, PhD, Postdoctoral Fellow
Saravana Murthy, PhD, Postdoctoral Fellow
Prabhuanand Selvaraj, PhD, Postdoctoral Fellow
Erwan Thouennon, PhD, Postdoctoral Fellow
Nigel Birch, PhD, Special Volunteer
Vipula Kolli, PhD, Special Volunteer
Xioyan Qin, MD, Special Volunteer
Alexander Chen, BS, Postbaccalaureate Fellow
Jane Huang, BS, Postbaccalaureate Fellow
Nikoletta Lendvai, MD, Predoctoral Student

We study the cell biology of neuroendocrine cells and the function of neuropeptides and their processing enzymes in health and disease. Our focus is three-fold, to: (i) investigate the mechanisms of biogenesis and intracellular trafficking of dense-core secretory granules containing neuropeptides and their processing enzymes; (ii) investigate the role of serpinin, a novel chromogranin A–derived peptide discovered in our lab, in neural and cardiac function; and (iii) determine the newly appreciated non-enzymatic neurotrophic role of the prohormone-processing enzyme carboxypeptidase E (CPE) in neuronal function and cancer. Our work has 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 as a trophic factor that mediates neuroprotection, neurodevelopment, and anti-depression. We also identified a splice variant of CPE (CPE-delta N) that drives metastasis in various cancer types. Using cell lines, primary cell cultures, and mouse models, our studies have deepened our 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 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, 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 in neuroprotection, stress, and neurodevelopment

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. We showed that, 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 these 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. The neurons, which are normally enriched in CPE, were normal at three weeks of age just before the animals were weaned. Interestingly, when weaning was delayed for a week, the degeneration was not observed until postnatal week 5 in the CPE–KO mice. Moreover, the anti-epileptic drug carbamezapine given i.p. at two weeks of age prevented the degeneration. 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. Indeed, when CPE was overexpressed and secreted or applied externally to cultured hippocampal or cortical neurons, they were protected from apoptosis after inducing oxidative stress with hydrogen peroxide. The mechanism of action of CPE as an extracellular signaling molecule in neuroprotection 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. Thus, CPE is a novel neuroprotective trophic factor, which we named Neurotrophic factor-alpha1 (NF-α1) (1). We further demonstrate that CPE has a neuroprotective role in vivo. During and after mild chronic restraint stress (CRS) for 1h/day for seven days, CPE mRNA and protein levels, as well as Bcl2, were significantly elevated in the hippocampus. In situ hybridization studies indicated especially elevated CPE 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 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 (2).

We also investigated the role of CPE/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 (3).

Recently, we found that NF-α1 plays a role during embryonic development. NF-α1/CPE mRNA was expressed in mouse embryos as early as day E5.5, rising each day, peaking at E8.5, and falling slightly at E9.5. CPE mRNA expression then declined sharply at E 10.5–11.5 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 somites in mouse embryos. To study neural stem cell proliferation, exogenous recombinant NF-α1 was added to E13.5 neocortex–derived neurospheres, which contains 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, which is known to enhance proliferation. This suggests that NF-α1’s inhibitory effect on proliferation is mediated by negatively regulating the wnt pathway. Differentiation studies were carried out using neurospheres from seven-day cultures that were dissociated into single cells and cultured for an additional five days. An increase in astrocytes in the presence of NF-α1, was observed without altering the percentage of neuronal and oligodendrocyte populations. Interestingly, dissociated cells from neurospheres derived from NF-α1–KO mouse embryos showed reduced astrocytes and increased neurons. Our results suggest a novel role of NF-α1 as an extracellular signal to differentiate neural stem cells into astrocytes.

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, 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. Stabilization of the granule proteins raises their levels in the Golgi, resulting in significantly enhanced 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 pituitary 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, suggesting its role as a neurotransmitter or neuromodulator. Additionally, pGlu-serpinin was found to exhibit 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. 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. In collaboration with Angelo Corti, we found that serpinin-RRG had an anti-angiogenesis effect (4).

A splice isoform of carboxypeptidase E is a tumor inducer and biomarker for predicting future metastasis.

Despite the numerous biomarkers reported, few are useful for predicting metastasis. In our recent studies, we discovered a novel splice isoform of CPE (CPE-deltaN) that is elevated in metastatic hepatocellular, colon, breast, head, and neck carcinoma. 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) 1/2. 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. In another study, we showed that CPE-deltaN can activate the canonical wnt pathway, resulting in elevated levels of β-catenin, which functions with T-cell factor/lymphoid enhancer factor in the nucleus to activate expression of Wnt target genes. It is well known that such a mechanism could lead to colorectal cancer progression. We also demonstrated that 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. Indeed, in vivo, the interplay between CPE-deltaN and WT-CPE could influence the metastatic potential of neuroendocrine tumors.

In retrospective clinical studies of 180 patients with HCC, CPE-deltaN mRNA quantification in primary HCC(T) tumor versus surrounding normal tissue HCC(N) established a T/N cut off level above which future metastasis within two years could be predicted with high sensitivity and specificity and independently of cancer stage (5). A recent prospective study on 120 stage I and stage II HCC patients further indicated that CPE-deltaN is an excellent biomarker for predicting future metastasis with greater than 80% positive predictive value for stage II patients who show no indication of lymph node invasion or metastasis at time of surgery. In a prospective clinical study on 42 patients with pheochromocytomas/paragangliomas, we were able to predict from the mRNA copy numbers of CPE-deltaN in the resected tumors with high accuracy those patients who would develop future metastasis, although they were diagnosed with benign tumors at the time of surgery. Additionally, in an ongoing prospective studies of papillary thyroid cancer and lung cancer, CPE-deltaN was found to be a good biomarker for diagnosis of metastasis and for identifying patients who have high or low risk of recurrence. Continued follow-up of these patients will substantiate our prediction. In a retrospective study on colorectal cancer, a ratio greater than two of CPE-deltaN levels in tumor (T) to that of normal tissue (N) accurately diagnosed metastatic disease. In a prospective study on patients with clear cell (ccRCC) and papillary renal cell carcinoma (pRCC), CPE-deltaN mRNA copy numbers distinguished metastatic ccRCC/pRCC from benign tumors with 65% specificity and 100% sensitivity. Thus, CPE-deltaN is a novel tumor inducer and a powerful diagnostic and prognostic marker for predicting future metastasis in various cancer types and appears to be superior to histopathological diagnosis. Additionally, we recently demonstrated that CPE-deltaN mRNA is secreted in exosomes derived from HCC, breast, and glioblastoma cell lines, paving the way to develop a non-invasive assay for this biomarker in body fluids for early detection of cancer in high-risk patients.

Publications

Cheng Y, Cawley NX, Loh YP. Carboxypeptidase E/NF-α1: a new neurotrophic factor against oxidative stress-induced apoptotic cell death mediated by ERK and PI3-K/AKT pathways. PLoS ONE 2013;8:e71578.
Murthy SRK, Thouennon E, Li W-S, Cheng Y, Bhupatkar J, Cawley NX, Lane M, Merchenthaler I, Loh YP. Carboxypeptidase E protects hippocampal neurons during stress in male mice by up-regulating pro-survival BCL2 protein expression. Endocrinology 2013;154:3284-3293.
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-α1 prevents stress-induced depression through enhancement of neurogenesis and is activated by rosiglitazone. Mol Psychiatry 2014;E-pub ahead of print.
Crippa L, Bianco M, Colombo B, Gasparri AM, Ferrero E, Loh YP, Curnis F, Corti A. A new chromogranin A-dependent angiogenic switch activated by thrombin. Blood 2013;121:392-402.
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.

Collaborators

Soyhun Ahn, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Jan Breza, MD, Comenius University School of Medicine, Bratislava, Slovakia
Angelo Corti, PhD, San Raffaele Scientific Institute, Milano, Italy
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
Maria Merino, MD, Laboratory of Pathology, NCI, Bethesda, 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
Tulin Yanik, PhD, Middle East Technical University, Ankara, Turkey
Y-Ching Wang, PhD, National Cheng Kung University, Tainin, Taiwan

Contact

For more information, email lohp@mail.nih.gov or visit scn.nichd.nih.gov or neuroscience.nih.gov/Faculty/Profile/y peng-loh.aspx.

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