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Neurosecretory Proteins in Neuroprotection and Neurodevelopment

• Y. Peng Loh, PhD, Head, Section on Cellular Neurobiology
• Hong Lou, MD, Senior Research Assistant
• Xuyu Yang, PhD, Staff Scientist
• Sangeetha Hareendran, PhD, Postdoctoral Visiting Fellow
• Vinay Sharma, PhD, Postdoctoral Visiting Fellow
• Ashley Xiao, PhD, Postdoctoral Intramural Research Training Award Fellow
• Areg Peltekian, MS, Postbaccalaureate Fellow
• Vida Falahatian, MD, Special Volunteer

The intracellular sorting of pro-neuropeptides and neurotrophins to the regulated secretory pathway (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, also known as 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 carboxypeptidase E (CPE) and then secreted. We showed that the sorting of prohormones to the RSP occurs by a receptor-mediated mechanism. Site-directed 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; 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. Overexpression of the CPE tail inhibited the movement of BDNF– and POMC/CPE–containing vesicles to the processes in hippocampal neurons and pituitary cells, respectively. The transmembrane CPE tails on the POMC/ACTH and BDNF vesicles anchor these organelles, which interact with dynactin and the microtubule-based motors KIF1A/KIF3A to effect anterograde vesicle movement to the plasma membrane. Recently, in collaboration with Josh Park, we showed that another player, snapin, binds directly to the cytoplasmic tail of CPE and connects to the microtubule motor complex, consisting of dynactin and kinesin-2, to mediate the post-Golgi transport of POMC/ACTH vesicles to the process terminals of AtT20 cells for activity-dependent secretion. Our study has thus uncovered a novel complex for secretory vesicle transport in neuroendocrine cells.

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 serpinin-RRG, a C-terminally extended form, 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 found that pGlu serpinin is a powerful cardio-protectant after ischemia. The mechanism involved the activation of the reperfusion injury salvage kinase (RISK) pathway. In collaboration with Angelo Corti, we showed that serpin-RRG had anti-angiogenic activity.

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

We generated a CPE (also known as Neurotrophic factor-α1, NF-α1) knock-out (KO) mouse to study the function of CPE/NF-α1 in vivo. The KO mouse exhibited obesity, infertility, and diabetes, as well as learning and memory deficits and depressive-like behavior. 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/NF-α1 in human disease. Using the Cpe-KO mice as a model in which to study its nervous system deficiencies, we showed defects in learning and memory by the Morris water maze and object-preference tests, and depressive-like behavior by the forced swim test. 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 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 or glutamate. Moreover, a non-enzymatically active form of CPE/NF-α1 (E342Q) applied extracellularly had the same neuroprotective effect, indicating that it acts independently of enzymatic activity. Hence, 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 binds to HT22 cells, an immortalized hippocampal neuronal cell line, in a saturable manner, and that the binding is specifically displaced by non-iodinated CPE/NF-α1, but not by bovine serum albumin, suggesting the existence of a receptor. Use of K235a, a Trk (tropomyosin receptor kinase) family inhibitor, and PD16285, a fibroblast growth factor receptor (FGFR1-3) inhibitor, did not prevent the neuroprotective action of CPE/NF-α1 in hippocampal neurons treated with H₂O₂, suggesting that the CPE/NF-α1 likely uses a different class of receptors than those of the Trk family or FGFRs. We screened a human G protein–coupled receptor (GPCR) library for binding activity to CPE and identified a promising receptor candidate.

The mechanism of action of CPE/NF-α1 in neuroprotection involves the activation of the ERK1/2 (extracellular-signal-regulated kinase) signaling pathway and the Akt signaling pathway (an intracellular signal transduction pathway) during stress, which then leads to enhanced expression of a pro-survival mitochondrial protein, Bcl2, inhibition of caspase 3 activation, and promotion of neuronal survival [Reference 1]. 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 the 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-apoptotic protein Bcl2, in the hippocampus. In situ hybridization studies indicated especially elevated CPE/NF-a1 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. The 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 after CRS, leading to the degeneration of the CA3 neurons [Reference 2]. Furthermore, CRS also elevated the expression of FGF2. We also demonstrated that primary hippocampal neurons treated with CPE/NF-α1 raised FGF2 expression. Thus, another pathway for CPE/NF-α1 may be through FGF2, which is known to have neuroprotective effect.

The relevance of CPE/NF-α1 in neuroprotection in humans was underscored by our studies on a mutation of the CPE gene found in an Alzheimer’s disease (AD) patient [Reference 3]. Our search in the GeneBank EST database identified a sequence entry from the cortex of an 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) CPE and CPE-QQ in Neuro2a cells resulted in degradation of both forms of the protein and reduction in secretion of WT CPE. Immuno-cytochemical studies show that CPE-QQ stains in the perinuclear region of the cells and co-stains with Calnexin, an ER marker, consistent with localization of the mutant protein in the ER. Moreover, many cells appear unhealthy, indicating that they might be undergoing ER stress, unlike the cells expressing WT CPE, which show staining in the cell body and neurites. CPE-QQ was not secreted and even prevented WT CPE from being secreted by aggregating with it. Overexpression of CPE-QQ in rat primary hippocampal neurons resulted in elevated levels of the ER stress marker CHOP, reduced levels of pro-survival protein Bcl-2, and increased neuronal cell death. Thus, 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 of age, the animals exhibited memory deficits and depressive-like behavior compared with WT mice, but that their spatial learning ability was unimpaired. The CPE-QQ mice were neither obese nor diabetic, likely owing to some CPE activity in these mice, given that the endogenous WT gene was not deleted. However, they had significantly fewer neurites in the CA3 region, the dentate gyrus of the hippocampus, and the medial prefrontal cortex, indicative of neurodegeneration. Moreover, they exhibited reduced neurogenesis in the subgranular zone and hyperphosphorylation of the microtubule-associated protein tau at ser³⁹⁵, a hallmark of AD. The studies identified a human mutation in the CPE gene resulting in expression of a CPE-QQ protein, which caused neurodegeneration and impairment of memory function in a mouse model, similar to AD, as well as depressive-like behavior.

Stress also induces depression. Huda Akil’s group (University of Michigan) reported that 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, (1h/day for 7 days) 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 at least in part, 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, we propose that CPE/NF-α1 prevents stress-induced depressive-like behavior 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)]. Rosiglitazone has previously been shown to be effective in treating diabetic patients with bi-polar disorders.

Role of CPE/NF-α1 and CPE-deltaN in embryonic brain development

Embryonic mouse brains express three forms of CPE/NF-α1 mRNA: 2.1kb, 1.9kb, 1.73kb in size, encoding a 53kD wild-type CPE/NF-α1, and 2 isoforms: 47kD and 40kD N-terminal truncated forms (CPE/NF-α1-DNs), respectively. The three mRNAs are expressed as early as E8.5 and increase significantly in two waves at E10.5 and postnatal day1 [Reference 5]. Interestingly, CPE/NF-α1-DNs are not expressed in adult mouse brain. 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 examined the effect of CPE/NF-α1 on E13.5 neocortex-derived neurospheres, which contain stem cells and neuroprogenitors. Application of recombinant CPE/NF-α1 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 brought about by negatively regulating the wnt pathway. We also 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 after CPE/NF-α1 treatment, without alteration in the percentage of neuronal and oligodendrocyte populations. We also observed this phenomenon when the cultured embryonic stem cells were treated with a non-enzymatic form of CPE, indicating that the effect was independent of enzymatic activity. Interestingly, dissociated cells from neurospheres derived from Cpe/Nf-α1–KO mouse embryos showed fewer astrocytes but more neurons, which was reversed with CPE/NF-α1 application. In vivo, Cpe/Nf-α1–KO mouse cortex (at P1, the time of astrocytogenesis) showed about half the astrocyte numbers compared with 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 6].

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.

We also studied the function of 40kD CPE/NF-α1-DN during development. Overexpression of 40kD CPE/NF-α1-DN in HT-22 cells, a hippocampal cell line, resulted in an increase in expression of IGFBP2, DAP1, and Ephrin 1A mRNAs and proteins; we demonstrated that the IGFBP2 is involved in proliferation in HT22 and mouse cortical neurons [Reference 5]. Further studies aimed at determining the role of CPE/NF-α1-DN in vivo are in progress.

Publications

1. 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.
2. 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.
3. 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.
4. Xiao L, Yang X, Sharma VK, Loh YP. Cloning, gene regulation and neuronal proliferation functions of a novel N-terminal truncated carboxypeptidase E/neurotrophic factor-1 variants in embryonic mouse brain. FASEB J 2018;33(1):808-820.
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 2017;35:557-571.

Collaborators

• Angelo Corti, MD, San Raffaele Scientific Institute, Milan, Italy
• Shiu-Feng Huang, MD, PhD, National Health Research Institutes, Zhunan, Taiwan
• Jennifer C. Jones, MD, PhD, Vaccine Branch, Center for Cancer Research, NCI, Bethesda, MD
• Jacqueline Jonklaas, MD, Georgetown University Medical Center, Washington, DC
• Beata Lecka-Czernik, PhD, University of Toledo, Toledo, OH
• Saravana Murthy, PhD, Life Magnetics Inc., Detroit, MI
• Karel Pacak, MD, PhD, Section on Medical Neuroendocrinology, NICHD, Bethesda, MD
• Joshua J. Park, PhD, University of Toledo, Toledo, OH
• Bruno Tota, MD, Università della Calabria, Cosenza, Italy
• Josef Troger, MD, Medizinische Universität Innsbruck, Innsbruck, Austria
• Y-Ching Wang, PhD, National Cheng Kung University, Tainan, Taiwan

Contact

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

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