Promoting effect and immunologic role of secretogranin II on bladder cancer progression via regulating MAPK and NF-κB pathways.

Bladder cancer (BLCA) is ranked among the top ten most prevalent cancers worldwide and is the second most common malignant tumor within the field of urology. The limited effectiveness of immune targeted therapy in treating BLCA, due to its high metastasis and recurrence rates, necessitates the identification of new therapeutic targets. Secretogranin II (SCG2), a member of the chromaffin granin/secreted granin family, plays a crucial role in the regulated release of peptides and hormones. The role of SCG2 in the tumor microenvironment (TME) of lung adenocarcinoma and colon cancer has been established, but its functional significance in BLCA remains uncertain. This study aimed to investigate SCG2 expression in 15 bladder cancer tissue samples and their corresponding adjacent control tissues. The potential involvement of SCG2 in BLCA progression was assessed using various techniques, including analysis of public databases, immunohistochemistry, Western Blotting, immunofluorescence, wound-healing assay, Transwell assay, and xenograft tumor formation experiments in nude mice. This study provided novel evidence indicating that SCG2 plays a pivotal role in facilitating the proliferation, migration, and invasion of BLCA by activating the MEK/Erk and MEK/IKK/NF-κB signaling pathways, as well as by promoting M2 macrophage polarization. These findings propose the potential of SCG2 as a molecular target for immunotherapy in human BLCA.

[High expression of secretogranin II increases oxaliplatin resistance of colorectal cancer cells].

OBJECTIVE: To investigate the expression of secretogranin II (SCG2) in colorectal cancer (CRC) tissues and its impact on oxaliplatin resistance of CRC cells. METHODS: We performed immunohistochemistry to detect the expression level of SCG2 on a tissue microarray containing 96 CRC and 84 adjacent tissues and analyzed the association of SCG2 expression with the clinical features of the CRC patients. SCG2 expression was also measured in DLD1 cells treated with oxaliplatin using immunoblotting and RT-qPCR analyses. The effects of SCG2 expression on oxaliplatin sensitivity and cell viability were evaluated in a DLD1 cell model of SCG2 knockout established using CRISPR-cas9 technique, and the expressions of apoptosis-related proteins were detected using Western blotting and RT-qPCR. We further examined SCG2 expression levels in an oxaliplatin-resistant DLD1 cell line and its parental DLD1 cells. RESULTS: SCG2 expression was significantly increased in CRC tissues as compared with the adjacent tissues (1.932±0.816 vs 1), and the tumor tissues in advanced stages showed higher SCG2 expression levels. In DLD1 cells, treatment with oxaliplatin significantly increased SCG2 expression, and SCG2 knockout obviously increased oxaliplatin sensitivity of the cells and enhanced the expressions of apoptosis-related proteins. Compared with the parental cells, oxaliplatin-resistant DLD1 cells showed a significant increase of SCG2 expression by 3.901±0.471 folds. CONCLUSION: SCG2 may serve as a risk gene in CRC, and its high expression increases oxaliplatin resistance of CRC cells.

Function of secretoneurin in regulating the expression of reproduction-related genes in ovoviviparous black rockfish (Sebastes schlegelii).

Secretoneurin (SN), a conserved peptide derived from secretogranin-2 (scg2), also known as secretogranin II or chromogranin C, plays an important role in regulating gonadotropin in the pituitary, which affects the reproductive system. This study aimed to clarify the mode of action of scg2 in regulating gonad development and maturation and the expression of mating behavior-related genes. Two scg2 cDNAs were cloned from the ovoviviparity teleost black rockfish (Sebastes schlegelii). In situ hybridization detected positive scg2 mRNA signals in the telencephalon and hypothalamus, where sgnrh and kisspeptin neurons were reported to be located and potentially regulated by scg2. In vivo, intracerebral ventricular injections of synthetic black rockfish SNa affected brain cgnrh, sgnrh, kisspeptin1, pituitary lh and fsh and gonad steroidogenesis-related gene expression levels with sex dimorphism. In vitro, a similar effect was found in primary cultured brain and pituitary cells. Thus, SN could contribute to the regulation of gonadal development, as well as reproductive behaviors, including mating and parturition.

Bone marrow-derived mesenchymal stem cells accelerate angiogenesis in pregnant experimentally induced deep venous thrombosis rat model via up-regulation of pro-angiogenic secretogranin II.

The present study investigated whether bone marrow-derived mesenchymal stem cells (BMMSCs) facilitate angiogenesis and improve outcomes of pregnancy with obstetric deep venous thrombosis (DVT) and explored the underlying mechanism. A pregnant DVT rat model was established using a "stenosis" method on the lower segment of the inferior vena cava (IVC). The extent of vascularization in thrombosed IVC was examined by immunohistochemistry. In addition, the effect of BMMSCs on DVT pregnancy outcomes was evaluated. We also characterized the effect of BMMSC-derived conditioned medium (BM-CM) on the impaired human umbilical vein endothelial cells (HUVECs). Thereafter, transcriptome sequencing was employed to identify the differentially expressed genes in thrombosed IVC tissues of DVT and DVT plus BMMSCs (thrice) groups. Lastly, the candidate gene's role in the promotion of angiogenesis was demonstrated in vitro and in vivo. The DVT model was successfully established using IVC stenosis. The injection of three consecutive BMMSC doses into pregnant SD rats with DVT was demonstrated to be the most effective treatment, which significantly reduced the length and weight of the thrombus, induced the highest level of angiogenesis, and ameliorated the embryo absorption rate. In vitro, BM-CM efficiently increased the abilities of impaired endothelial cells to proliferate, migrate, invade, and form vessel-like tubes, while inhibiting their apoptosis. Transcriptome sequencing revealed that BMMSCs induced a prominent upregulation of a variety of pro-angiogenic genes, including secretogranin II (SCG2). When SCG2 expression was knocked down by lentivirus, the BMMSCs' and BM-CM-induced pro-angiogenic effects on pregnant DVT rats and HUVECs were markedly attenuated. In conclusion, the study results suggest that BMMSCs enhance angiogenesis via up-regulation of SCG2, providing an effective alternative regenerative agent and novel target for the therapy of obstetric DVT.

Tuning the Size of Large Dense-Core Vesicles and Quantal Neurotransmitter Release via Secretogranin II Liquid-Liquid Phase Separation.

Large dense-core vesicles (LDCVs) are larger in volume than synaptic vesicles, and are filled with multiple neuropeptides, hormones, and neurotransmitters that participate in various physiological processes. However, little is known about the mechanism determining the size of LDCVs. Here, it is reported that secretogranin II (SgII), a vesicle matrix protein, contributes to LDCV size regulation through its liquid-liquid phase separation in neuroendocrine cells. First, SgII undergoes pH-dependent polymerization and the polymerized SgII forms phase droplets with Ca2+ in vitro and in vivo. Further, the Ca2+ -induced SgII droplets recruit reconstituted bio-lipids, mimicking the LDCVs biogenesis. In addition, SgII knockdown leads to significant decrease of the quantal neurotransmitter release by affecting LDCV size, which is differently rescued by SgII truncations with different degrees of phase separation. In conclusion, it is shown that SgII is a unique intravesicular matrix protein undergoing liquid-liquid phase separation, and present novel insights into how SgII determines LDCV size and the quantal neurotransmitter release.

Differential Expression of Secretogranins II and III in Canine Adrenal Chromaffin Cells and Pheochromocytomas.

Secretogranin II (SgII) and III (SgIII) function within peptide hormone-producing cells and are involved in secretory granule formation. However, their function in active amine-producing cells is not fully understood. In this study, we analyzed the expression profiles of SgII and SgIII in canine adrenal medulla and pheochromocytomas by immunohistochemical staining. In normal adrenal tissues, the intensity of coexpression of these two secretogranins (Sgs) differed from each chromaffin cell, although a complete match was not observed. The coexpression of vesicular monoamine transporter 2 (VMAT2) with SgIII was similar to that with chromogranin A, but there was a subpopulation of VMAT2-expressing cells that were negative or hardly detectable for SgII. These results are the first to indicate that there are distinct expression patterns for SgII and SgIII in adrenal chromaffin cells. Furthermore, the expression of these two Sgs varied in intensity among pheochromocytomas and did not necessarily correlate with clinical plasma catecholamine levels in patients. However, compared with SgIII, the expression of SgII was shown to be strong at the single-cell level in some tumor tissues. These findings provide a fundamental understanding of the expression differences between SgII and SgIII in normal adrenal chromaffin cells and pheochromocytomas.

Bidirectional perisomatic inhibitory plasticity of a Fos neuronal network.

Behavioural experiences activate the FOS transcription factor in sparse populations of neurons that are critical for encoding and recalling specific events1-3. However, there is limited understanding of the mechanisms by which experience drives circuit reorganization to establish a network of Fos-activated cells. It is also not known whether FOS is required in this process beyond serving as a marker of recent neural activity and, if so, which of its many gene targets underlie circuit reorganization. Here we demonstrate that when mice engage in spatial exploration of novel environments, perisomatic inhibition of Fos-activated hippocampal CA1 pyramidal neurons by parvalbumin-expressing interneurons is enhanced, whereas perisomatic inhibition by cholecystokinin-expressing interneurons is weakened. This bidirectional modulation of inhibition is abolished when the function of the FOS transcription factor complex is disrupted. Single-cell RNA-sequencing, ribosome-associated mRNA profiling and chromatin analyses, combined with electrophysiology, reveal that FOS activates the transcription of Scg2, a gene that encodes multiple distinct neuropeptides, to coordinate these changes in inhibition. As parvalbumin- and cholecystokinin-expressing interneurons mediate distinct features of pyramidal cell activity4-6, the SCG2-dependent reorganization of inhibitory synaptic input might be predicted to affect network function in vivo. Consistent with this prediction, hippocampal gamma rhythms and pyramidal cell coupling to theta phase are significantly altered in the absence of Scg2. These findings reveal an instructive role for FOS and SCG2 in establishing a network of Fos-activated neurons via the rewiring of local inhibition to form a selectively modulated state. The opposing plasticity mechanisms acting on distinct inhibitory pathways may support the consolidation of memories over time.

TRPV1, TRPA1, and TRPM8 are expressed in axon terminals in the cornea: TRPV1 axons contain CGRP and secretogranin II; TRPA1 axons contain secretogranin 3.

Purpose: The cornea is highly enriched in sensory neurons expressing the thermal TRP channels TRPV1, TRPA1, and TRPM8, and is an accessible tissue for study and experimental manipulation. The aim of this work was to provide a concise characterization of the expression patterns of various TRP channels and vesicular proteins in the mammalian cornea. Methods: Immunohistochemistry (IHC) was performed using wholemount and cryostat tissue preparations of mouse and monkey corneas. The expression patterns of TRPV1 and TRPA1 were determined using specific antisera, and further colocalization was performed with antibodies directed against calcitonin-related gene protein (CGRP), neurofilament protein NF200, and the secretogranins ScgII and SCG3. The expression of TRPM8 was determined using corneas from mice expressing EGFP under the direction of a TRPM8 promoter (TRPM8EGFP mice). Laser scanning confocal microscopy and image analysis were performed. Results: In the mouse cornea, TRPV1 and TRPM8 were expressed in distinct populations of small diameter C fibers extending to the corneal surface and ending either as simple or ramifying terminals, or in the case of TRPM8, as complex terminals. TRPA1 was expressed in large-diameter NF200-positive Aδ axons. TRPV1 and TRPA1 appeared to localize to separate intracellular vesicular structures and were primarily found in axons containing components of large dense vesicles with TRPV1 colocalizing with CGRP and ScgII, and TRPA1 colocalizing with SCG3. Monkey corneas showed similar colocalization of CGRP and TRPV1 on small-diameter axons extending to the epithelial surface. Conclusions: The mouse cornea is abundant in sensory neurons expressing TRPV1, TRPM8, and TRPA1, and provides an accessible tissue source for implementing a live tissue preparation useful for further exploration of the molecular mechanisms of hyperalgesia. This study showed that surprisingly, these TRP channels localize to separate neurons in the mouse cornea and likely have unique physiological functions. The similar TRPV1 expression pattern we observed in the mouse and monkey corneas suggests that mice provide a reasonable initial model for understanding the role of these ion channels in higher mammalian corneal physiology.

Secretoneurin is a secretogranin-2 derived hormonal peptide in vertebrate neuroendocrine systems.

Secretogranin-2 (SCG2) is a large precursor protein that is processed into several potentially bioactive peptides, with the 30-43 amino acid central domain called secretoneurin (SN) being clearly evolutionary conserved in vertebrates. Secretoneurin exerts a diverse array of biological functions including regulating nervous, endocrine, and immune systems in part due to its wide tissue distribution. Expressed in some neuroendocrine neurons and pituitary cells, SN is a stimulator of the synthesis and release of luteinizing hormone from both goldfish pituitary cells and the mouse LßT2 cell line. Neuroendocrine, paracrine and autocrine signaling pathways for the stimulation of luteinizing hormone release indicate hormone-like activities to regulate reproduction. Mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. A single injection of the SNa peptide enhanced reproductive outcomes in scg2a/scg2b double mutant zebrafish. Evidence in goldfish suggests a new role for SN to stimulate food intake by actions on other feeding-related neuropeptides. Expression and regulation of the Scg2a precursor mRNA in goldfish gut also supports a role in feeding. In rodent models, SN has trophic-like properties promoting both neuroprotection and neuronal plasticity and has chemoattractant properties that regulate neuroinflammation. Data obtained from several cellular models suggest that SN binds to and activates a G-protein coupled receptor (GPCR), but a bona fide SN receptor protein needs to be identified. Other signaling pathways for SN have been reported which provides alternatives to the GPCR hypothesis. These include AMP-activated protein kinase (AMPK), extracellular signal-regulated kinases (ERK), mitogen-activated protein kinase (MAPK)and calcium/calmodulin-dependent protein kinase II in cardiomyocytes, phosphatidylinositol 3-kinase (PI3K) and Akt/Protein Kinase B (AKT, and MAPK in endothelial cells and Janus kinase 2/signal transducer and activator of transcription protein (JAK2-STAT) signaling in neurons. Some studies in cardiac cells provide evidence for cellular internalization of SN by an unknown mechanism. Many of the biological functions of SN remain to be fully characterized, which could lead to new and exciting applications.

Targeted mutation of secretogranin-2 disrupts sexual behavior and reproduction in zebrafish.

The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. We previously reported that secretoneurin-a, a neuropeptide derived from the processing of secretogranin-2a (Scg2a), stimulates luteinizing hormone release, suggesting a role in reproduction. Here we provide evidence that mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. Large-scale spawning within-line crossings (n = 82 to 101) were conducted. Wild-type (WT) males paired with WT females successfully spawned in 62% of the breeding trials. Spawning success was reduced to 37% (P = 0.006), 44% (P = 0.0169), and 6% (P < 0.0001) for scg2a-/- , scg2b-/- , and scg2a-/-;scg2b-/- mutants, respectively. Comprehensive video analysis indicates that scg2a-/-;scg2b-/- mutation reduces all male courtship behaviors. Spawning success was 47% in saline-injected WT controls compared to 11% in saline-injected scg2a-/-;scg2b-/- double mutants. For these mutants, spawning success increased 3-fold following a single intraperitoneal (i.p.) injection of synthetic secretoneurin-a (P = 0.0403) and increased 3.5-fold with injection of human chorionic gonadotropin (hCG). Embryonic survival at 24 h remained on average lower in scg2a-/-;scg2b-/- fish compared to WT injected with secretoneurin-a (P < 0.001). Significant reductions in the expression of gonadotropin-releasing hormone 3 in the hypothalamus, and luteinizing hormone beta and glycoprotein alpha subunits in the pituitary provide evidence for disrupted hypothalamo-pituitary function in scg2a and scg2b mutant fish. Our results indicate that secretogranin-2 is required for optimal reproductive function and support the hypothesis that secretoneurin is a reproductive hormone.

Secretogranin III upregulation is involved in parkinsonian toxin-mediated astroglia activation.

Environmental neurotoxins such as paraquat (PQ), manganese, and 1-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are associated with a higher risk of Parkinson's disease (PD). These parkinsonian toxins exert certain common toxicological effects on astroglia; however, their role in the regulatory functions of astroglial secretory proteins remains unclear. In a previous study, we observed that secretogranin II (SCG2) and secretogranin III (SCG3), which are important components of the regulated secretory pathway, were elevated in PQ-activated U118 astroglia. In the current study, we used the parkinsonian toxins dopamine (DA), active metabolite of MPTP (MPP+), MnCl2, and lipopolysaccharide (LPS) as inducers, and studied the potential regulation of SCG2 and SCG3. Our results showed that all the parkinsonian toxins except LPS affected astroglial viability but did not cause apoptosis. Exposure to DA, MPP+, and MnCl2 upregulated glial fibrillary acidic protein (GFAP), a marker for astrocyte activation, and stimulated the levels of several astrocytic-derived factors. Further, DA, MPP+, and MnCl2 exposure impeded astroglial cell cycle progression. Moreover, the expression of SCG3 was elevated, while its exosecretion was inhibited in astroglia activated by parkinsonian toxins. The level of SCG2 remained unchanged. In combination with our previous findings, the results of this study indicate that SCG3 may act as a cofactor in astrocyte activation stimulated by various toxins, and the regulation of SCG3 could be involved in the toxicological mechanism by which parkinsonian toxins affect astroglia.

Fasting before or after wound injury accelerates wound healing through the activation of pro-angiogenic SMOC1 and SCG2.

Healing of the chronic diabetic ulceration and large burns remains a clinical challenge. Therapeutic fasting has been shown to improve health. Our study tested whether fasting facilitates diabetic and burn wound healing and explored the underlying mechanism. Methods: The effects of fasting on diabetic and burn wound healing were evaluated by analyzing the rates of wound closure, re-epithelialization, scar formation, collagen deposition, skin cell proliferation and neovascularization using histological analyses and immunostaining. In vitro functional assays were conducted to assess fasting and refeeding on the angiogenic activities of endothelial cells. Transcriptome sequencing was employed to identify the differentially expressed genes in endothelial cells after fasting treatment and the role of the candidate genes in the fasting-induced promotion of angiogenesis was demonstrated. Results: Two times of 24-h fasting in a week after but especially before wound injury efficiently induced faster wound closure, better epidermal and dermal regeneration, less scar formation and higher level of angiogenesis in mice with diabetic or burn wounds. In vitro, fasting alone by serum deprivation did not increase, but rather reduced the abilities of endothelial cell to proliferate, migrate and form vessel-like tubes. However, subsequent refeeding did not merely rescue, but further augmented the angiogenic activities of endothelial cells. Transcriptome sequencing revealed that fasting itself, but not the following refeeding, induced a prominent upregulation of a variety of pro-angiogenic genes, including SMOC1 (SPARC related modular calcium binding 1) and SCG2 (secretogranin II). Immunofluorescent staining confirmed the increase of SMOC1 and SCG2 expression in both diabetic and burn wounds after fasting treatment. When the expression of SMOC1 or SCG2 was down-regulated, the fasting/refeeding-induced pro-angiogenic effects were markedly attenuated. Conclusion: This study suggests that fasting combined with refeeding, but not fasting solely, enhance endothelial angiogenesis through the activation of SMOC1 and SCG2, thus facilitating neovascularization and rapid wound healing.

Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia.

BACKGROUND: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. METHODS: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. RESULTS: SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. CONCLUSIONS: SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.

Lipid-dependent deposition of alpha-synuclein and Tau on neuronal Secretogranin II-positive vesicular membranes with age.

This report demonstrates insoluble alpha-synuclein (aSYN)+ aggregates in human sporadic Parkinson's disease (PD) midbrain that are linearly correlated with loss of glucocerebrosidase (GCase) activity. To identify early protein-lipid interactions that coincide with loss of lipid homeostasis, an aging study was carried out in mice with age-dependent reductions in GCase function. The analysis identified aberrant lipid-association by aSYN and hyperphosphorylated Tau (pTau) in a specific subset of neurotransmitter-containing, Secretogranin II (SgII)+ large, dense-core vesicles (LDCVs) responsible for neurotransmission of dopamine and other monoamines. The lipid vesicle-accumulation was concurrent with loss of PSD-95 suggesting synaptic destabilization. aSYN overexpression in the absence of lipid deregulation did not recapitulate the abnormal association with SgII+ vesicles. These results show lipid-dependent changes occur with age in neuronal vesicular membrane compartments that accumulate lipid-stabilized aSYN and pTau.

The Neuropeptide Secretoneurin Exerts a Direct Effect on Arteriogenesis In Vivo and In Vitro.

It is well known that nerves modulate the development and remodeling of blood vessels by releasing different neuropeptides and neurotransmitters. Secretoneurin (SN), a neuropeptide located in nerve fibers along blood vessels, acts as a pro-angiogenic agent and induces postnatal vasculogenesis. However, little is known about its involvement in arteriogenesis. In the present study, we tested the hypothesis that SN promotes arteriogenesis in a rat model of hind limb ischemia, as such, we evaluated the effect of this neuropeptide on proliferation and the production of adhesion and chemotaxis molecules in vascular smooth muscle cells (VSMCs), the main component that carries the burden of the transformation of a small arteriole into a large collateral vessel. In vivo, SN-immunoreactive nerve fibers were abundantly distributed in the adventitia of the collateral vessel. Moreover, administration of SN induced cell proliferation in the vascular wall and the infiltration of inflammatory cells/macrophages to promote collateral vessel growth. This was shown by an increased density of arterioles/arteries, together with a well-developed network of collateral vessels, and well-preserved skeletal muscles. In vitro, SN exerted proliferative effects on VSMCs and stimulated these cells to express adhesion molecules. In conclusion, our data demonstrate for the first time that SN acts as a mediator of inflammation, contributing to collateral vessel growth, in addition to directly stimulating cell proliferation in the vascular wall to promote collateral vessel growth in a rat model of hind limb ischemia. Anat Rec, 301:1917-1927, 2018. © 2018 Wiley Periodicals, Inc.

Gene therapy with the angiogenic neuropeptide secretoneurin ameliorates experimental diabetic neuropathy.

The pathogenesis of diabetic neuropathy remains enigmatic. Damage to the vasa nervorum may be responsible for this disorder. Recently, we showed that secretoneurin (SN) induces angiogenesis in hindlimb and myocardial ischemia. Moreover, beneficial effects were observed in wound healing. We therefore hypothesized that SN therapy may ameliorate diabetic neuropathy. We used db/db mice as animal model for neuropathy. Gene therapy was accomplished by intramuscular injection of SN plasmid along the sciatic nerve. Sciatic nerve motor and sensory conduction velocities were then measured for 9 wk. Nerve conduction velocities showed normal values in heterozygous mice for the observational period, but were severely reduced in homozygous mice in which velocities were significantly improved by SN, but not by control plasmid gene therapy. The reaction time in the tail-flick test improved significantly in SN-treated animals. The induction of growth of vasa nervorum seems to be part of the underlying mechanism. In addition, SN positively affected Schwann cell function in vitro and induced activation of important signaling pathways. Our observations suggest that SN exerts beneficial effects on nerve function in vivo and on Schwann cells in vitro. It therefore may be a promising treatment option for diabetic neuropathy.-Theurl, M., Lener, D., Albrecht-Schgoer, K., Beer, A., Schgoer, W., Liu, Y., Stanzl, U., Fischer-Colbrie, R., Kirchmair, R. Gene therapy with the angiogenic neuropeptide secretoneurin ameliorates experimental diabetic neuropathy.

Secretogranin-II plays a critical role in zebrafish neurovascular modeling.

Secretoneurin (SN) is a neuropeptide derived from specific proteolytic processing of the precursor secretogranin II (SgII). In zebrafish and other teleosts, there are two paralogs named sgIIa and sgIIb. Our results showed that neurons expressing sgIIb were aligned with central arteries in the hindbrain, demonstrating a close neurovascular association. Both sgIIb-/- and sgIIa-/-/sgIIb-/- mutant embryos were defective in hindbrain central artery development due to impairment of migration and proliferation of central artery cells. Further study revealed that sgIIb is non-cell autonomous and required for central artery development. Hindbrain arterial and venous network identities were not affected in sgIIb-/- mutant embryos, and the mRNA levels of Notch and VEGF pathway-related genes were not altered. However, the activation of MAPK and PI3K/AKT pathways was inhibited in sgIIb-/- mutant embryos. Reactivation of MAPK or PI3K/AKT in endothelial cells could partially rescue the central artery developmental defects in the sgIIb mutants. This study provides the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling of the hindbrain. Targeting the SgII system may, therefore, represent a new avenue for the treatment of vascular defects in the central nervous system.

Ovulatory Induction of SCG2 in Human, Nonhuman Primate, and Rodent Granulosa Cells Stimulates Ovarian Angiogenesis.

The luteinizing hormone (LH) surge is essential for ovulation, but the intrafollicular factors induced by LH that mediate ovulatory processes (e.g., angiogenesis) are poorly understood, especially in women. The role of secretogranin II (SCG2) and its cleaved bioactive peptide, secretoneurin (SN), were investigated as potential mediators of ovulation by testing the hypothesis that SCG2/SN is induced in granulosa cells by human chorionic gonadotropin (hCG), via a downstream LH receptor signaling mechanism, and stimulates ovarian angiogenesis. Humans, nonhuman primates, and rodents were treated with hCG in vivo resulting in a significant increase in the messenger RNA and protein levels of SCG2 in granulosa cells collected early during the periovulatory period and just prior to ovulation (humans: 12 to 34 hours; monkeys: 12 to 36 hours; rodents: 4 to 12 hours post-hCG). This induction by hCG was recapitulated in an in vitro culture system utilizing granulosa-lutein cells from in vitro fertilization patients. Using this system, inhibition of downstream LH receptor signaling pathways revealed that the initial induction of SCG2 is regulated, in part, by epidermal growth factor receptor signaling. Further, human ovarian microvascular endothelial cells were treated with SN (1 to 100 ng/mL) and subjected to angiogenesis assays. SN significantly increased endothelial cell migration and new sprout formation, suggesting induction of ovarian angiogenesis. These results establish that SCG2 is increased in granulosa cells across species during the periovulatory period and that SN may mediate ovulatory angiogenesis in the human ovary. These findings provide insight into the regulation of human ovulation and fertility.

Identification and functional characterization of two Secretogranin II genes in orange-spotted grouper (Epinephelus coioides).

Secretoneurin (SN) is an important stimulator of pituitary luteinizing hormone (LH) synthesis and secretion in goldfish. It is unknown whether this neuropeptide performs the same role in other fish species. In this study, the full-length cDNAs encoding Secretogranin IIa (SgIIa) and b (SgIIb) were cloned from the brain of orange-spotted grouper. Sequence analysis showed that a 34-amino acid SN peptide (SNa) is present in SgIIa proprotein, and a 33-amino acid SN peptide (SNb) is present in SgIIb proprotein. The two SN peptides share a low degree of similarity but contain the signature YTPQ-X-LA-X7-EL sequence. Real-time PCR showed that two SgII genes are mainly expressed in the brain and pituitary. During ovarian development, the expression levels of two SgII genes in the hypothalamus and pituitary were significantly reduced at the stage when the ovary contained full-grown oocytes. The biological functions of the two SN peptides were further investigated in vitro and in vivo. Both SN peptides could significantly elevate the mRNA levels of Gonadotropin-Releasing Hormone 1 (GnRH1) and 3 (GnRH3) in the hypothalamic fragments and upregulated the expression of Follicle-Stimulating Hormone beta (FSHb) and Luteinizing Hormone beta (LHb) in the pituitary cells. The stimulatory effects on the expression of GnRHs and Gonadotropins were also observed after intraperitoneal injection of SN peptides. Our study indicated that the SgII/SN system has stimulatory effects on the reproductive axis of orange-spotted grouper.

Effects of PQ's cytotoxicity on secretory vesicles in astroglia: Expression alternation of secretogranin II and its potential interaction with intracellular factors.

It has been extensively characterized that paraquat (PQ) selectively targets to the substantia nigra and exerts neurotoxic actions on dopaminergic neurons. However, a little knowledge is available about astroglia in PQ exposure, especially its complex secretory machinery. To explore this point, we built up a PQ-induced model in cultural U118 astrocyte. Since the granin family is considered as a master regulator of cargo sorting and large dense core vesicles (LDCVs) biogenesis in the regulated secretory pathway of nervous and neuroendocrine cells, the current study focused on one member, secretogranin II (SCG2) and investigated its alternation and potential relationship with other astrocyte-derived factors under PQ insult. We found that PQ upregulated SCG2 expression on both RNA and protein levels and stimulated the mRNA expression of neurotrophic factors, cytokines and glutamine synthetase (GS) simultaneously. RNAi knockdown of SCG2 did not rescue the cell cycle arrest induced by PQ but affected expressions of IL-6 and GS on mRNA and protein levels. Further studies on subcellular location showed that SCG2-positive secretory granules were partially colocalized with IL-6 but not GS in PQ exposure astrocyte. Taken together, our findings indicate that the expression alternation of SCG2 under astroglial activation by PQ may be necessary compensation for cargo sorting and LDCV biogenesis. The involvement of the IL-6 and GS suggests that the SCG2 may potentially regulate inflammatory factors and excitatory neurotransmitter to the cytotoxicity of PQ on astroglia.