Calcium-binding Protein Secretagogin Research Articles

Secretagogin as a marker to distinguish between different neuron types in human frontal and temporal cortex.

The principal aim of the present work was to chemically characterize the population of neurons labeled for the calcium binding protein secretagogin (SCGN) in the human frontal and temporal cortices (Brodmann's area 10 and 21, respectively). Both cortical regions are involved in many high cognitive functions that are especially well developed (or unique) in humans, but with different functional roles. The pattern of SCGN immunostaining was rather similar in BA10 and BA21, with all the labeled neurons displaying a non-pyramidal morphology (interneurons). Although SCGN cells were present throughout all layers, they were more frequently observed in layers II, III and IV, whereas in layer I they were found only occasionally. We examined the degree of colocalization of SCGN with parvalbumin (PV) and calretinin (CR), as well as with nitric oxide synthase (nNOS; the enzyme responsible for the synthesis of nitric oxide by neurons) by triple immunostaining. We looked for possible similarities or differences in the coexpression patterns of SCGN with PV, CR and nNOS between BA10 and BA21 throughout the different cortical layers (I-VI). The percentage of colocalization was estimated by counting the number of all labeled cells through columns (1,100-1,400 μm wide) across the entire thickness of the cortex (from the pial surface to the white matter) in 50 μm-thick sections. Several hundred neurons were examined in both cortical regions. We found that SCGN cells include multiple neurochemical subtypes, whose abundance varies according to the cortical area and layer. The present results further highlight the regional specialization of cortical neurons and underline the importance of performing additional experiments to characterize the subpopulation of SCGN cells in the human cerebral cortex in greater detail.

Secretagogin Immunoreactivity Reveals Lugaro Cells in the Pigeon Cerebellum.

Lugaro cells are inhibitory interneurons found in the upper granular layer of the cerebellar cortex, just below or within the Purkinje cell layer. They are characterized by (1) a fusiform soma oriented in the parasagittal plane, (2) two pairs of dendrites emanating from opposite ends of the soma, (3) innervation from Purkinje cell collaterals, and (4) an axon that projects into the molecular layer akin to granular cell parallel fibers. Lugaro cells have been described in mammals, but not in other vertebrate classes, save one report in teleost fish. Here, we propose the existence of Lugaro cells in the avian cerebellum based on the morphological characteristics and connectivity described above. Immunohistochemical staining against the calcium binding protein secretagogin (SCGN) revealed Lugaro-like cells in the pigeon cerebellum. Some SCGN-labeled cells exhibit fusiform somata and dendrites parallel to the Purkinje cell layer in the parasagittal plane, as well as long axons that project into the molecular layer and travel alongside parallel fibers in the coronal plane. While mammalian Lugaro cells are known to express calretinin, the SCGN-labeled cells in the pigeon do not. SCGN-labeled cells also express glutamic acid decarboxylase, confirming their inhibitory function. Calbindin labeling revealed Purkinje cell terminals surrounding the SCGN-expressing cells. Our results suggest that Lugaro cells are more widespread among vertebrates than previously thought and may be a characteristic of the cerebellum of all vertebrates.

Secretagogin is Expressed by Developing Neocortical GABAergic Neurons in Humans but not Mice and Increases Neurite Arbor Size and Complexity.

The neocortex of primates, including humans, contains more abundant and diverse inhibitory neurons compared with rodents, but the molecular foundations of these observations are unknown. Through integrative gene coexpression analysis, we determined a consensus transcriptional profile of GABAergic neurons in mid-gestation human neocortex. By comparing this profile to genes expressed in GABAergic neurons purified from neonatal mouse neocortex, we identified conserved and distinct aspects of gene expression in these cells between the species. We show here that the calcium-binding protein secretagogin (SCGN) is robustly expressed by neocortical GABAergic neurons derived from caudal ganglionic eminences (CGE) and lateral ganglionic eminences during human but not mouse brain development. Through electrophysiological and morphometric analyses, we examined the effects of SCGN expression on GABAergic neuron function and form. Forced expression of SCGN in CGE-derived mouse GABAergic neurons significantly increased total neurite length and arbor complexity following transplantation into mouse neocortex, revealing a molecular pathway that contributes to morphological differences in these cells between rodents and primates.

Characterization of secretagogin‐immunoreactive amacrine cells in marmoset retina

The retina contains at least 30 different types of amacrine cells but not many are well characterized. In the present study the calcium-binding protein secretagogin was localized in a population of regular and displaced amacrine cells in the retina of the common marmoset Callithrix jacchus. Irrespective of their soma location, the dendrites of secretagogin amacrine cells occupy strata 2, 3, and 4 of the inner plexiform layer, between the two bands formed by cholinergic amacrine cells. Segretagogin amacrine cells are also immunopositive to antibodies against glutamic acid decarboxylase, suggesting that they use γ-aminobutyric acid (GABA) as their neurotransmitter. The spatial density of secretagogin amacrine cells decreases from a peak of about 400 cells/mm(2) near 1 mm eccentricity to less than 100 cells/mm(2) in peripheral retina; these densities account for about 1% of amacrine cells in the inner nuclear layer and for up to 27% of displaced amacrine cells. The cell bodies form a regular mosaic, suggesting that they constitute a single amacrine cell population. Secretagogin cells have varicose dendrites, which are decorated with small spines. Intracellular injection of DiI into secretagogin cells revealed an average dendritic field diameter of 170 μm and an average coverage factor of 3.2. In summary, secretagogin cells in marmoset retina are medium-field amacrine cells that share their stratification pattern with narrow-field amacrine cells and their neurotransmitter with wide-field amacrine cells. They may mediate spatial inhibition spanning the centralmost (on and off) bands of the inner plexiform layer.

Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

Secretagogin is a calcium binding protein (CBP) highly expressed in neuroendocrine cells. It has been shown to be involved in insulin secretion from pancreatic beta cells and is a strong candidate as a biomarker for endocrine tumors, stroke, and eventually psychiatric conditions. Secretagogin has been hypothesized to exert a neuroprotective role in neurodegenerative diseases like Alzheimer’s disease. The expression pattern of Secretagogin is not conserved from rodents to humans. We used brain tissue and primary neuronal cell cultures from rat to further characterize this CBP in rodents and to perform a few functional assays in vitro. Immunohistochemistry on rat brain slices revealed a high density of Secretagogin-positive cells in distinct brain regions. Secretagogin was found in the cytosol or associated with subcellular compartments. We tested primary neuronal cultures for their suitability as model systems to further investigate functional properties of Secretagogin. These cultures can easily be manipulated by treatment with drugs or by transfection with test constructs interfering with signaling cascades that might be linked to the cellular function of Secretagogin. We show that, like in pancreatic beta cells and insulinoma cell lines, also in neurons the expression level of Secretagogin is dependent on extracellular insulin and glucose. Further, we show also for rat brain neuronal tissue that Secretagogin interacts with the microtubule-associated protein Tau and that this interaction is dependent on Ca2+. Future studies should aim to study in further detail the molecular properties and function of Secretagogin in individual neuronal cell types, in particular the subcellular localization and trafficking of this protein and a possible active secretion by neurons.

Expression of secretagogin in clear-cell renal cell carcinomas is associated with a high metastasis rate

Renal cell carcinomas are divided into several subgroups according to their histopathologic characteristics. The outcome, therapy responses, and the applicability of molecular-targeted therapies depend on the tumor classification and on the tumor stage. Recent advances within the biomarker research facilitated the exact classification of the molecular character of the renal tumor. For example, the calcium-binding proteins parvalbumin and S-100A1 are characteristically expressed in renal cell carcinoma subgroups. This led us to investigate the expression of the novel calcium-binding protein secretagogin in renal cell carcinomas. Tissue microarray cylinders including 94 clear-cell renal cell carcinomas, 61 non-clear-cell renal cell carcinomas (37 papillary renal cell and 24 chromophobe carcinomas), and 30 oncocytomas were analyzed by immunohistochemistry. This showed remarkable secretagogin expression in 37% of the clear-cell renal cell carcinomas. Non-clear-cell renal cell carcinomas and oncocytomas were completely negative. Consequently performed immunoblotting analyses confirmed this expression profile. Because publicly available data direct toward a formation of a hierarchical cluster of secretagogin overexpressing clear-cell renal cell carcinomas, we conducted a clinical follow-up of the patients with clear-cell renal cell carcinoma. This revealed significantly more metastasis within the secretagogin-positive clear-cell renal cell carcinoma subgroup (49% versus 28%; P < .05). In conclusion, we report on detection of the novel calcium-binding protein secretagogin within a subgroup of clear-cell renal cell carcinomas. The increased metastasis rates within the secretagogin-positive subgroup of clear-cell renal cell carcinomas direct toward a clinical impact of our findings.

Calcium-binding protein secretagogin is a novel neuroendocrine marker

借助免疫组织化学标志对神经内分泌肿瘤的诊断非常重要,但现有的标志物并不能满足诊断和鉴别诊断的需要.促泌素(secretagoin)作为神经内分泌标志物已受到重视.促泌素首先由Wagner等[1-2]克隆,并认为是一种胰岛特异性神经内分泌标志物.我们通过对比研究,探讨促泌素在神经内分泌肿瘤临床病理诊断中的应用价值。

Expression of TAU in insulin-secreting cells and its interaction with the calcium-binding protein secretagogin

Tauopathies have been associated with Alzheimer's disease (AD), which frequently manifests together with diabetes mellitus type 2. Calcium-binding proteins such as the recently identified secretagogin (SCGN) might exert protective effects. As pancreatic beta-cells and neurons share common electrophysiological properties, we investigated the appearance of TAU (listed as MAPT in the HUGO and MGI Databases) protein at the islets of Langerhans and beta-cell-derived cell lines which highly express the neuroendocrine-specific protein SCGN. Six predominant TAU isoforms could be identified by immunoblotting, which formed TAU deposits detectable by immunofluorescence and sarkosyl-insoluble pellets. Using GST-SCGN pull-down assays, a calcium-dependent SCGN-TAU interaction was found. In this line, sucrose density gradient fractionation and differential ultracentrifugation studies of TAU and SCGN revealed co-appearance of both proteins. Co-localization of TAU and SCGN within insulinoma cells and islets of Langerhans mainly restricted to insulin-positive beta-cells was demonstrated by confocal microscopy. Motivated by these findings, we looked if SCGN overexpression could exert protective function on Rin-5F cells, which showed differences in TAU levels. Testing the vulnerability of Rin-5F clones by MTT assay, we revealed that high TAU levels going along with highest TAU aggregates could not be antagonized by high levels of SCGN protein. Our findings demonstrated for the first time the association of TAU and the calcium-binding protein SCGN and support earlier results implicating that beta-cells might represent an extra cerebral site of tauopathy.

Localization of the calcium‐binding protein secretagogin in cone bipolar cells of the mammalian retina

Secretagogin, a recently cloned member of the EF-hand family of calcium binding proteins, was localized in the mouse, rat, and rabbit retina using immunofluorescence immunohistochemistry. Secretagogin is expressed in subpopulations of ON and OFF cone bipolar cells; however, no immunoreactivity was observed in rod bipolar cells in any of these species. Using subtype-specific markers and mice expressing green fluorescent protein (GFP) within specific cell classes, we found that secretagogin is expressed in Types 2, 3, 4, 5, 6 and possibly Type 8 cone bipolar cells in the mouse retina. The expression pattern in the rat retina differs slightly with expression in cone bipolar cell Types 2, 5, 6, 7, and 8. Evaluation of secretagogin in the developing mouse retina revealed expression as early as postnatal day 6, with OFF cone bipolar cells showing secretagogin expression prior to the ON cone bipolar cells. Secretagogin is a useful marker of cone bipolar cells for studying alterations in bipolar cell morphology during development and degeneration. Further work will be necessary to elucidate the functional role of this protein in bipolar cells.

New Algorithm for the Identification of Intact Disulfide Linkages Based on Fragmentation Characteristics in Tandem Mass Spectra

Identifying the sites of disulfide bonds in a protein is essential for thorough understanding of a protein's tertiary and quaternary structures and its biological functions. Disulfide linked peptides are usually identified indirectly by labeling free sulfhydryl groups with alkylating agents, followed by chemical reduction and mass spectral comparison or by detecting the expected masses of disulfide linked peptides on mass scan level. However, these approaches for determination of disulfide bonds become ambiguous when the protein is highly bridged and modified. For accurate identification of disulfide linked peptides, we present here an algorithmic solution for the analysis of tandem mass (MS/MS) spectra of disulfide bonded peptides under nonreducing condition. A new algorithm called "DBond" analyzes disulfide linked peptides based on specific features of disulfide bonds. To determine disulfide linked sites, DBond takes into account fragmentation patterns of disulfide linked peptides in nucleoside diphosphate kinase (NDPK) as a model protein, considering fragment ions including cysteine, cysteine thioaldehyde (-2 Da, C(T)), cysteine persulfide (+32 Da, C(S)) and dehydroalanine (-34 Da, C(Delta)). Using this algorithm, we successfully identified about a dozen novel disulfide bonds in a hexa EF-hand calcium binding protein secretagogin and in a methionine sulfoxide reductase. We believe that DBond, taking into account the disulfide bond fragmentation characteristics and post-translational modifications, offers a novel approach for automatic identification of unknown disulfide bonds and their sites in proteins from MS/MS spectra.

Involvement of the calcium-binding protein secretagogin in pituitary adenoma hormone production

Calcium is a potent intracellular signal transducer whose fluctuations control numerous cellular processes among them hormone production. Secretagogin (SCGN) is an intracytoplasmatic protein belonging to the EF-hand calcium binding protein superfamily, which was initially isolated from a human pancreatic cDNA library and was shown to be involved in the regulation of insulin synthesis and secretion. Since SCGN was also detected in neuroendocrine cells in the anterior pituitary, we have studied the expression of SCGN both in normal and tumoral pituitary and have started to elucidate its role in pituitary cells. Our preliminary results obtained by Real time PCR analysis showed the expression of SCGN in a group of 4 normal human anterior pituitary glands and in 26 different pituitary adenomas. In the tumours, SCGN mRNA was downregulated in 2/4 somatotrophinomas, 6/7 nonfunctioning pituitary adenomas, 4/6 prolactinomas, 3/6 corticotrophinomas and 2/2 thyrotrophinomas when compared to the expression in normal pituitary tissue. The co-transfection of a rat SCGN expression plasmid with prolactin-luc reporter plasmid, GH-luc plasmid and POMC-luc reporter plasmid, respectively in mammosomatotroph GH3 cells and in corticotroph AtT-20 cells, showed that SCGN overexpression was able to induce a 46% increase in the activity of the prolactin gene promoter and a 60% increase in the activity of the GH gene promoter, while in AtT-20 cells the activity of POMC gene promoter showed an increase of 66%. The analysis of the different promoter elements showed an activation of CRE element in GH3 cells after SCGN plasmid co-transfection and an increase in cAMP production. In AtT-20 cells SCGN plasmid co-transfection was able to induce an increase in the activities of AP-1, Nbr-E and NurrRE elements, which was also accompanied by an increase in cAMP production. These preliminary findings suggest the involvement of SCGN in controlling the synthesis of pituitary hormones, therefore constituting a potential target for inhibiting the hormone production.

Involvement of the calcium-binding protein Secretagogin in pituitary adenoma hormone secretion

Involvement of the calcium-binding protein Secretagogin in pituitary adenoma hormone secretion

Immunoreactivity of calcium binding protein secretagogin in the human hippocampus is restricted to pyramidal neurons

Disturbed calcium homeostasis plays a crucial role in the aetiology of Alzheimer’s disease (AD) and the aging process. We evaluated immunoreactivity of secretagogin, a recently cloned calcium binding protein, in hippocampus and adjacent entorhinal cortex of 30 neuropathologically examined post mortem brains (m:f = 12:18; mean age, 79.8 ± 15.1 years). The study group consisted of 15 cases fulfilling the criteria for high probability of AD according to the NIA-Reagan Institute Criteria and 15 cases with no to medium probability. Sections were incubated with secretagogin-specific antibodies and the number of immunoreactive neurons as well as staining intensities in both neurons and neuropil were assessed. Both cellular and neuropil immunoreactivity were restricted to subiculum and Ammons horn. Cellular immunoreactivity was further restricted to pyramidal neurons and showed a hierarchical distribution: the mean percentage of immunoreactive neurons was highest in sector CA3 (64.41%), followed by CA2 (44.09%), CA4 (34.38%), CA1 (10.9%), and the subiculum (2.92%; P < 0.001, except CA2–CA4, P > 0.05), while it did not differ significantly between groups with different degrees of AD pathology. The pattern of secretagogin immunoreactivity resembles that of calcium sensor proteins as it is restricted to a subset of neurons and therefore secretagogin could serve highly specialized tasks in neuronal calcium signalling.

P3-183: Cellular immunoreactivity of calcium-binding protein secretagogin in human hippocampus is restricted to pyramidal neurons

P3-183: Cellular immunoreactivity of calcium-binding protein secretagogin in human hippocampus is restricted to pyramidal neurons

Setagin and secretagogin-R22: Posttranscriptional modification products of the secretagogin gene

We describe two new variants of the recently identified hexa-EF-hand calcium binding protein secretagogin. The first variant (secretagogin-R22) is characterized by one single amino acid exchange (Q/R) at codon 22, most likely due to RNA editing. The second variant of secretagogin (setagin) consists of 49 amino acids. Due to a frame shift, only the first 27 amino acids are identical to secretagogin. We demonstrate that this protein truncation results in complete loss of the calcium binding capacity. Setagin expression was found in considerable amounts in the pancreas whereas secretagogin and secretagogin-R22 were also found in the central nervous system and organs containing neurendocrine cells.