Aspects of the neuroendocrine cerebellum: expression of secretogranin II,chromogranin A and chromogranin B in mouse cerebellar unipolar brush cells

Morphologically distinct neuron classes can be subdivided in sublineages by differential chemical phenotypes that correlate with functional diversity. Here we show by immunocytochemistry that chromogranin A (CgA) chromogranin B (CgB) and secretogranin II (SgII), the principal granins situated in neuronal secretory granules and large dense-core vesicles, are widely but differentially expressed in cells of the mouse cerebellum and terminals of cerebellar afferents. While CgA and CgB were nearly panneuronal, SgII was more restricted in distribution. The cells most intensely immunoreactive for SgII were a class of small, excitatory interneurons enriched in the granular layer of the vestibulocerebellum, the unipolar brush cells (UBCs), although larger neurons likely to be a subset of the Golgi–Lugaro–globular cell population were also distinctly immunopositive; by contrast, Purkinje cells and granule cells were, at best, faintly stained and, stellate, basket cells were unstained. SgII was also present in subsets of mossy fibers, climbing fibers and varicose fibers. Neurons in the cerebellar nuclei and inferior olive were distinctly positive for the three granins. Double-labeling with subset-specific cell class markers indicated that, while both CgA and CgB were present in most UBCs, SgII immunoreactivity was present in the calretinin (CR)-expressing subset, but lacked in metabotropic glutamate receptor 1alpha (mGluR1α)-expressing UBCs. Thus, we have identified an additional cell class marker, SgII, which serves to study subtype properties in the UBC population. The abundance of SgII in only one of the two known subsets of UBCs is remarkable, as its expression in other neurons of the cortex was moderate or altogether lacking. The data suggest that the CR-positive UBCs represent a unique neuroendocrine component of the mammalian cerebellar cortex, presumably endowed with transynaptically regulated autocrine or paracrine action/s. Because of the well-known organization of the cerebellar system, several of its neuron classes may represent valuable cellular models to analyze granin functions in situ, in acute slices and in dissociated cell and organotypic slice cultures.     

Phylogenetic study on distribution and chromogranin/secretogranin content of histamine immunoreactive elements in the gut

The distribution of histamine-immunoreactive (HA-IR) elements and possible coexistence of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII) were immunohistochemically studied in gut specimens of various vertebrate species. In fish, HA-IR cells were distributed mainly within the gastric and duodenal mucosa, and nerve fibres in the gastric myenteric plexus. Only the gastric HA-IR cells co-stored SgII. In frog specimens, HA-IR endocrine cells and nerve fibres were found in the distal stomach wall, but SgII coexisted only in the nerve fibres. In lizard, HA-IR endocrine cells were widely distributed from the oesophagus to the small intestine, but only those in the stomach co-stored CgA, CgB and SgII. In chick, HA-IR cells were found in the proventriculus and almost all co-stored all three proteins. In rat, HA-IR cells were accumulated in the oxyntic mucosa and all of them constantly immunostained for CgA only.     

Granins as disease-biomarkers: translational potential for psychiatric and neurological disorders

The identification of biomarkers represents a fundamental medical advance that can lead to an improved understanding of disease pathogenesis, and holds the potential to define surrogate diagnostic and prognostic endpoints. Because of the inherent difficulties in assessing brain function in patients and objectively identifying neurological and cognitive/emotional symptoms, future application of biomarkers to neurological and psychiatric disorders is extremely desirable. This article discusses the biomarker potential of the granin family, a group of acidic proteins present in the secretory granules of a wide variety of endocrine, neuronal and neuroendocrine cells: chromogranin A (CgA), CgB, Secretogranin II (SgII), SgIII, HISL-19 antigen, 7B2, NESP55, VGF and ProSAAS. Their relative abundance, functional significance, and secretion into the cerebrospinal fluid (CSF), saliva, and the general circulation have made granins tractable targets as biomarkers for many diseases of neuronal and endocrine origin, recently impacting diagnosis of a number of neurological and psychiatric disorders including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia, and schizophrenia. Although research has not yet validated the clinical utility of granins as surrogate endpoints for the progression or treatment of neurological or psychiatric disease, a growing body of experimental evidence indicates that the use of granins as biomarkers might be of great potential clinical interest. Advances that further elucidate the mechanism(s) of action of granins, coupled with improvements in biomarker technology and direct clinical application, should increase the translational effectiveness of this family of proteins in disease diagnosis and drug discovery.     

Secretogranin II expression in Ewing's sarcomas and primitive neuroectodermal tumors.

Production of chromogranins, the acidic components of the chromaffin granules regarded as specific neuroendocrine markers, was analyzed by immunocytochemistry and hybridization (Northern blotting and in situ hybridization) in primary lesions and cell lines of Ewing's sarcomas, primitive neuroectodermal tumors (PNETs), and neuroblastomas. Antibodies and probes specific for chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) were used. Ewing's sarcomas and PNETs, unlike neuroblastomas, were negative for CgA and CgB. Two primary Ewing's sarcomas, one primary PNET (an Askin tumor), and one PNET cell line (TC32) were found to strongly express the SgII gene, as shown by the presence of specific mRNA. This result supports the hypothesis that some Ewing's sarcomas represent a most primitive form of neuroectodermal tumor; in addition, it indicates a diagnostic role of SgII in cases of Ewing's sarcomas and PNETs.     

Molecular approaches for the analysis of chromogranins and secretogranins.

Recent molecular analyses have contributed to our knowledge about the chromogranin/secretogranin (Cg/Sg) family and their utility in diagnostic pathology. The genes for five of these proteins have been cloned, and the deduced amino acid sequences have provided insights into the structure and possible functions of the Cgs/Sgs, including their role as prohormones. Northern hybridization and in situ hybridization histochemistry have provided a great deal of information about the tissue distribution of the Cg/Sg gene products. Some neoplasms such as small cell lung carcinomas, which have little stored Cg/Sg protein, have abundant cytoplasmic mRNAs that can be readily detected by hybridization studies. Some other neoplasms such as neuroblastomas have decreased CgA and increased SgII mRNAs during maturation to ganglioneuromas. There is also a differential expression of Cgs/Sgs in some endocrine neoplasms such as parathyroid adenomas, which express abundant CgA mRNA and little CgB mRNA, and in pituitary prolactinomas, which express CgB mRNA but not CgA mRNA. The mRNA for CgA has been found unexpectedly in some neoplasms such as 15% of colonic adenocarcinomas. Thus, molecular approaches in the analysis of Cgs/Sgs should contribute to the diagnosis of endocrine neoplasms and may provide support for a molecular classification of neoplasms in diagnostic pathology.     

Chromogranin A and chromogranin B in noninvasive and invasive breast carcinoma

Recent progress in the study of chromogranins has revealed that there are many novel peptides derived from chromogranin with their multiple pathophysiologic roles. To learn the possible roles of chromogranin in breast carcinoma, we immunohistochemically investigated tissue localization of chromogranin A (CgA) and chromogranin B (CgB) in 10 normal breast tissues, 23 noninvasive ductal carcinomas (NIDCs), and 169 invasive ductal carcinomas (IDCs) and compared their expression with estrogen receptor (ER), progesterone receptor (PR), and Ki67. CgA and CgB were sporadically detected in normal cells of the ducts, acini, and luminal secretion. The expression of CgA and CgB was higher in NIDCs than in IDCs: CgA=70% of NIDC vs 22% of IDC and CgB=65% of NIDC vs 30% of IDC. There was a statistical correlation between the expression of CgA and PR (p < 0.05) and CgB and ER (p < 0.05) in IDCs without lymph node metastasis. On the other hand, there was a significant correlation between expression of CgB and PR and an inverse correlation between CgA and Ki67 in IDCs of overall cases. The data suggest that CgA and CgB may play some role in the early phase of neoplastic progression.     

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the endocrine pancreas of reptiles.

The endocrine pancreas of four reptile species belonging to the turtles, lizards and snakes was investigated immunohistochemically for the occurrence and cellular distribution of chromogranin A (CgA) and of two synthetic secretonin II (SgII)-peptides (C23-3 and C26-3). CgA-immunoreactivity was found only in the turtle pancreas, whereas that for SGIIC23-3 appeared both in the turtle and snake. None of the species studied displayed immunoreactivity for SgIIC26-3. The two detected granins showed different distributions in relation to the endocrine cell types. Conspicuous variations of the immunostaining density for either granin in the same endocrine cell population and even complete lack of the immunoreaction were recorded. The findings suggest that, despite the restricted presence in the endocrine pancreas of the reptiles investigated, the granins are relatively well conserved during phylogeny; they do not confirm, however, the previously accepted usefulness of the granin protein family as common markers of neuroendocrine cells.     

Chromogranin A, chromogranin B and secretogranin II mRNAs in the pituitary and adrenal glands of various mammals. Regulation of chromogranin A, chromogranin B and secretogranin II mRNA levels by estrogen.

BACKGROUND: The chromogranin/secretogranin (Cg/Sg) acidic proteins are widely distributed in vertebrate species. They are thought to play a role in hormone packaging within secretory granules, in hormone secretion, and serve as prohormones for various proteolytic cleavage products. The genes for most members of the Cg/Sg family have been cloned, so hybridization analysis can be used to analyze the distribution and regulation of Cg/Sg mRNAs in various vertebrate species. EXPERIMENTAL DESIGN: The method of in situ hybridization was used to localize chromogranin A, chromogranin B, and secretogranin II in adrenal and pituitary tissues from laboratory animals and from humans in order to analyze the distribution of various Cg/Sg mRNAs in these tissues. To gain some insight into the regulation and possible functions of specific Cg/Sg members, female rats were ovariectomized for different periods with and without estrogen replacement and the pituitaries were subsequently analyzed by in situ hybridization and Northern hybridization analyses. Combined ISH and immunohistochemistry were used to localize the specific cell types in normal rat pituitary that expressed the mRNA for chromogranin A, chromogranin B, and secretogranin II. RESULTS: All three Cg/Sg mRNAs were detected in pituitary and adrenal tissues of rats, mice, dogs, monkeys, and humans. Combined in situ hybridization and immunohistochemistry using rat pituitary revealed that the glycoprotein hormone-secreting cells expressed all three Cg/Sg mRNAs in approximately equal amounts. Ovariectomy followed by estrogen replacement resulted in decreased levels of CgA and SgII mRNAs. In contrast, the level of CgB mRNA, that was not changed by ovariectomy, was increased after estrogen treatment, probably secondary to prolactin cell hyperplasia. CONCLUSIONS: The three principal Cg/Sg mRNAs are present in the adrenal and pituitary of various vertebrates. Estrogen plays a significant role in regulating the mRNA levels of different Cgs/Sgs suggesting functional and regulatory differences in Cg/Sg proteins.     

Immunohistochemical expression of chromogranins A and B, prohormone convertases 2 and 3, and amidating enzyme in carcinoid tumors and pancreatic endocrine tumors.

Although chromogranin A (CgA) is widely distributed in neuroendocrine tumors, the distribution of chromogranin B (CgB) has not been elucidated. Hormones produced by tumors are sometimes prohormones and not necessarily bioactive hormones. Prohormones have to be processed into bioactive peptides by prohormone convertases (PCs), and some of them have to be amidated by peptidylglycine a-amidating monooxygenase (PGM). Whether PCs and PGM are present or not in tumors may explain why some tumors are functioning and some are nonfunctioning. We investigated 45 carcinoids and 16 pancreatic endocrine tumors. Of the carcinoids, CgA was expressed in most of the tumors, except for the rectal and ovarian carcinoids, which expressed CgB strongly. The expressions of PC2, PC3, and PGM were 31%, 100%, and 87%, respectively. In the pancreatic tumors, CgA was expressed in all tumors, whereas CgB was not expressed in any tumor. The expressions of PC2, PC3, and PGM were 63%, 88%, and 63%, respectively. PC3 was expressed in all of the functioning tumors but not in two of the four nonfunctioning tumors. PC2 and PGM were not expressed in three of the four nonfunctioning tumors. In conclusion, expression of CgA and CgB was different depending on the tumor location. High frequency of PCs and PGM may explain why even nonfunctioning tumors produce some inconspicuous peptides.     

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the intestine of reptiles

The occurrence, distribution and the possible cellular co-localizations of chromogranin A (CgA) and of two synthetic secretogranin II-peptides (SgIIC23-3 and SgIIC26-3) with several enteric neuropeptides and serotonin have been investigated immunohistochemically in turtles, lizards and snakes. The distribution of CgA-immunoreactivity was restricted only to the enteroendocrine cells in all the reptiles studied. SgII-immunoreactivity--absent in turtle--revealed nerve cells and fibers, besides enteroendocrine cells in lizard and snake guts. Moreover, the two antisera (C23-3 and C26-3) raised against the different regions of the SgII-molecule yielded distinct distribution patterns of immunoreactivity both in the lizard and snake organs. Small amounts of enteric serotonin cells co-stored CgA or SgIIC23-3 in lizards and snakes and only SgIIC26-3-peptide in snakes. CgA was found co-stored with somatostatin in a few enterocytes of the turtle duodenum. In the same gut segment of lizards and throughout the snake organ, neurotensin and the SgIIC23-3-peptide co-existed in a small number of endocrine cells. The pancreatic polypeptide-containing cells were devoid of immunoreactivity both for CgA and SgII. Bombesin immunopositive cells were absent throughout the intestines of the reptiles investigated. The above findings entirely support the heterogenous distribution of granins in neuroendocrine organs and tissues and also within the same neuroendocrine cell population. They further support the concept of a good conservation of granins during phylogeny.     

Expression of members of the chromogranin family in primary neuroblastomas.

Expression of the members of the chromogranin family [i.e., chromogranin A (CgA), chromogranin B, and secretogranin II (SgII)], the acidic proteins of the matrix of the chromaffin granules presently regarded as specific neuroendocrine markers, was investigated at gene and protein levels in a series of 14 cases of primary untreated neuroblastomas. Oligonucleotides and cRNA probes were employed for hybridization analysis of specific mRNAs (both by Northern blots and nonradioactive in situ hybridization); proteins were localized by immunocytochemistry. Expression of different amounts of each type of chromogranin was determined in all tumors. Cases found immunocytochemically negative were all positive by Northern blot and in situ hybridization. A better prognosis was associated with a higher relative expression of SgII; on the contrary, a worse outcome was observed in cases with a higher expression of CgA. These findings are consistent with the hypothesis that SgII is preferentially expressed in neuroblastomas undergoing neuronal differentiation. In cases of neuroblastomas, determination of expression levels of the different chromogranins in the tissues (and in the serum) can provide parameters of high diagnostic and prognostic value.     

Immunohistochemical staining of human islet cells with region-specific antibodies against secretogranins II and III

Chromogranins and secretogranins belong to the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In earlier publications we have described the development of region-specific antibodies against CgA and CgB. In this study we describe antibodies to SgII and SgIII and their usefulness for immunohistochemical staining. Peptides homologous to defined parts of secretogranins II and III were selected and synthesized. Antibodies were raised and immunostainings were performed on normal human pancreas. The SgII 154-165 (N-terminal secretoneurin), SgII 172-186 (C-terminal secretoneurin) and SgIII antibodies immunostained all insulin-immunoreactive cells, most of the glucagon cells and some of the pancreatic polypeptide cells. The SgII 225-242 antibody immunostained only the insulin-containing cells. None of the antibodies immunostained the somatostatin cells. This study is the first observation of the expression of SgIII in human tissues, where we show expression of SgIII in three of the four major islet cell types in human pancreas.     

Calcitonin immunoreactivity in neoplastic and hyperplastic parathyroid glands: an immunohistochemical study

Background Previous studies have established that calcitonin (CT) and the calcitonin generelated peptide (CGRP) are synthesized and stored in subsets of hyperplastic parathyroid cells that also contain chromogranin B (Schmid, KW, et al. Lab Invest 73:90, 1995). The purpose of the current study was to determine whether other generic but variably expressed neuroendocrine markers, i.e., synaptophysin (SYN) and CD57 (Leu7), are also present in normal, hyperplastic, and neoplastic parathyroid tissue and to assess their relationships to the presence of CT. Design Immunoperoxidase stains for chromogranin A (CgA), chromogranin B (CgB), SYN CD57, and CT were performed on 54 hyperplastic, 17 neoplastic (adenoma), and 16 normal parathyroid glands. Sequential sections were stained with antibodies to CgA, CgB, SYN, CD57, and CT using standard avidin-biotin-peroxidase techniques. Results: CgA was diffusely expressed in all normal, hyperplastic, and neoplastic glands. In hyperplasia, CgB was variably expressed in 6 cases (11%), SYN in 6 (11%), CD57 in 15 (28%), and CT in 8 (15%). In adenomas, CgB was variably expressed in 3 (17%), SYN in 3 (17%)., CD57 in 4 (23%), and CT in 4 (23%). All normal glands were negative for CgB, SYN, and CT, while CD57 was variably expressed in 17%. Of the 12 glands that were CT positive, 8 were also positive for CgB, 2 for SYN, and 9 for CD57. Four glands that were strongly and diffusely positive for CT were CgB and SYN negative. Conclusions: CgB, SYN, and CD57 are markers for subsets of hyperplastic and neoplastic parathyroid glands. CT is also expressed in a significant proportion of hyperplastic and neoplastic parathyroid glands, and may be independent of the presence of CgB, SYN, or DD57. The significance of these findings in relationship to the abnormal calcium metabolism in patients with parathyroid hyperplasia remains to be determined.     

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the endocrine pancreas of amphibians

The occurrence and cellular distribution of chromogranin A (CgA) and of two synthetic secretogranin II (SgII)-fragments (termed C23-3 and C26-3) has been investigated immunohistochemically in the endocrine pancreas of five amphibian species. Immunoreactivity for CgA was detected only in specimens of the genus Rana, whereas for SgII it was found in all the urodeles and anurans studied. Either CgA or the SgII-fragment displayed its own cellular distribution patterns in the endocrine pancreas of a given species. Moreover, immunoreactivity for both regions (C23-3 and C26-3) of the SgII-molecule exhibited by the same endocrine cell population have been encountered in newt and frog organs. Besides the interspecific heterogeneous distribution of CgA and of the two SgII-fragments in relation to the insular cell types, a striking heterogeneity of their immunostaining density among the endocrine cells of the same type was also revealed. The above findings entirely support the concept of a good conservation of granins during phylogeny; they do not support, however, the previously ascribed usefulness of these anionic glycoproteins as markers for all neuro-endocrine cells.     

Chromogranin A and B gene expression in carcinomas of the breast. Correlation of immunocytochemical, immunoblot, and hybridization analyses.

Chromogranins (Cg) are regarded as specific neuroendocrine (NE) markers in cells and tumors. Expression of CgA and CgB genes has been demonstrated by correlative immunocytochemical, immunoblotting, in situ hybridization, and Northern blot procedures in seven argyrophilic breast carcinomas, while eight control cases of ductal carcinomas, not otherwise specified, were negative. A high degree of correlation was observed between the various techniques revealing CgA and/or CgB gene expression at different levels; minor discrepancies might be related to tumor heterogeneity or to technical factors. The present study, confirming previous investigations, establishes NE differentiation in a group of human breast cancers. The identification of this type of tumors, especially by testing chromogranin(s) production, appears to be of both biologic and clinical interest.     

Pancreastatin secretion by pituitary adenomas and regulation of chromogranin B mRNA expression.

Pancreastatin, a carboxyl-terminal amidated peptide derived from chromogranin (Cg)A, inhibits secretion of insulin and parathyroid hormone. Our recent studies found significant amounts of immunoreactive pancreastatin in all pituitary adenomas except prolactin adenomas. To analyze the effects of pancreastatin on pituitary cell function, 17 cultured pituitary adenomas were examined for immunoreactive pancreastatin and pancreastatin secretion by the tumors. The effects of pancreastatin on pituitary hormone secretion and on pituitary hormone (follicle-stimulating hormone and prolactin), CgA, and CgB mRNA levels were also examined. Immunoreactive pancreastatin and CgA were present diffusely in gonadotroph and null cell adenomas, but only a few prolactin adenoma cells expressed pancreastatin or CgA. When cells were treated with hypothalamic peptides, gonadotroph adenomas were the only group that released increased amounts of pancreastatin in response to gonadotropin-releasing hormone (10(-7) mol/L). Pancreastatin (10(-7) mol/L) treatment did not stimulate pituitary hormone secretion significantly. In situ hybridization analyses showed that gonadotropin-releasing hormone and pancreastatin treatment led to significant increases in CgB and follicle-stimulating hormone mRNAs in gonadotroph adenomas, whereas CgA mRNA levels did not change significantly. These results show that there is a differential distribution of pancreastatin secretion in pituitary adenomas and that the hypothalamic hormone gonadotropin-releasing hormone and the CgA-derived peptide pancreastatin can regulate CgB mRNA in gonadotroph adenomas, suggesting an autocrine effect of pancreastatin on pituitary tumor function.     

Analysis of the chromogranin A post-translational cleavage product pancreastatin and the prohormone convertases PC2 and PC3 in normal and neoplastic human pituitaries.

Several members of the chromogranin/secretogranin (Cg/Sg) family are post-translationally processed in neuroendocrine cells and tumors to smaller peptides, some of which are biologically active. For example, CgA is processed to pancreastatin, parastatin, and other peptides. We analyzed the distribution of pancreastatin and CgA proteins in normal and neoplastic pituitaries as well as the prohormone convertases PC2 and PC3/1 (PC3), the putative processing enzymes for the Cg/Sg family, in 35 pituitary adenomas and 4 non-neoplastic pituitaries by immunohistochemistry and immunoblotting with highly specific antisera. CgA and CgB mRNAs were also examined. Pancreastatin was present in all subtypes of pituitary tumors, although prolactin-secreting adenomas expressed this peptide less frequently than did other tumor types. CgA protein and CgA mRNA expression were also restricted in prolactin adenomas and in normal prolactin cells, as shown by combined in situ hybridization and immunostaining. The prohormone convertases PC2 and PC3 were present in pituitary tumors and in non-neoplastic pituitaries. Immunoblot analysis and immunostaining showed a principal approximately 69-kd PC3 band and a approximately 68-kd PC2 band. Adrenocorticotrophic hormone-secreting adenomas expressed mainly PC3 as determined by immunoblotting and immunohistochemistry, whereas all other adenoma groups expressed predominantly PC2. These results indicate that the enzymes capable of processing CgA and other members of the Cg/Sg family to peptides with biological activity such as pancreastatin are widely expressed in human pituitary adenomas and in non-neoplastic pituitaries, with adrenocorticotrophic hormone tumors expressing predominantly PC3 and other adenomas expressing mainly PC2. The infrequent expression of CgA protein and pancreastatin peptides in normal and neoplastic prolactin cells suggests a unique role of CgA in these tumors.     

Signal-mediated sorting to the regulated pathway of protein secretion

The existence of specific sorting signals which direct regulated secretory proteins to secretory granules (SGs) was hypothesized two decades ago and since then has been addressed in numerous studies. The discovery that aggregation of regulated secretory proteins is involved in their sorting to SGs questioned the existence of specific sorting signals. In this short review we summarize the identification of a specific sorting signal for chromogranin B (CgB), a regulated secretory protein which undergoes Ca2+/pH-dependent aggregation. This signal is represented by the N-terminal disulfide-bonded loop of CgB encoded by exon 3 and is necessary to direct CgB to SGs. Its essential role was revealed only by the expression of a loopless deletion mutant in the absence of endogenous protein synthesis to preclude aggregative sorting of the former with the latter. The signal is also sufficient to direct a reporter protein to SGs, but only its multiple presence on the reporter leads to high sorting efficiency. Importantly, the identified signal functions at the level of the TGN by binding to membrane components that give rise to SGs. Furthermore, these studies lead to further insights into the mechanism of sorting. First, conclusive evidence is provided that regulated secretory proteins lacking a specific signal, can be sorted via coaggregation with proteins containing a specific sorting signal. Second, the data support an additional function of aggregation in the TGN which is multimerization of sorting signals per sorting unit leading to highly efficient sorting to SGs.