Three-dimensional mapping of tyrosine hydroxylase in the transparent brain and adrenal of prenatal and pre-weaning mice: Comprehensive methodological flowchart and quantitative aspects of 3D mapping

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The pro-hormone secretogranin II regulates dense-core secretory granule biogenesis in catecholaminergic cells

Processes underlying the formation of dense core secretory granules (DCGs) of neuroendocrine cells are poorly understood. Here, we present evidence that DCG biogenesis is dependent on the secretory protein secretogranin (Sg) II, a member of the granin family of pro-hormone cargo of DCGs in neuroendocrine cells. Depletion of SgII expression in PC12 cells leads to a decrease in both the number and size of DCGs and impairs DCG trafficking of other regulated hormones. Expression of SgII fusion proteins in a secretory-deficient PC12 variant rescues a regulated secretory pathway. SgII-containing dense core vesicles share morphological and physical properties with bona fide DCGs, are competent for regulated exocytosis, and maintain an acidic luminal pH through the V-type H+-translocating ATPase. The granulogenic activity of SgII requires a pH gradient along this secretory pathway. We conclude that SgII is a critical factor for the regulation of DCG biogenesis in neuroendocrine cells, mediating the formation of functional DCGs via its pH-dependent aggregation at the trans-Golgi network

Immunohistochemical distribution of the secretogranin II-derived peptide EM66 in the rat hypothalamus: A comparative study with jerboa

International audienceEM66 is a 66-amino acid peptide derived from secretogranin II, a member of granin acidic secretory protein family, by proteolytic processing. EM66 has been previously characterized in the jerboa (Jaculus orientalis) hypothalamus and its potential implication in the neuroendocrine regulation of feeding behaviour has been demonstrated. In the present study, an immunohistochemical analysis of the localization of EM66 within hypothalamic structures of rat was performed and compared to the distribution of EM66 in the jerboa hypothalamus. In the rat hypothalamus, as in the jerboa, EM66 immunostaining was detected in the parvocellular paraventricular, preoptic and arcuate nuclei, as well as the lateral hypothalamus which displayed an important density of EM66-producing neurones. However, unlike jerboa, the suprachiasmatic and supraoptic nuclei of the rat hypothalamus were devoid of cellular EM66-immunolabeling. Thus, the novel peptide EM66 may exert common neuroendocrine activities in rat and jerboa, e.g. control of food intake, and species-specific roles in jerboa such as the regulation of biological rhythms and hydromineral homeostasis. These results suggest the existence of differences between jerboas and rats in neuroendocrine regulatory mechanisms involving EM66

Chromogranin A Induces the Biogenesis of Granules with Calcium- and Actin-Dependent Dynamics and Exocytosis in Constitutively Secreting Cells

International audienceChromogranins are a family of acidic glycoproteins that play an active role in hormone and neuro-peptide secretion through their crucial role in secretory granule biogenesis in neuroendocrine cells. However, the molecular mechanisms underlying their granulogenic activity are still not fully understood. Because we previously demonstrated that the expression of the major component of secretory granules, chromogranin A (CgA), is able to induce the formation of secretory granules in nonendocrine COS-7 cells, we decided to use this model to dissect the mechanisms triggered by CgA leading to the biogenesis and trafficking of such granules. Using quantitative live cell imaging, we first show that CgA-induced organelles exhibit a Ca 2ϩ-dependent trafficking, in contrast to native vesicle stomatitis virus G protein-containing constitutive vesicles. To identify the proteins that confer such properties to the newly formed granules, we developed CgA-stably-expressing COS-7 cells, purified their CgA-containing granules by subcellular fractionation, and analyzed the granule proteome by liquid chromatography tandem mass spectrometry. This analysis revealed the association of several cytosolic proteins to the granule membrane, including GTPases, cytoskeleton-based molecular motors, and other proteins with actin-and/or Ca 2ϩ-binding properties. Furthermore, disruption of cytoskeleton affects not only the distribution and the transport but also the Ca 2ϩ-evoked exocytosis of the CgA-containing granules, indicating that these granules interact with microtubules and cortical actin for the regulated release of their content. These data demonstrate for the first time that the neuroendocrine factor CgA induces the recruitment of cytoskeleton-, GTP-, and Ca 2ϩ-binding proteins in constitutively secreting COS-7 cells to generate vesicles endowed with typical dynamics and exocytotic properties of neuroen-docrine secretory granules. (Endocrinology 153: 0000-0000, 2012

Myosin 1b and F-actin are involved in the control of secretory granule biogenesis

Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.This work was supported by Institut National de la Santé et de la Recherche Médicale, the University of Rouen Normandy, the Conseil Régional de Normandie and the Ministère de l’Enseignement Supérieur et de la RecherchePeer Reviewe

The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells

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Normotensive Incidentally Discovered Pheochromocytomas Display Specific Biochemical, Cellular, and Molecular Characteristics

International audienceDesign: This was a retrospective cohort recruited from 2001 to 2011 in 2 tertiary care medical departments.Patients and Methods: Clinical, biological, and radiological investigations performed in 96 consecutive patients with sporadic unilateral pheochromocytomas were examined; 47 patients had overt pheochromocytomas responsible for hypertension. Among the patients with incidental pheochromocytomas, 28 had hypertension and 21 were normotensive (NIPs). A total of 62 tumors were examined to determine the Pheochromocytoma of the Adrenal Gland Scale Score, and 29 were studied for the expression of 16 genes involved in chromaffin cell function.Context: A number of incidentally discovered pheochromocytomas are not associated with hypertension. The characteristics of normotensive incidentally discovered pheochromocytomas (NIPs) are poory known.Objective: The purpose of this work was to assess the clinical, hormonal, histological, and molecular features of NIPs.Results: Tumor size and metaiodobenzylguanidine (MIBG) scintigraphy results were similar for hypertensive pheochromocytomas (HPs) and NIPs. Patients with NIPs displayed reduced summed levels of urinary catecholamines and metanephrines and, more specifically, reduced levels of adrenaline and metadrenaline compared with those of patients with HPs (P < .001). Urinary metanephrines had 98% diagnostic sensitivity in patients with HPs and only 75% in patients with NIPs (P < .01). Tumor diameter positively correlated with the total amount of urinary concentrations of metanephrines in patients with HPs (P < .001) but not in patients with NIPs. NIPs displayed global decreased chromaffin gene expression (reaching significance for 5 of them) and 2 corresponding proteins (phenylethanolamine N-methyltransferase and secretogranin II) and a significant increase in the cellularity, mitotic activity, and presence of atypical mitosis (P < .05).Conclusions: NIPs differ from pheochromocytomas responsible for hypertension and display features of altered chromaffin differentiation. These tumors may be misdiagnosed with the use of the usual biological diagnostic tools

Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytoma.

We have previously demonstrated that measurement of tissue concentration of the novel secretogranin II-derived peptide EM66 may help to discriminate between benign and malignant pheochromocytomas. The aim of the present study was to characterize EM66 in plasma and urine of healthy volunteers and pheochromocytoma patients, in order to further evaluate the usefulness of this peptide as a circulating marker for the management of the tumors. HPLC analysis of plasma and urine samples demonstrated that the EM66-immunoreactive material coeluted with the recombinant peptide. In healthy volunteers, plasma and urinary EM66 levels were, respectively, 2.6 (1.9-3.7) ng/ml and 2.9 (1.9-4.6) ng/ml. In patients with pheochromocytoma, plasma EM66 levels were 10-fold higher than those of healthy volunteers (26.9 (7.3-44) ng/ml), and returned to normal values after removal of the tumor. In contrast, urinary EM66 levels were not significantly different from those of healthy volunteers (3.2 (2.2-3.9) ng/ml). Measurement of total or free plasma metanephrines and 24 hr urinary metanephrines in our series of patients revealed that these tests, taken separately, are less sensitive than the EM66 determination. Pheochromocytes in primary culture secreted high levels of EM66, suggesting that the chromaffin tumor was actually responsible for the increased plasma peptide concentrations in the patients. These data indicate that EM66 is secreted in the general circulation and that elevated plasma EM66 levels are correlated with the occurrence of pheochromocytoma. Thus, EM66 is a sensitive plasma marker that should be considered as a complementary tool in the management of pheochromocytoma