The intracellular sorting of pro-neuropeptides, prohormones and neurotrophins to the regulated secretory pathway (RSP) is essential for processing, storage and release of active proteins and peptides in the neuroendocrine cell. The sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), pro-insulin and brain derived neurotrophic factor (BDNF) to the RSP was investigated. Such studies have led to the better understanding of diseases related to defects in hormone and neuropeptide targeting, obesity, diabetes, memory and learning. We show that these pro-proteins undergo homotypic oligomerization as a concentration step, as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network (TGN), where they are sorted into dense-core granules of the RSP for processing and secretion. Site-directed mutagenesis studies identified consensus sorting motifs which are necessary for sorting these pro-proteins to the RSP. A RSP sorting receptor that was specific for the sorting signal of POMC, pro-insulin and BDNF was identified as membrane carboxypeptidase E (CPE). Using a CPE knockout (KO) mouse model, we showed missorting of endogenous POMC and proinsulin in pituitary and pancreatic islet cells, respectively, in these animals. BDNF which modulates synaptic plasticity was missorted in cortical and hippocampal neurons, and that may account for the memory deficits observed in CPE KO mice. These studies provide evidence for a sorting signal/receptor mediated mechanism for targeting prohormones, neuropeptides and BDNF, to the regulated secretory pathway in endocrine cells and neurons. We have used our knowledge of the sorting motif of hormones to engineer biologically active mutant hormones, e.g. mutant proinsulin that we have been able to express in salivary glands and redirect to the constitutive pathway for systemic secretion. The ultimate aim is to apply such technology to gene therapy in the treatment of diseases such as diabetes.? The sorting and processing of a mutant form of CART (CART Leu34Phe), found in a family of obese patients was investigated. CART, found in brain, is an anorexigenic peptide that has several physiological effects such as inhibiting feeding, regulating energy expenditure, and stress. CART acts downstream of leptin in the obesity controlling signaling pathway. We showed that all members of the family bearing the (Phe34Leu) mutation had only pro - and intermediate CART in their circulation, but no mature CART. In contrast , normal humans and the unaffected sibling have significant amounts of circulating CART. Cell biological studies demonstrated that mutant pro-CART was partially missorted, poorly processed to bioactive CART and was secreted via the constitutive pathway, in contrast to wild type proCART which was properly sorted, processed and secreted in the regulated secretory pathway. The reduction in levels of bioactive CART in humans is likely due to cellular missorting to the constitutive pathway which does not have the appropriate enzymes to process proCART. Thus the hyperphagia and obesity observed in humans bearing the CART (Leu34Phe) mutation is likely attributed to a putative deficiency of bioactive CART in the brain of these patients. Recently, the role of CART in bone remodeling was investigated. We show that the CPE KO mouse lack plasma and hypothalamic CART, unlike WT mice. Bone density scans revealed that CPE KO mice had lower bone density compared to WT mice in both males and females. Furthermore, the osteoblast numbers were significantly reduced, while the osteoclast numbers were significantly increased in the CPE KO animals. This finding is consistent with the literature showing that CART-/- mice have increased osteoclast numbers and further supports a role of CART in bone remodeling. However, the CART-/- mice did not show a decrease in osteoblast number suggesting the exciting possibility that othher neuropeptides or peptide hormones known to be missing in the CPE KO mouse may play a role in osteoblast formation or survival. ? The role of CPE in the nervous system was investigated. Behavioral analyses revealed that CPE KO animals had diminished reactivity to stimuli, delayed learning in the water-maze test and abnormal neurotransmission from the photoreceptors to the inner retina, showing a loss of the b wave in their retinogram. Electron microscopic studies indicated that there were significantly decreased numbers of synaptic vesicles in the synaptic butons of the photo receptors of CPE KO mice, suggesting a defect in transport of synaptic vesicles. Indeed our recent studies indicate that CPE plays a role in transport and movement of peptidergic vesicles to the release site in endocrine cells and neurons. Absence of CPE in the KO mice could lead to failure in neurotransmission, deficits in learning and memory, and abnormal behavior.? The importance of cholesterol in secretory granule biogenesis was investigated in vivo, in cholesterol-deficient mouse models of Smith-Lemli Opitz Syndrome (SLOS) and lathosterolosis (Sc5d-/-). SLOS and lathosterolosis are human disorders, respectively defective in 7-dehydrocholesterol reductase and lathosterol 5-desaturase, enzymes necessary for the final steps of cholesterol synthesis. Morphological analysis of neonatal pancreas zymogen granules showed a marked decrease in the number of granules in both SLOS and Sc5d-/- versus control mice. Of the granules present in SLOS and Sc5d-/-animals, most were of an immature phenotype as compared to control animals, appearing as partially formed spheres. Furthermore, in primary cultures of cholesterol deficient secretory cells in the exocrine pancreas, granule biogenesis and regulated secretion of amylase was impaired. Addition of exogenous cholesterol to these cells rescued the phenotype. Granule biogenesis was also impaired in endocrine tissues of Sc5d-/- mice. We show that the defect in granule biogenesis and maturation is due to different physical contributions of sterols to membrane curvature. Lathosterol and 7-dehydro-cholesterol has a lower binding rigidity than cholesterol and exhibit poorer ability to form curvature. Thus genetic inhibition of cholesterol synthesis in SLOS and Sc5d-/- mice impairs regulated secretory pathway granule biogenesis and maturation, leading to deficits in the secretory function in the exocrine and endocrine systems. ? We have also investigated the protein factors governing the formation of large dense-core granules (LDCG)which is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our studies demonstrate that chromogranin A (CgA) controls the formation of LDCG in neuroendocrine cells. An antisense mRNA transgenic mouse model deficient in CgA had severe aberrant granule formation quantitatively and qualitatively in the adrenal medulla. Depletion of CgA in rat PC12 cells resulted in the loss of LDCG, regulated secretion, and degradation of granule proteins. Overexpression of bovine CgA in these cells rescued the wild type phenotype. In a mutant endocrine cell line, 6T3, lacking CgA, there was a deficit in LDCGs, regulated hormone secretion and degradation of granule proteins. We found a protease inhibitor, protease nexin-1 (PN-1) in the Golgi in 6T3 cells that is transcriptionally activated by CgA. Transfection of PN-1 into 6T3 cells lacking CgA prevented LDCG protein degradation and rescued granule biogenesis. Furthermore, 6T3 cells incubated with conditioned medium from 6T3-bCgA cells expressing CgA, increased PN-1 mRNA and granule biogenesis. Thus CgA/CgA derived peptides secreted into the medium may bind to a receptor and transcriptionally activate expression of a protease inhibitor, PN-1, which then stabilizes granule proteins at the TGN to promote LDCG biogenesis in endocrine cells.
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