Regulated Pathway Of Secretion Research Articles

Cell-specific secretory granule sorting mechanisms: the role of MAGEL2 and retromer in hypothalamic regulated secretion.

Intracellular protein trafficking and sorting are extremely arduous in endocrine and neuroendocrine cells, which synthesize and secrete on-demand substantial quantities of proteins. To ensure that neuroendocrine secretion operates correctly, each step in the secretion pathways is tightly regulated and coordinated both spatially and temporally. At the trans-Golgi network (TGN), intrinsic structural features of proteins and several sorting mechanisms and distinct signals direct newly synthesized proteins into proper membrane vesicles that enter either constitutive or regulated secretion pathways. Furthermore, this anterograde transport is counterbalanced by retrograde transport, which not only maintains membrane homeostasis but also recycles various proteins that function in the sorting of secretory cargo, formation of transport intermediates, or retrieval of resident proteins of secretory organelles. The retromer complex recycles proteins from the endocytic pathway back to the plasma membrane or TGN and was recently identified as a critical player in regulated secretion in the hypothalamus. Furthermore, melanoma antigen protein L2 (MAGEL2) was discovered to act as a tissue-specific regulator of the retromer-dependent endosomal protein recycling pathway and, by doing so, ensures proper secretory granule formation and maturation. MAGEL2 is a mammalian-specific and maternally imprinted gene implicated in Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. In this review, we will briefly discuss the current understanding of the regulated secretion pathway, encompassing anterograde and retrograde traffic. Although our understanding of the retrograde trafficking and sorting in regulated secretion is not yet complete, we will review recent insights into the molecular role of MAGEL2 in hypothalamic neuroendocrine secretion and how its dysregulation contributes to the symptoms of Prader-Willi and Schaaf-Yang patients. Given that the activation of many secreted proteins occurs after they enter secretory granules, modulation of the sorting efficiency in a tissue-specific manner may represent an evolutionary adaptation to environmental cues.

A dominant negative variant of RAB5B disrupts maturation of surfactant protein B and surfactant protein C

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.

Dense-core vesicle biogenesis and exocytosis in neurons lacking chromogranins A and B.

Chromogranin A and B (Cgs) are considered to be master regulators of cargo sorting for the regulated secretory pathway (RSP) and dense‐core vesicle (DCV) biogenesis. To test this, we analyzed the release of neuropeptide Y (NPY)‐pHluorin, a live RSP reporter, and the distribution, number, and appearance of DCVs, in mouse hippocampal neurons lacking expression of CHGA and CHGB genes. qRT‐PCR analysis showed that expression of other granin family members was not significantly altered in CgA/B−/− neurons. As synaptic maturation of developing neurons depends on secretion of trophic factors in the RSP, we first analyzed neuronal development in standardized neuronal cultures. Surprisingly, dendritic and axonal length, arborization, synapse density, and synaptic vesicle accumulation in synapses were all normal in CgA/B−/− neurons. Moreover, the number of DCVs outside the soma, stained with endogenous marker Secretogranin II, the number of NPY‐pHluorin puncta, and the total amount of reporter in secretory compartments, as indicated by pH‐sensitive NPY‐pHluorin fluorescence, were all normal in CgA/B−/− neurons. Electron microscopy revealed that synapses contained a normal number of DCVs, with a normal diameter, in CgA/B−/− neurons. In contrast, CgA/B−/− chromaffin cells contained fewer and smaller secretory vesicles with a smaller core size, as previously reported. Finally, live‐cell imaging at single vesicle resolution revealed a normal number of fusion events upon bursts of action potentials in CgA/B−/− neurons. These events had normal kinetics and onset relative to the start of stimulation. Taken together, these data indicate that the two chromogranins are dispensable for cargo sorting in the RSP and DCV biogenesis in mouse hippocampal neurons.

Lifecycle of Weibel-Palade bodies.

Weibel-Palade bodies (WPBs) are rod or cigar-shaped secretory organelles that are formed by the vascular endothelium. They contain a diverse set of proteins that either function in haemostasis, inflammation, or angiogenesis. Biogenesis of the WPB occurs at the Golgi apparatus in a process that is dependent on the main component of the WPB, the haemostatic protein von Willebrand Factor (VWF). During this process the organelle is directed towards the regulated secretion pathway by recruiting the machinery that responds to exocytosis stimulating agonists. Upon maturation in the periphery of the cell the WPB recruits Rab27A which regulates WPB secretion. To date several signaling pathways have been found to stimulate WPB release. These signaling pathways can trigger several secretion modes including single WPB release and multigranular exocytosis. In this review we will give an overview of the WPB lifecycle from biogenesis to secretion and we will discuss several deficiencies that affect the WPB lifecycle.

60 YEARS OF POMC: Biosynthesis, trafficking, and secretion of pro-opiomelanocortin-derived peptides.

Pro-opiomelanocortin (POMC) is a prohormone that encodes multiple smaller peptide hormones within its structure. These peptide hormones can be generated by cleavage of POMC at basic residue cleavage sites by prohormone-converting enzymes in the regulated secretory pathway (RSP) of POMC-synthesizing endocrine cells and neurons. The peptides are stored inside the cells in dense-core secretory granules until released in a stimulus-dependent manner. The complexity of the regulation of the biosynthesis, trafficking, and secretion of POMC and its peptides reflects an impressive level of control over many factors involved in the ultimate role of POMC-expressing cells, that is, to produce a range of different biologically active peptide hormones ready for action when signaled by the body. From the discovery of POMC as the precursor to adrenocorticotropic hormone (ACTH) and β-lipotropin in the late 1970s to our current knowledge, the understanding of POMC physiology remains a monumental body of work that has provided insight into many aspects of molecular endocrinology. In this article, we describe the intracellular trafficking of POMC in endocrine cells, its sorting into dense-core secretory granules and transport of these granules to the RSP. Additionally, we review the enzymes involved in the maturation of POMC to its various peptides and the mechanisms involved in the differential processing of POMC in different cell types. Finally, we highlight studies pertaining to the regulation of ACTH secretion in the anterior and intermediate pituitary and POMC neurons of the hypothalamus.

Carboxypeptidase E and Secretogranin III Coordinately Facilitate Efficient Sorting of Proopiomelanocortin to the Regulated Secretory Pathway in AtT20 Cells.

Proopiomelanocortin (POMC) is a multivalent prohormone that can be processed into at least 7 biologically active peptide hormones. Processing can begin in the trans-Golgi network (TGN) and continues in the secretory granules of the regulated secretory pathway (RSP). Sorting of POMC into these granules is a complex process. Previously, a membrane-associated form of carboxypeptidase E (CPE) was shown to bind to POMC and facilitate its trafficking into these granules. More recently, secretogranin III (SgIII) was also found to affect POMC trafficking. Here, we show by RNA silencing that CPE and SgIII play a synergistic role in the trafficking of POMC to granules of the RSP in AtT20 cells. Reduction of either protein resulted in increased constitutive secretion of POMC and chromogranin A, which was increased even further when both proteins were reduced together, indicative of missorting at the TGN. In SgIII-reduced cells, POMC accumulated in a compartment that cofractionated and colocalized with syntaxin 6, a marker of the TGN, on sucrose density gradients and in immunocytochemistry, respectively, indicating an accumulation of this protein in the presumed sorting compartment. Regulated secretion of ACTH, as a measure of sorting and processing of POMC in mature granules, was reduced in the SgIII down-regulated cells but was increased in the CPE down-regulated cells. These results suggest that multiple sorting systems exist, providing redundancy to ensure the important task of continuous and accurate trafficking of prohormones to the granules of the RSP for the production of peptide hormones.

Insulin Secreted From Genetically Engineered Intestinal Cells Reduces Blood Glucose Levels in Diabetic Mice

Poorly controlled diabetes mellitus can result in serious complications. Gene therapy is increasingly being considered as an alternative approach to treat diabetes, because of its ability to induce physiological insulin secretion and it allows patients to escape insulin injections. The properties of gut K and L-cells, including glucose sensitivity, the ability to process insulin and a regulated secretion pathway support their use as surrogate β-cells. Previous in vitro studies have provided sufficient evidence supporting the use of these cells for gene therapy studies. Therefore, we examined the ability of K and L-cells to produce insulin in diabetic mice. Chitosan nanoparticles were used to transfer the insulin gene into intestinal cells via oral administration. The efficiency of chitosan as a gene vehicle was investigated through the use of reporter gene. Insulin mRNA and protein expression levels were measured by RT-PCR and ELISA, respectively. Blood glucose testing revealed that this treatment reduced glucose levels in diabetic mice. The decrease in blood glucose level in the first week of treatment was greater in mice with K-cell specific insulin expression compared with mice with L-cell-specific insulin expression. These results indicate that inducing insulin secretion in K-cells conferred a quicker response to gene therapy.

Complementation of juxtaglomerular cell granulation in Ren‐ 1d null mice by human renin

Mice null for the Ren‐1d gene lack renin storage granules in their juxtaglomerular cells and exhibit altered macula densa morphology. Three lines of transgenic mice carrying a 55kbp transgene encompassing the human renin (hRen) locus were generated and crossed onto a Ren1d‐null background. Expression of hRen was seen to restore granulation in juxtaglomerular (JG) cells in a transgene expression level‐dependent manner, suggesting that a threshold level of renin expression is required for the formation of dense renin‐containing granules. Complementation of the phenotype by the human enzyme suggests that hRen and mouse Ren1d share conserved epitopes required for trafficking renin into the regulated secretion pathway and hence for granulopoiesis. To dissect the granulation phenotype in detail, 2D electron microscopic (EM) images were taken of kidney sections, which were then reconstructed in 3D to show the full extent of renin granular structure. As expected, the expression of hRen, which does not effectively cleave mouse angiotensinogen, was unable to restore macula densa morphology, indicating that a locally active RAS is required for normal macular densa structure and function.

Intracellular trafficking and secretion of cerebral dopamine neurotrophic factor in neurosecretory cells

Cerebral dopamine neurotrophic factor (CDNF) is a novel evolutionary conserved protein which can protect and restore the function of dopaminergic neurons in the rat model of Parkinson's disease, suggesting that CDNF might be beneficial for the treatment of Parkinson's disease. CDNF is widely expressed in neurons in several brain regions including cerebral cortex, hippocampus, substantia nigra, striatum and cerebellum. Human CDNF is glycosylated and secreted from transiently transfected cells; however, the mechanism underlying CDNF secretion is currently unclear. In this study, we found that CDNF could be secreted primarily via the regulated secretion pathway in PC12 cells. The glycosylation of CDNF is not required for its secretion. Moreover, we identified two key subdomains in CDNF which are important for its intracellular localization and secretion. Disrupting helix-1 of CDNF significantly reduces its constitutive and regulated secretion and the helix-1 mutant is retained in the endoplasmic reticulum. Although helix-7 mutation only decreases CDNF regulated secretion and has no effect on its constitutive secretion, which is further supported by the reduction in co-localization of helix-7 mutant with secretory granules. In all, these findings will advance our understanding of the molecular mechanism of CDNF trafficking and secretion.

RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway

The regulated release of proteins depends on their inclusion within large dense-core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network (TGN), but the mechanism for protein sorting to this regulated secretory pathway (RSP) and the cytosolic machinery involved in this process have remained poorly understood. Using an RNA interference screen in Drosophila melanogaster S2 cells, we now identify a small number of genes, including several subunits of the heterotetrameric adaptor protein AP-3, which are required for sorting to the RSP. In mammalian neuroendocrine cells, loss of AP-3 dysregulates exocytosis due to a primary defect in LDCV formation. Previous work implicated AP-3 in the endocytic pathway, but we find that AP-3 promotes sorting to the RSP within the biosynthetic pathway at the level of the TGN. Although vesicles with a dense core still form in the absence of AP-3, they contain substantially less synaptotagmin 1, indicating that AP-3 concentrates the proteins required for regulated exocytosis.

Snapshot Peptidomics of the Regulated Secretory Pathway

Neurons and endocrine cells have the regulated secretory pathway (RSP) in which precursor proteins undergo proteolytic processing by prohormone convertase (PC) 1/3 or 2 to generate bioactive peptides. Although motifs for PC-mediated processing have been described ((R/K)X(n)(R/K) where n = 0, 2, 4, or 6), actual processing sites cannot be predicted from amino acid sequences alone. We hypothesized that discovery of bioactive peptides would be facilitated by experimentally identifying signal peptide cleavage sites and processing sites. However, in vivo and in vitro peptide degradation, which is widely recognized in peptidomics, often hampers processing site determination. To obtain sequence information about peptides generated in the RSP on a large scale, we applied a brief exocytotic stimulus (2 min) to cultured endocrine cells and analyzed peptides released into supernatant using LC-MSMS. Of note, 387 of the 400 identified peptides arose from 19 precursor proteins known to be processed in the RSP, including nine peptide hormone and neuropeptide precursors, seven granin-like proteins, and three processing enzymes (PC1/3, PC2, and peptidyl-glycine alpha-amidating monooxygenase). In total, 373 peptides were informative enough to predict processing sites in that they have signal sequence cleavage sites, PC consensus sites, or monobasic cleavage sites. Several monobasic cleavage sites identified here were previously proved to be generated by PCs. Thus, our approach helps to predict processing sites of RSP precursor proteins and will expedite the identification of unknown bioactive peptides hidden in precursor sequences.

Sorting of growth hormone–erythropoietin fusion proteins in rat salivary glands

Neuroendocrine and exocrine cells secrete proteins in either a constitutive manner or via the regulated secretory pathway (RSP), but the specific sorting mechanisms involved are not fully understood. After gene transfer to rat salivary glands, the transgenic model proteins human growth hormone (hGH) and erythropoietin (hEpo) are secreted primarily into saliva (RSP; exocrine) and serum (constitutive; endocrine), respectively. We hypothesized that fusion of hGH at either the C-terminus or the N-terminus of hEpo would re-direct hEpo from the bloodstream into saliva. We constructed and expressed two fusion proteins, hEpo-hGH and hGH-hEpo, using serotype 5-adenoviral vectors, and delivered them to rat submandibular glands in vivo via retroductal cannulation. Both the hEpo-hGH and hGH-hEpo fusion proteins, but not hEpo alone, were secreted primarily into saliva ( p < 0.0001 and p = 0.0083, respectively). These in vivo studies demonstrate for the first time that hGH, in an N- as well as C-terminal position, influences the secretion of a constitutive pathway protein.

Spontaneous fibrillation of the native neuropeptide hormone Somatostatin-14

Natural Somatostatin-14 is a small cyclic neuropeptide hormone with broad inhibitory effects on endocrine secretions. Here we show that natural Somatostatin-14 spontaneously self-assembles in water and in 150mM NaCl into liquid crystalline nanofibrils, which follow characteristic structural features of amyloid fibrils. These non-covalent highly stable structures are based on the Somatostatin native backbone conformation and are formed under non-denaturing conditions. Our results support the hypothesis that self-assembly into amyloid fibrils is a generic property of the polypeptide chain under appropriate conditions. Given recent advances on the mechanisms of biological storage and sorting modes of peptide/protein hormones into secretory granules, we propose that Somatostatin-14 fibrillation could be relevant to the regulated secretion pathway of this neuropeptide hormone. Such a hypothesis is consistent with the emerging concept of the existence of non-disease related but functional amyloids.

Sorting of transgenic secretory proteins in miniature pig parotid glands following adenoviral‐mediated gene transfer

Gene transfer to salivary glands for use in treating both systemic and upper gastrointestinal tract diseases shows considerable potential. Numerous studies in rodents demonstrate that salivary glands can secrete transgenic secretory proteins either into saliva, primarily via the regulated secretory pathway (RSP), or into the bloodstream, primarily by the constitutive secretory pathway (CSP). The purpose of the present study was to assess the sorting characteristics of human growth hormone (hGH), a RSP protein, and human erythropoietin (hEpo), a CSP protein, in a large animal model of salivary gland gene transfer, the miniature pig. Recombinant serotype 5 adenoviral (Ad5; 10(11) particles/gland) vectors encoding either hGH (AdCMVhGH) or hEpo (AdCMVhEpo) were administered to both parotid glands of male miniature pigs by intraductal cannulation. The secretion of hGH or hEpo was measured in both saliva and serum on days 3, 7 and 14 following administration. Detailed serum chemistry and hematological analyses were performed, and the presence of serum antibodies to hGH and hEpo was measured. For AdCMVhEpo-treated minipigs vector distribution in multiple tissues was determined by quantitative polymerase chain reaction (QPCR). The RSP protein hGH was secreted entirely into saliva, while the CSP protein hEpo was secreted into both saliva and serum. Most hEpo was found in saliva, but serum hEpo levels were sufficient to significantly increase hematocrit levels in treated animals by approximately 10%. Expression of both transgenes was maximal on day 3 and declined to near background by day 14. The amount of vector found in the targeted glands was 100 x more than in other tissues. Secretion of transgenic hGH from minipig parotid glands occurred principally into saliva via the RSP, as seen in rodents, while hEpo was secreted into both saliva and serum, the latter presumably via the CSP. Even though hEpo secretion into the bloodstream was not to the extent previously observed in rodents, serum hEpo levels were considerable and the hEpo was biologically active. Ad5 vector distribution was highly restricted to the parotid glands with little vector detected elsewhere. While the results in this large animal model support the established notion that salivary gland gene transfer can be used for treating systemic single protein deficiency disorders, they also highlight differences in transgenic CSP protein sorting between rodents and miniature pigs.

Leptin secretion by white adipose tissue and gastric mucosa.

Leptin is a hormone that plays a central role in the regulation of food intake and energy expenditure. Originally discovered in mature white adipocytes, it was subsequently isolated from the gastric mucosa. This tissue contains a large number of epithelial endocrine and exocrine cells secreting leptin in the blood stream and in the gastric lumen, respectively. Light and electron microscopy have shown that adipocytes and gastric epithelial cells contain leptin along their rough endoplasmic reticulum-Golgi-granules secretory pathway. Both tissues synthesize a soluble form of the leptin receptor that is secreted bound to leptin in the blood and into the gastric juice. This soluble receptor protect leptin and enhances its half-life. Despite the similarities in the mechanisms of leptin secretion by adipocytes and gastric epithelial cells, they are in fact radically different. In gastric cells leptin follows a rapid regulated secretion pathway whereas adipocytes secrete leptin in a constitutive slow fashion. These differences can be explained by the specific roles play by leptin originating from these two different tissues. Gastric leptin is involved in the short-term regulation of digestion, including delay of gastric emptying, absorption of nutrients by the intestinal wall and secretion of gastric, intestinal and pancreatic hormones. On the other hand, leptin secreted by white adipocytes acts primarily on the hypothalamus for the long-term regulation of food intake. Therefore, the coordination of adipose and gastric leptins ensures the proper management of food processing and energy storage.

Identification of a novel targeting sequence for regulated secretion in the serine protease inhibitor neuroserpin

Ns (neuroserpin) is a member of the serpin (serine protease inhibitor) gene family that is primarily expressed within the central nervous system. Its principal target protease is tPA (tissue plasminogen activator), which is thought to contribute to synaptic plasticity and to be secreted in a stimulus-dependent manner. In the present study, we demonstrate in primary neuronal cultures that Ns co-localizes in LDCVs (large dense core vesicles) with the regulated secretory protein chromogranin B. We also show that Ns secretion is regulated and can be specifically induced 4-fold by secretagogue treatment. A novel 13-amino-acid sorting signal located at the C-terminus of Ns is identified that is both necessary and sufficient to target Ns to the regulated secretion pathway. Its deletion renders Ns no longer responsive to secretagogue stimulation, whereas PAI-Ns [Ns (neuroserpin)-PAI-1 (plasminogen activator inhibitor-1) chimaera appending the last 13 residues of Ns sequence to the C-terminus of PAI-1] shifts PAI-1 secretion into a regulated secretory pathway.

The transcriptional repressor REST is a critical regulator of the neurosecretory phenotype

Release of distinct cellular cargoes in response to specific stimuli is a process fundamental to all higher eukaryotes and controlled by the regulated secretory pathway (RSP). However, the mechanism by which genes involved in the RSP are selectively expressed, leading to the establishment and appropriate functioning of regulated secretion remaining largely unknown. Using the rat pheochromocytoma cell line PC12, we provide evidence that, by controlling expression of many genes involved in the RSP, the transcriptional repressor REST can regulate this pathway and hence the neurosecretory phenotype. Introduction of REST transgenes into PC12 cells leads to the repression of many genes, the products of which are involved in regulated secretion. Moreover, chromatin immunoprecipitation assays show that many of the repressed genes recruit the recombinant REST protein to RE1 sites within their promoters and abrogation of REST function leads to reactivation of these transcripts. In addition to the observed transcriptional effects, PC12 cells expressing REST have fewer secretory granules and a reduction in the ability to store and release noradrenaline. Furthermore, an important trigger for synaptic release, influx of calcium through voltage-operated calcium channels, is compromised. This is the first demonstration of a transcription factor that directly controls expression of many major components of the RSP and provides further insight into the function of REST.

Disruption of disulfide bond formation alters the trafficking of prothyrotropin releasing hormone (proTRH)-derived peptides

Rat prothyrotropin releasing hormone (proTRH) is processed in the regulated secretory pathway (RSP) of neuroendocrine cells yielding five TRH peptides and several non-TRH peptides. It is not understood how these peptides are targeted to the RSP. We show here that a disulfide bond in the carboxy-terminus of proTRH plays an important role in the trafficking of this prohormone. Recombinant proTRH was observed to migrate faster on a native gel when treated with dithiothreitol (DTT) suggesting the presence of a disulfide bond. In vitro disulfide bond formation was prevented either by DTT treatment or by mutating cysteines 213 and 219 to glycines. In both cases the peptides derived from these mutants exhibited increased constitutive release and processing defects when expressed in AtT20 cells, a neuroendocrine cell line used in our prior studies on proTRH processing. Immunocytochemistry revealed that wild-type proTRH and mutant proTRH localized in a punctate pattern typical of proteins sorted to the regulated secretory pathway. These data suggest that the proposed disulfide bond of proTRH is involved in sorting of proTRH-derived peptides and in their retention within maturing secretory granules. This is the first evidence of structural motifs being important for the sorting of proTRH.

Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins

Neurotrophins are a family of secreted neuronal survival and plasticity factors comprising NGF, BDNF, neurotrophin-3 (NT-3), and NT-4. Whereas synaptic secretion of BDNF has been described, the routes of intracellular targeting and secretion of NGF, NT-3, and NT-4 in neurons are poorly understood. To allow for a direct comparison of intracellular targeting and release properties, all four mammalian neurotrophins were expressed as green fluorescent protein fusion proteins in cultured rat hippocampal neurons. We show that BDNF and NT-3 are targeted more efficiently to dendritic secretory granules of the regulated pathway of secretion (BDNF, in 98% of cells; NT-3, 85%) than NGF (46%) and NT-4 (23%). For all NTs, the remaining cells showed targeting to the constitutive secretory pathway. Fusing the BDNF pre-pro sequence to NT-4 directed NT-4 more efficiently to the regulated pathway of secretion. All neurotrophins, once directed to the regulated secretion pathway, were detected near synapsin I-positive presynaptic terminals and colocalized with PSD-95-DsRed (postsynaptic density-95-Discosoma red), suggesting postsynaptic targeting of the neurotrophins to glutamatergic synapses. Depolarization-induced release of all neurotrophins from synaptic secretory granules was slow (delay in onset, 10-30 s; tau = 120-307 s) compared with transmitter release kinetics monitored with FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide] destaining (onset, <5 s; tau = 13 +/- 2 s). Among the neurotrophins, NT-4 secretion was most rapid but still proceeded 10 times more slowly than transmitter secretion. Preincubation of neurons with monensin (neutralizing intragranular pH, thus solubilizing the peptide core) increased the speed of secretion of BDNF, NGF, and NT-3 to the value of NT-4. These data suggest that peptide core dissolution in secretory granules is the critical determinant of the speed of synaptic secretion of all mammalian NTs and that the speed of release is not compatible with fast transmitter-like actions of neurotrophins.

EGFP-tagged vasopressin precursor protein sorting into large dense core vesicles and secretion from PC12 cells.

1. Hypothalamic magnocellular neurons synthesize, store, and secrete large quantities of the neuropeptides, vasopressin (VP) and oxytocin (OT), which are synthesized as protein precursors also containing proteins called neurophysins. These protein precursors are sorted through the regulated secretory pathway (RSP), packaged into large dense core vesicles LDCVs, and their peptide products are secreted from nerve terminals in the posterior pituitary. 2. It has been hypothesized that this efficient packaging is dependent on the interaction of the peptide with neurophysin in a complex that forms the granule core. To test this, PC12 cells were transfected with vasopressin precursor DNA constructs that either contained or deleted the neurophysin moiety and tagged with enhanced green fluorescent protein (EGFP) as reporters. The intracellular routing and secretion of the EGFP-tagged VP precursor proteins were studied by in differentiated PC12 cells by fluorescence microscopy, electron microscopic immunocytochemistry, and fluorescent imaging techniques. 3. The data showed that only when the neurophysin was present in the VP precursor construct did the fluorescent fusion protein become routed to the RSP and get efficiently packaged into LDCVs and secreted. These data are consistent with the view that routing of the precursor to LDCVs requires the amino acids that encode the intravesicular chaperone, neurophysin.