Large Dense-core Vesicles Research Articles

Using protein structure prediction with molecular phylogenetic analysis to understand membrane interactions in synaptotagmin-like proteins

Protein structure prediction is emerging as a core technology for understanding biomolecules and their interactions. Here, we have combined structure prediction with molecular phylogenetic analysis in order to provide information on the evolution of electrostatic membrane binding in synaptotagmin-like proteins (Slps). Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles in vertebrates. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS) found in the cytoplasmic leaflet of the plasma membrane.

A two-subpopulation model that reflects heterogeneity of large dense core vesicles in exocytosis

ABSTRACT Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell excitation demonstrates large heterogeneity. However, there are currently no models that clearly reflect such heterogeneity. Here, we develop a novel model based on single vesicle release events from amperometry recordings of PC12 cells using carbon fiber microelectrode. In this model, releasable vesicles can be grouped into two subpopulations, namely, SP1 and SP2. SP1 vesicles replenish quickly, with kinetics of ~0.0368 s−1, but likely undergo slow fusion pore expansion (amperometric signals rise at ~2.5 pA/ms), while SP2 vesicles demonstrate slow replenishment (kinetics of ~0.0048 s−1) but prefer fast dilation of fusion pore, with an amperometric signal rising rate of ~9.1 pA/ms. Phorbol ester enlarges the size of SP2 partially via activation of protein kinase C and conveys SP1 vesicles into SP2. Inhibition of Rho GTPase-dependent actin rearrangement almost completely depletes SP2. We also propose that the phorbol ester-sensitive vesicle subpopulation (SP2) is analogous to the subset of superprimed synaptic vesicles in neurons. This model provides a meticulous description of the architecture of the readily releasable vesicle pool and elucidates the heterogeneity of the vesicle priming mechanism.

Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging.

The absolute concentration and the compartmentalization of analytes in cells and organelles are crucial parameters in the development of drugs and drug delivery systems, as well as in the fundamental understanding of many cellular processes. Nanoscale secondary ion mass spectrometry (NanoSIMS) imaging is a powerful technique which allows subcellular localization of chemical species with high spatial and mass resolution, and high sensitivity. In this study, we combined NanoSIMS imaging with spatial oversampling with transmission electron microscopy (TEM) imaging to discern the compartments (dense core and halo) of large dense core vesicles in a model cell line used to study exocytosis, and to localize 13C dopamine enrichment following 4–6 h of 150 μM 13C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of 13C dopamine in distinct vesicle domains as well as in entire single vesicles were quantified and validated by comparison to electrochemical data. We found concentrations of 87.5 mM, 16.0 mM and 39.5 mM for the dense core, halo and the whole vesicle, respectively. This approach adds to the potential of using combined TEM and NanoSIMS imaging to perform absolute quantification and directly measure the individual contents of nanometer-scale organelles.

Proteomic characterization of secretory granules in dopaminergic neurons indicates chromogranin/secretogranin-mediated protein processing impairment in Parkinson's disease.

Parkinson’s disease (PD) is an aging disorder related to vesicle transport dysfunctions and neurotransmitter secretion. Secretory granules (SGs) are large dense-core vesicles for the biosynthesis of neuropeptides and hormones. At present, the involvement of SGs impairment in PD remains unclear. In the current study, we found that the number of SGs in tyrosine hydroxylase-positive neurons and the marker proteins secretogranin III (Scg3) significantly decreased in the substantia nigra and striatum regions of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) exposed mice. Proteomic study of SGs purified from the dopaminergic SH-sy5Y cells under 1-methyl-4-phenylpyridinium (MPP+) treatments (ProteomeXchange PXD023937) identified 536 significantly differentially expressed proteins. The result indicated that disabled lysosome and peroxisome, lipid and energy metabolism disorders are three characteristic features. Protein-protein interaction analysis of 56 secretory proteins and 140 secreted proteins suggested that the peptide processing mediated by chromogranin/secretogranin in SGs was remarkably compromised, accompanied by decreased candidate proteins and peptides neurosecretory protein (VGF), neuropeptide Y, apolipoprotein E, and an increased level of proenkephalin. The current study provided an extensive proteinogram of SGs in PD. It is helpful to understand the molecular mechanisms in the disease.

Synaptophysin Regulates Fusion Pores and Exocytosis Mode in Chromaffin Cells.

Synaptophysin (syp) is a major integral membrane protein of secretory vesicles. Previous work has demonstrated functions for syp in synaptic vesicle cycling, endocytosis, and synaptic plasticity, but the role of syp in the process of membrane fusion during Ca2+-triggered exocytosis remains poorly understood. Furthermore, although syp resides on both large dense-core and small synaptic vesicles, its role in dense-core vesicle function has received less attention compared with synaptic vesicle function. To explore the role of syp in membrane fusion and dense-core vesicle function, we used amperometry to measure catecholamine release from single vesicles in male and female mouse chromaffin cells with altered levels of syp and the related tetraspanner protein synaptogyrin (syg). Knocking out syp slightly reduced the frequency of vesicle fusion events below wild-type (WT) levels, but knocking out both syp and syg reduced the frequency 2-fold. Knocking out both proteins stabilized initial fusion pores, promoted fusion pore closure (kiss-and-run), and reduced late-stage fusion pore expansion. Introduction of a syp construct lacking its C-terminal dynamin-binding domain in syp knock-outs (KOs) increased the duration and fraction of kiss-and-run events, increased total catecholamine release per event, and reduced late-stage fusion pore expansion. These results demonstrated that syp and syg regulate dense-core vesicle function at multiple stages to initiate fusion, control the choice of mode between full-fusion and kiss-and-run, and influence the dynamics of both initial and late-stage fusion pores. The transmembrane domain (TMD) influences small initial fusion pores, and the C-terminal domain influences large late-stage fusion pores, possibly through an interaction with dynamin.SIGNIFICANCE STATEMENT The secretory vesicle protein synaptophysin (syp) is known to function in synaptic vesicle cycling, but its roles in dense-core vesicle functions, and in controlling membrane fusion during Ca2+-triggered exocytosis remain unclear. The present study used amperometry recording of catecholamine release from endocrine cells to assess the impact of syp and related proteins on membrane fusion. A detailed analysis of amperometric spikes arising from the exocytosis of single vesicles showed that these proteins influence fusion pores at multiple stages and control the choice between kiss-and-run and full-fusion. Experiments with a syp construct lacking its C terminus indicated that the transmembrane domain (TMD) influences the initial fusion pore, while the C-terminal domain influences later stages after fusion pore expansion.

Structure Prediction and Molecular Phylogenetic Analysis of Membrane Interactions in Synaptotagmin-Like Proteins

Membrane trafficking and exocytosis are evolutionarily conserved throughout eukaryotes and involve many interactions between proteins and membrane surfaces. In vertebrates, synaptotagmin-like proteins (Slp) play key roles in trafficking of large dense-core secretory vesicles. Slp family proteins promote secretory vesicle docking to the plasma membrane through their N-terminal Slp homology domains, which bind to vesicular Rab proteins, and their tandem C-terminal C2 domains (C2A and C2B), which bind plasma membrane lipids independently of calcium. Our previous experimental and computational studies showed that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to membrane surfaces containing phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). A conserved PIP2 binding site centered on three lysine residues in the beta3-beta4 region specifically anchors the domain to PIP2 lipids, while many other basic residues surrounding the Lys-cluster enhance interactions with PS. We hypothesize that the positive electrostatic potential of the membrane binding surface is a key determinant of its membrane affinity and function, and therefore the net charge on the protein surface is likely to be evolutionarily conserved. To test this hypothesis, the C2A domain sequences for Slp-4 and Slp-2 in vertebrates were compared to assess the evolution of their respective polybasic surfaces. Using a molecular phylogenetic approach, clades were identified and the net charges of individual and consensus sequences were calculated. Structural homology models were generated using a crystal structure of Slp-4 C2A, and Poisson-Boltzmann calculations were carried out to quantify the positive electrostatic surfaces. This approach may help characterize electrostatic interactions with charged membrane surfaces and identify conserved differences that govern affinity and function of membrane-binding proteins.

Biogenesis of large dense core vesicles in mouse chromaffin cells.

Large dense core vesicle (LDCVs) biogenesis in neuroendocrine cells involves: (a) production of cargo peptides processed in the Golgi; (b) fission of cargo loaded LDCVs undergoing maturation steps; (c) movement of these LDCVs to the plasma membrane. These steps have been resolved over several decades in PC12 cells and in bovine chromaffin cells. More recently, the molecular machinery involved in LDCV biogenesis has been examined using genetically modified mice, generating contradictory results. To address these contradictions, we have used NPY-mCherry electroporation combined with immunolabeling and super-resolution structured illumination microscopy. We show that LDCVs separate from an intermediate Golgi compartment, mature in its proximity for about 1 hour and then travel to the plasma membrane. The exocytotic machinery composed of vSNAREs and synaptotagmin1, which originate from either de novo synthesis or recycling, is most likely acquired via fusion with precursor vesicles during maturation. Finally, recycling of LDCV membrane protein is achieved in less than 2 hours. With this comprehensive scheme of LDCV biogenesis we have established a framework for future studies in mouse chromaffin cells.

Spatial and Temporal Aspects of Exocytosis Studied on the Isolated Plasma Membranes.

Exocytosis of large-dense core vesicles in neuroendocrine cells is a highly regulated, calcium-dependent process, mediated by networks of interrelated proteins and lipids. Here, I describe experimental procedures for studies of selective spatial and temporal aspects of exocytosis at the plasma membrane, or in its proximity, using adrenal chromaffin cells. The assay utilizes primary cells subjected to a brief ultrasonic pulse, resulting in the formation of thin, flat inside-out plasma membranes with attached secretory vesicles and elements of cell cytoskeleton. In this model, secretion of plasma membrane-attached secretory vesicles was found to be dependent on calcium and sensitive to clostridial neurotoxins. Depending on the probe selected for secretory vesicle cargo, protein, and/or lipid detection, this simple assay is versatile, fast and inexpensive, and offers excellent spatial resolution.

HPS1 Regulates the Maturation of Large Dense Core Vesicles and Lysozyme Secretion in Paneth Cells.

HPS1, a BLOC-3 subunit that acts as a guanine nucleotide exchange factor of Rab32/38, may play a role in the removal of VAMP7 during the maturation of large dense core vesicles of Paneth cells. Loss of HPS1 impairs lysozyme secretion and alters the composition of intestinal microbiota, which may explain the susceptibility of HPS-associated inflammatory bowel disease. Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding tendency, and other chronic organ lesions due to defects in tissue-specific lysosome-related organelles (LROs). For some HPS subtypes, such as HPS-1, it is common to have symptoms of HPS-associated inflammatory bowel disease (IBD). However, its underlying mechanism is largely unknown. HPS1 is a subunit of the BLOC-3 complex which functions in the biogenesis of LROs. Large dense core vesicles (LDCVs) in Paneth cells of the intestine are a type of LROs. We here first report the abnormal LDCV morphology (increased number and enlarged size) in HPS1-deficient pale ear (ep) mice. Similar to its role in melanosome maturation, HPS1 plays an important function in the removal of VAMP7 from LDCVs to promote the maturation of LDCVs. The immature LDCVs in ep mice are defective in regulated secretion of lysozyme, a key anti-microbial peptide in the intestine. We observed changes in the composition of intestinal microbiota in both HPS-1 patients and ep mice. These findings provide insights into the underlying mechanism of HPS-associated IBD development, which may be implicated in possible therapeutic intervention of this devastating condition.

Anatomical and ultrastructural analysis of the posterior optic tubercle in the locust Schistocerca gregaria

Locusts, like other insects, partly rely on a sun compass mechanism for spatial orientation during seasonal migrations. To serve as a useful guiding cue throughout the day, however, the sun's apparent movement has to be accounted for. In locusts, a neural pathway from the accessory medulla, the circadian pacemaker, via the posterior optic tubercle, to the protocerebral bridge, part of the internal sky compass, has been proposed to mediate the required time compensation. Toward a better understanding of neural connectivities within the posterior optic tubercle, we investigated this neuropil using light and electron microscopy. Based on vesicle content, four types of synaptic profile were distinguished within the posterior optic tubercle. Immunogold labeling showed that pigment-dispersing hormone immunoreactive neurons from the accessory medulla, containing large dense-core vesicles, have presynaptic terminals in the posterior optic tubercle. Ultrastructural examination of two Neurobiotin-injected tangential neurons of the protocerebral bridge revealed that these neurons are postsynaptic in the posterior optic tubercle. Our data, therefore, support a role of the posterior optic tubercles in mediating circadian input to the insect sky compass.

Isolation of large dense-core vesicles from bovine adrenal medulla for functional studies

Large dense-core vesicles (LDCVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRNAs (miRNAs). Isolation of LDCVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against RNase A. This protocol can be useful in research on the biological functions of LDCVs.

A rare case of pancreatic neuroendocrine tumor

Pancreatic neuroendocrine tumors (PNETs) are a group of endocrine neoplasms that exhibit neuroendocrine phenotypes. These include the production of neuropeptides, large dense-core secretory vesicles, and a lack of neural structures. They arise in the pancreas and are among the most common neuroendocrine tumors. A case of 38 years old female presented with a painless progressive swelling in the right upper abdomen since one month which is insidious in onset, gradually increasing in size with a history of significant weight loss and patient is not a known diabetic. On Examination, a swelling of size 5x7cm noted involving epigastric and right hypochondrium, which is not moving with respiration and dull on percussion. Plane of swelling is Intra-abdominal& retroperitoneal. Computed tomography abdomen (CECT) showed well defined rounded iso dense lesion showing heterogeneous enhancement in arterial and venous phase. Magnetic resonance cholangiopancreatography (MRCP) showed well defined lobulated heterogeneous solid lesion with central areas of necrosis seen in the pancreaticoduodenal groove (PDG) involving main pancreatic duct (MPD) and loss of fat planes with surrounding structures most likely malignant etiology. The patient underwent pancreaticoduodenectomy surgery (Whipple procedure) and had an uneventful recovery. Post-operative biopsy report confirmed pancreatic neuroendocrine tumor grade II (well-differentiated type), stage II a – pT3 N0 Mx.

Aptamer-Modified Microelectrodes for the Electrochemical Impedance Spectroscopy Detection of Neuropeptide Y

The design novel biosensors is a broad field including a wide range of applications including medical diagnostics and environmental monitoring. Small sizes of the electrodes have advantages used for selective identification of a biological target, such as voltammetric time scales, lower background noise and higher spatial resolution. Platinum and Carbon nanofiber has been some promising materials for fabrication of electrodes because of their electrical conductivity, chemical stability and high surface area. The intended application is to the possibility to create point of care diagnostics for monitoring the health of astronauts. Neuropeptides are short amino acid chains that are synthesized in the soma and dendrites, and they are stored in large dense core vesicles until released from nerve endings as well as from dendrites. Also, it’s not recycled and there is no reuptake system, but they are broken by peptidases, which require the cell to always synthesize new molecules to replace them by sending them from the soma to the nerve endings and dendrites. Other small neurotransmitters are normally released together with neuropeptides; therefore, it is essential to develop a technique for their detection as selective as possible and to have a strategy to cancel out the signal exerted by other neurotransmitters. The structural similarity between neuropeptides makes their detection impossible using common electrochemical techniques. Their study is currently done measuring the expression of neuropeptides in-vitro or using microdialysis coupled with mass spectrometry, which gives temporal resolution of tens of minutes. In this project, we have developed a modification protocol that reproducibly adds highly selective aptamers to the microelectrodes and the adsorption of neuropeptide Y differentially detected using microelectrodes. Based on the results, changes in concentration due to the release and absorption of neuropeptide Y can be followed with continuous electrochemical impedance measurements using microelectrodes, with the specificity provided by the used of aptamers in the surface of the microelectrodes.

Changes of Protein Phosphorylation Are Associated with Synaptic Functions during the Early Stage of Alzheimer's Disease.

Alzheimer's disease is an irreversible neurodegenerative disorder for which we have limited knowledge of the mechanisms underlying its pathogenesis, especially the molecular events that trigger the deterioration of neuronal functions in the early stage. Protein phosphorylation and dephosphorylation are highly dynamic and reversible post-translational modifications that control protein signaling and hence neuronal functions, aberrations of which are implicated in various neurodegenerative diseases including Alzheimer's disease. We conducted a quantitative phosphoproteomic analysis in the brains of APP/PS1 mice, an Aβ-deposition transgenic mouse model, at 3 months old, the stage at which amyloid pathology just initiates. Compared to the wild-type mouse brains, we found that changes in serine phosphorylation were predominant in the APP/PS1 mouse brains, and that the occurrence of proline-directed phosphorylation was most common among the overrepresented phosphopeptides. Further analysis of the 167 phosphoproteins that were significantly up- or downregulated in APP/PS1 mouse brains revealed the enrichment of these proteins in synapse-related pathways. In particular, Western blot analysis validated the increased phosphorylation of chromogranin B, a protein enriched in large dense-core vesicles, in APP/PS1 mouse brains. These findings collectively suggest that changes in the phosphoprotein network may be associated with the deregulation of synaptic functions during the pathogenesis of Alzheimer's disease.

Membrane Binding of Synaptotagmin-Like Protein 4: Insight from Molecular Dynamics Simulations

Synaptotagmin-like proteins (Slps) are a family of Rab GTPase effectors involved in secretory vesicle trafficking and membrane docking. Slps possess an N-terminal domain that binds Rab27 on large dense-core secretory vesicles and one or two C-terminal C2 domains that dock to plasma membranes via interaction with anionic lipid molecules such as phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). In particular, Slp-4, also known as granuphilin, docks secretory vesicles to the plasma membrane primarily via its C2A domain, which has a high affinity for anionic lipid bilayers. Although the structure of the Slp-4 C2A domain is known and some of its key PIP2-binding residues have been identified, the full structural mechanism of its strong membrane binding remains unclear. Here, we employ molecular dynamics simulations of the Slp-4 C2A domain to determine contributions of particular residues in binding to a lipid bilayer containing physiologically relevant levels of the anionic target lipids PS and PIP2. Simulations include mutations of two important residues: Lys398, which is known to participate in PIP2 coordination, and Phe452, which is suspected to insert into target membranes hydrophobically. Preliminary results support the experimental finding that both the K398A and F452A mutants are capable of binding membranes but suggest a different balance of lipid interactions in the two mutants. Overall, experimental and computational results indicate that high-affinity membrane binding of the Slp-4 C2A domain is sustained via many lipid-interacting residues distributed over a large electropositive protein surface.

Two ancient neuropeptides, PACAP and AVP, modulate motivated behavior at synapses in the extrahypothalamic brain: a study in contrast.

We examine evolutionary aspects of two primordial neuropeptides, arginine vasopressin (AVP) and pituitary adenylate cyclase-activating polypeptide (PACAP); the distribution of AVP and PACAP and their receptors in mammals; AVP and PACAP release patterns relevant to their roles in neuroendocrine control in brain and periphery; and finally the intricate interlocking of homeostatic and allostatic regulation created by extrahypothalamic AVP and PACAP projections to brain circuit nodes important in controlling appetitive, avoidance and aggressive motor responses. A cardinal feature of peptide neurotransmission important in regulatory control of organismic responses and emphasized in this review, is that neuropeptides are released from large dense-core vesicles docked not only within axonal varicosities and dendrites but also at presynaptic nerve terminal sites, along with small clear synaptic vesicles, at active zones. Peptide transmitter nerve terminals, from hypothalamic and other projections, are distributed widely to multiple brain areas important in integrative control of behavior. They converge with heterologous inputs that release other transmitters, including other peptides, in the same areas. The concept of a quasi-hormonal effect of peptide neurotransmission through coordinated release at multiple synapses throughout the brain echoes earlier conceptualizations of "action-at-a-distance" by diffusion following peptide release at non-synaptic sites. Yet, it recognizes that peptide delivery occurs with neuroanatomical precision, from discrete peptide-containing brain nuclei, via highly distributed projections to multiple extrahypothalamic nodes, registering multiple homeostatic, hedonistic, aversive and reproductive drives that modulate real-time motor decisions. There is paradigmatic value in the discussion of these two particular ancient neuropeptides, for peptide-centric translational neuroendocrinology and peptide GPCR-based neurotherapeutics.

An Alternative Exon of CAPS2 Influences Catecholamine Loading into LDCVs of Chromaffin Cells

The calcium-dependent activator proteins for secretion (CAPS) are priming factors for synaptic and large dense-core vesicles (LDCVs), promoting their entry into and stabilizing the release-ready state. A modulatory role of CAPS in catecholamine loading of vesicles has been suggested. Although an influence of CAPS on monoamine transporter function and on vesicle acidification has been reported, a role of CAPS in vesicle loading is disputed. Using expression of naturally occurring splice variants of CAPS2 into chromaffin cells from CAPS1/CAPS2 double-deficient mice of both sexes, we show that an alternative exon of 40 aa is responsible for enhanced catecholamine loading of LDCVs in mouse chromaffin cells. The presence of this exon leads to increased activity of both vesicular monoamine transporters. Deletion of CAPS does not alter acidification of vesicles. Our results establish a splice-variant-dependent modulatory effect of CAPS on catecholamine content in LDCVs.SIGNIFICANCE STATEMENT The calcium activator protein for secretion (CAPS) promotes and stabilizes the entry of catecholamine-containing vesicles of the adrenal gland into a release-ready state. Expression of an alternatively spliced exon in CAPS leads to enhanced catecholamine content in chromaffin granules. This exon codes for 40 aa with a high proline content, consistent with an unstructured loop present in the portion of the molecule generally thought to be involved in vesicle priming. CAPS variants containing this exon promote serotonin uptake into Chinese hamster ovary cells expressing either vesicular monoamine transporter. Epigenetic tuning of CAPS variants may allow modulation of endocrine adrenaline and noradrenaline release. This mechanism may extend to monoamine release in central neurons or in the enteric nervous system.

Lipid-dependent deposition of alpha-synuclein and Tau on neuronal Secretogranin II-positive vesicular membranes with age

This report demonstrates insoluble alpha-synuclein (aSYN)+ aggregates in human sporadic Parkinson’s disease (PD) midbrain that are linearly correlated with loss of glucocerebrosidase (GCase) activity. To identify early protein-lipid interactions that coincide with loss of lipid homeostasis, an aging study was carried out in mice with age-dependent reductions in GCase function. The analysis identified aberrant lipid-association by aSYN and hyperphosphorylated Tau (pTau) in a specific subset of neurotransmitter-containing, Secretogranin II (SgII)+ large, dense-core vesicles (LDCVs) responsible for neurotransmission of dopamine and other monoamines. The lipid vesicle-accumulation was concurrent with loss of PSD-95 suggesting synaptic destabilization. aSYN overexpression in the absence of lipid deregulation did not recapitulate the abnormal association with SgII+ vesicles. These results show lipid-dependent changes occur with age in neuronal vesicular membrane compartments that accumulate lipid-stabilized aSYN and pTau.

Ultrastructure of Substance P Immunopositive Axons in the Human Dental Pulp

There exists very little information on the ultrastructure of substance P immunopositive (+) fibers in the human dental pulp, which may help in understanding the mechanism for substance P associated pulpal inflammatory pain. To address this issue, we investigated the presence of substance P+ fibers in the human dental pulp by light- and electron-microscopic immunohistochemistry. Light microscopy revealed that substance P+ fibers ran within neurovascular bundles in the radicular pulp and in the core of coronal pulp. They were also frequently present in the peripheral pulp. Substance P+ fibers showed beads like swellings interconnected by thin axonal strand, in a manner similar to bouton en passants and interconnecting axonal strand in the spinal cord. Electron microscopy revealed that almost all the substance P+ axons were unmyelinated. The axonal swellings of the substance P+ contained numerous clear round vesicles (40-50 nm in diameter) and many large dense-cored vesicles (80-110 nm in diameter) as well as many mitochondria. The vesicles and mitochondria were rarely observed in the thin axonal strand interconnecting the swellings. Intimate interrelationship or synaptic structure between the swellings of substance P+ axon and nearby pulpal cells or axons was not found. These findings suggest co-release of substance P and glutamate from the substance P+ pulpal axons and its action on nearby structures in a paracrine manner.

Paralogs of the Calcium-Dependent Activator Protein for Secretion Differentially Regulate Synaptic Transmission and Peptide Secretion in Sensory Neurons.

The two paralogs of the calcium-dependent activator protein for secretion (CAPS) are priming factors for synaptic vesicles (SVs) and neuropeptide containing large dense-core vesicles (LDCVs). Yet, it is unclear whether CAPS1 and CAPS2 regulate exocytosis of these two vesicle types differentially in dorsal root ganglion (DRG) neurons, wherein synaptic transmission and neuropeptide release are of equal importance. These sensory neurons transfer information from the periphery to the spinal cord (SC), releasing glutamate as the primary neurotransmitter, with co-transmission via neuropeptides in a subset of so called peptidergic neurons. Neuropeptides are key components of the information-processing machinery of pain perception and neuropathic pain generation. Here, we compared the ability of CAPS1 and CAPS2 to support priming of both vesicle types in single and double knock-out mouse (DRG) neurons using a variety of high-resolution live cell imaging methods. While CAPS1 was localized to synapses of all DRG neurons and promoted synaptic transmission, CAPS2 was found exclusively in peptidergic neurons and mediated LDCV exocytosis. Intriguingly, ectopic expression of CAPS2 empowered non-peptidergic neurons to drive LDCV fusion, thereby identifying CAPS2 as an essential molecular determinant for peptidergic signaling. Our results reveal that these distinct functions of both CAPS paralogs are based on their differential subcellular localization in DRG neurons. Our data suggest a major role for CAPS2 in neuropathic pain via control of neuropeptide release.