Dense-core Vesicles In Neurons Research Articles

Genetically-encoded markers for confocal visualization of single dense core vesicles.

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

Genetically-encoded markers for confocal visualization of single dense core vesicles.

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

Ca2+ and cAMP open differentially dilating synaptic fusion pores.

Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins that affect synaptic growth and plasticity. Rather than using full collapse exocytosis that commonly mediates peptide hormone release by endocrine cells, DCVs at the Drosophila neuromuscular junction release their contents via fusion pores formed by kiss-and-run exocytosis. Here, we used fluorogen-activating protein (FAP) imaging to reveal the permeability range of synaptic DCV fusion pores and then show that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca2+-independent full fusions require PKA-R2, a PKA phosphorylation site on Complexin and the acute presynaptic function of Rugose, the homolog of mammalian neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca2+-independent cAMP signaling opens dilating fusion pores to release large cargoes that cannot pass through the narrower fusion pores that mediate spontaneous and activity-dependent neuropeptide release. These results imply that the fusion pore is a variable filter that differentially sets the composition of proteins released at the synapse by independent exocytosis triggers responsible for routine peptidergic transmission (Ca2+) and synaptic development (cAMP).

Tetanus insensitive VAMP2 differentially restores synaptic and dense core vesicle fusion in tetanus neurotoxin treated neurons

The SNARE proteins involved in the secretion of neuromodulators from dense core vesicles (DCVs) in mammalian neurons are still poorly characterized. Here we use tetanus neurotoxin (TeNT) light chain, which cleaves VAMP1, 2 and 3, to study DCV fusion in hippocampal neurons and compare the effects on DCV fusion to those on synaptic vesicle (SV) fusion. Both DCV and SV fusion were abolished upon TeNT expression. Expression of tetanus insensitive (TI)-VAMP2 restored SV fusion in the presence of TeNT, but not DCV fusion. Expression of TI-VAMP1 or TI-VAMP3 also failed to restore DCV fusion. Co-transport assays revealed that both TI-VAMP1 and TI-VAMP2 are targeted to DCVs and travel together with DCVs in neurons. Furthermore, expression of the TeNT-cleaved VAMP2 fragment or a protease defective TeNT in wild type neurons did not affect DCV fusion and therefore cannot explain the lack of rescue of DCV fusion by TI-VAMP2. Finally, to test if two different VAMPs might both be required in the DCV secretory pathway, Vamp1 null mutants were tested. However, VAMP1 deficiency did not reduce DCV fusion. In conclusion, TeNT treatment combined with TI-VAMP2 expression differentially affects the two main regulated secretory pathways: while SV fusion is normal, DCV fusion is absent.

PTPN21 and Hook3 relieve KIF1C autoinhibition and activate intracellular transport

The kinesin-3 KIF1C is a fast organelle transporter implicated in the transport of dense core vesicles in neurons and the delivery of integrins to cell adhesions. Here we report the mechanisms of autoinhibition and release that control the activity of KIF1C. We show that the microtubule binding surface of KIF1C motor domain interacts with its stalk and that these autoinhibitory interactions are released upon binding of protein tyrosine phosphatase PTPN21. The FERM domain of PTPN21 stimulates dense core vesicle transport in primary hippocampal neurons and rescues integrin trafficking in KIF1C-depleted cells. In vitro, human full-length KIF1C is a processive, plus-end directed motor. Its landing rate onto microtubules increases in the presence of either PTPN21 FERM domain or the cargo adapter Hook3 that binds the same region of KIF1C tail. This autoinhibition release mechanism allows cargo-activated transport and might enable motors to participate in bidirectional cargo transport without undertaking a tug-of-war.

SNAP-25 gene family members differentially support secretory vesicle fusion.

Neuronal dense-core vesicles (DCVs) transport and secrete neuropeptides necessary for development, plasticity and survival, but little is known about their fusion mechanism. We show that Snap-25-null mutant (SNAP-25 KO) neurons, previously shown to degenerate after 4 days in vitro (DIV), contain fewer DCVs and have reduced DCV fusion probability in surviving neurons at DIV14. At DIV3, before degeneration, SNAP-25 KO neurons show normal DCV fusion, but one day later fusion is significantly reduced. To test if other SNAP homologs support DCV fusion, we expressed SNAP-23, SNAP-29 or SNAP-47 in SNAP-25 KO neurons. SNAP-23 and SNAP-29 rescued viability and supported DCV fusion in SNAP-25 KO neurons, but SNAP-23 did so more efficiently. SNAP-23 also rescued synaptic vesicle (SV) fusion while SNAP-29 did not. SNAP-47 failed to rescue viability and did not support DCV or SV fusion. These data demonstrate a developmental switch, in hippocampal neurons between DIV3 and DIV4, where DCV fusion becomes SNAP-25 dependent. Furthermore, SNAP-25 homologs support DCV and SV fusion and neuronal viability to variable extents - SNAP-23 most effectively, SNAP-29 less so and SNAP-47 ineffectively.

Super-resolution Imaging of Neuronal Dense-core Vesicles

Super-resolution Imaging of Neuronal Dense-core Vesicles

Super-resolution imaging of neuronal dense-core vesicles.

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization. Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

Regulation of vesicle trafficking: GTPases and friends

With the 2013 Nobel Prize awarded to scientists who uncovered mechanisms of vesicular trafficking, the next big question in the field is how this process is regulated. Talks in the Minisymposium on “Regulation of Vesicle Trafficking” highlighted ways in which GTPases from the highly conserved Ypt/Rab and Arf families regulate and coordinate vesicle trafficking steps. The roles of luminal phosphorylation and calcium signaling in this process were also underlined (see Figure 1). FIGURE 1: Regulation of vesicle trafficking. Vesicles carrying luminal or membrane protein cargo (blue) are formed on one compartment and fuse with the next. Regulation processes discussed here are shown at the top (see the text for details). Vesicular trafficking ... How do downstream effectors of GTPases mediate transport steps? When stimulated, GTPases recruit their effectors to mediate the various steps of vesicular transport. Three talks described how these effectors function. Bruno Goud (Institut Curie) talked about two molecular motors, an actin-based myosin and a microtubule-based kinesin, that act as Rab6 effectors to drive membrane fission during vesicle formation. Michael Ailion (University of Washington) described the identification of two Rab2 effectors in a genetic screen for Caenorhabditis elegans mutants defective in the maturation of neuronal dense-core vesicles. These effectors are part of a complex that contains Rab2, at least three Rab2 effectors, and a Rab2 GTPase-activating protein, underscoring the importance of large multiprotein complexes in vesicular trafficking. Anne Spang (University of Basel) talked about another protein complex, Exomer, which acts as an Arf effector to traffic cargo from the trans-Golgi network (TGN) to the plasma membrane (PM). The interesting twist in this story is the suggestion that prion domain–dependent aggregation might serve as a reversible signal for stalling of cargo in a compartment. GTPases and coordination GTPases are in a perfect position not only to regulate individual transport steps but also to coordinate them. Two talks described how different GTPases that function sequentially in the same pathway coordinate successive vesicular transport steps. In such cascades, the effector of one GTPase acts as a guanine-nucleotide exchange factor (GEF) of the next GTPase. Wei Guo (University of Pennsylvania) described two mechanisms that regulate recycling endosome-to-PM transport involving the stimulation of the GEF activity of Rabin8 on Rab8 by Rab11 and ERK1/2 phosphorylation. Chris Fromme (Cornell University) talked about a Rab-Arf cascade in which two Rabs regulate the Arf-GEF Sec7 at the TGN. Whereas Ypt1 (Rab1) helps recruit Sec7 to the Golgi, Ypt31/32 (Rab11) stimulates its GEF activity on Arf to stimulate vesicle formation. In another talk, Nava Segev (University of Illinois at Chicago) described how one Rab GTPase functions in two different GEF-GTPase-effector modules to coordinate transport from one compartment to two different destinations. When stimulated by one GEF, Ypt1 (Rab1) recruits certain effectors to regulate endoplasmic reticulum (ER)-to-Golgi transport in the secretory pathway. When stimulated by another GEF, the same Ypt recruits an autophagy-specific effector to shuttle membranes loaded with excess or misfolded proteins from the ER for degradation in the lysosome. Calcium signaling Calcium is a known signal of regulated secretion. However, its role in constitutive vesicular trafficking has been elusive. Jared Helm (Hay Laboratory, University of Montana) suggested a mechanism for the dependence of ER-to-Golgi transport on luminal calcium. On localized release of calcium from the ER, the calcium-binding protein ALG-2 interacts with the COPII coat component Sec31. This interaction ensures assembly of a functional vesicle and/or proper timing of its uncoating.

Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena

Secretory granules, such as neuronal dense core vesicles, are specialized for storing cargo at high concentration and releasing it via regulated exocytosis in response to extracellular stimuli. Here, we used expression profiling to identify new components of the machinery for sorting proteins into mucocysts, secretory granule-like vesicles in the ciliate Tetrahymena thermophila. We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis. In particular, the delivery of nonaggregated, but not aggregated, cargo proteins requires classical receptors of the sortilin/VPS10 family, which indicates that dual mechanisms are involved in sorting to this secretory compartment. In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation. Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

Munc13 controls the location and efficiency of dense-core vesicle release in neurons

Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2-null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically.

Release notes for dense-core vesicles

![Figure][1] DCV secretion events (green) occur both inside (filled arrowhead) and outside (open arrowhead) synapses (red). Van de Bospoort et al. describe how Munc13 proteins control when and where dense-core vesicles (DCVs) are released from neurons. Neuronal DCVs contain neuropeptides

Expression of the dominant-negative tail of myosin Va enhances exocytosis of large dense core vesicles in neurons.

Regulated exocytosis of secretory vesicles is a fundamental process in neurotransmission and the release of hormones and growth factors. The F-actin-binding motor protein myosin Va was recently shown to be involved in exocytosis of peptide-containing large dense core vesicles of neuroendocrine cells. It has not previously been discussed whether it plays a similar role in neurons. We performed live-cell imaging of cultured hippocampal neurons to measure the exocytosis of large dense core vesicles containing fluorescently labelled neuropeptide Y. To address the role of myosin Va in this process, neurons were transfected with the dominant-negative tail domain of myosin Va (myosinVa-tail). Under control conditions, about 0.75% of the labelled large dense core vesicles underwent exocytosis during 5 min of stimulation. This value was doubled to 1.80% of the vesicles when myosinVa-tail was expressed. Depolymerization of F-actin using latrunculin B resulted in a similar increase in exocytosis in both control and myosinVa-tail expressing cells. Interestingly, the increase in exocytosis caused by myosinVa-tail expression was completely abolished in the presence of KN-62, an inhibitor of calcium-calmodulin-dependent kinase II. We suggest that myosinVa-tail causes the liberation of large dense core vesicles from the actin cytoskeleton, leading to an increase in exocytosis in the cultured hippocampal neurons.

Neuropeptides in cnidarians

Cnidarians are the lowest animal group having a nervous system. In the primitive nervous systems of cnidarians, peptides play important roles as neurotransmitters or neurohormones. So far, we have isolated and sequenced about 35 neuropeptides from different cnidarian classes (Hydrozoa, Scyphozoa, Anthozoa). All these neuropeptides have a C-terminal amide group, which protects against C-terminal degradation, but which also is important for receptor recognition. Also the N-termini of the cnidarian neuropeptides often contain different kinds of protecting groups (such as <Glu residues, L-3-phenyllactyl groups, and X-Pro or X-Pro-Pro sequences). Cnidarian neuropeptides are located in neuronal dense-core vesicles and are synthesized as preprohormones, which can contain up to 41 copies of a neuro peptide sequence. From Hydra, six different neuropeptide genes have been cloned so far. Each gene is expressed by a specific population of neurons, but in two instances coexpression of neuropeptide genes has been found. We have also cloned some of the cnidarian prohormone processing enzymes, among them the enzymes necessary for C-terminal amidation. These enzymes are closely related to their mammalian counterparts. All these data show that the primitive nervous systems of cnidarians have already acquired some of the sophisticated principles that we know from higher animals.

Synaptic proteins in Alzheimer's disease.

Chromogranin A, chromogranin B, and secretogranin II are acidic proteins which are stored in large dense core vesicles of neurons. An antiserum, raised against a synthetic peptide (PE-11), present in the chromogranin B molecule, and an antiserum raised against secretoneurin contained in the secretogranin II sequence, was used to localize these peptides together with chromogranin A in the human hippocampal formation. The distribution of these peptides was investigated in Alzheimer's disease and compared to control subjects. Chromogranin A, chromogranin B, and secretogranin II are distinctly distributed with an overlap in their distribution patterns. They were only detected in neuronal structures. The highest density of immunoreactivity was found for chromogranin B. A layer specific distribution was especially obvious in the inner molecular layer of the dentate gyrus as secretoneurin-like immunoreactivity was restricted to its innermost part whereas that of chromogranin B was highly concentrated throughout the inner molecular layer. In Alzheimer's disease, about 10 to 20% of the amyloid-immunoreactive plaques contained either chromogranin A, chromogranin B or secretoneurin. The density of secretoneurin-and chromogranin B-like immunoreactivity was significantly reduced in the inner molecular layer of the dentate gyyrs, the CA1 area, the subiculum and in layers I, III and V of the entorhinal cortex. The present study demonstrates that chromogranin peptides are markers for human hippocampal pathways. Thee are particularly suitable to study nerve fibers, terminating at the inner molecular layer of the dentate gyrus. Chromogranin peptides have a potential as neuronal markers for synaptic degeneration in Alzheimer's disease.

Activation and Routing of Membrane-tethered Prohormone Convertases 1 and 2

Many peptide hormones and neuropeptides are processed by members of the subtilisin-like family of prohormone convertases (PCs), which are either soluble or integral membrane proteins. PC1 and PC2 are soluble PCs that are primarily localized to large dense core vesicles in neurons and endocrine cells. We examined whether PC1 and PC2 were active when expressed as membrane-tethered proteins, and how tethering to membranes alters the biosynthesis, enzymatic activity, and intracellular routing of these PCs. PC1 and PC2 chimeras were constructed using the transmembrane domain and cytoplasmic domain of the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The membrane-tethered PCs were rerouted from large dense core vesicles to the Golgi region. In addition, the chimeras were transiently expressed at the cell surface and rapidly internalized to the Golgi region in a fashion similar to PAM. Membrane-tethered PC1 and PC2 exhibited changes in pro-domain maturation rates, N-glycosylation, and in the pH and calcium optima required for maximal enzymatic activity against a fluorogenic substrate. In addition, the PC chimeras efficiently cleaved endogenous pro-opiomelanocortin to the correct bioactive peptides. The PAM transmembrane domain/cytoplasmic domain also prevented stimulated secretion of pro-opiomelanocortin products in AtT-20 cells.

Neuropeptides in coelenterates: a review

Coelenterate neurones produce peptides containing an Arg-Phe-NH2(RF-amide)-like carboxyterminus. RF-amide-like peptides are located in neuronal dense-cored vesicles, indicating that they are released by exocytosis and that they might function as neurotransmitters or neurohormones. Using a radioimmunoassay for the sequence RF-amide, 3 peptides were isolated from the sea anemone Anthopleura elegantissima: < Glu-Gly-Arg-Phe-NH2(Antho-RF-amide), <Glu-Ser-Leu-Arg-Trp-NH2(Antho-RWamide I) and <Glu-Gly-Leu-Arg-Trp-NH2(Antho-RW-amide II). The general structure of these peptides can be described as <Glu...Arg-X-NH2, where X is an aromatic amino acid. From the hydromedusa Polyorchis penicillatus, the peptide <Glu-Leu-Leu-Gly-Gly-Arg-Phe-NH2(Pol-RF-amide I) was isolated, which also belongs to the <Glu...Arg-X-NH2 family. Using specific antisera, it was shown that all 4 peptides were located in neurones, many of which were associated with smooth muscle fibres. Application of low doses of Antho-RF-amide or of Antho-RW-amide I and II induced contractions of endodermal muscles of sea anemones. This suggests that these peptides are transmitters or modulators at neuromuscular junctions.

Hypophysiotrophic neurons are capable of altering the ratio of co-packaged neurohormones

Neuronal dense-core vesicles provide a mechanism whereby peptide messengers are secreted in discrete quanta. Here we report on the capacity of rat hypophysiotrophic corticotropin releasing factor-41 neurons to alter the peptide content as well as the size of dense-core vesicles after removal of glucocorticoid negative feedback by adrenalectomy. We demonstrate, using quantitative immunoelectron microscopy, that long-term adrenalectomy induces a progressive increase in the ratio of vasopressin to corticotropin releasing factor-41-immunoreactive sites in the dense-core vesicle compartment. The intravesicular concentration of vasopressin appeared to be the variable parameter while that of the corticotropin releasing factor-41 remained stable at all survival times after adrenalectomy. Moreover, observations for up to 5 weeks indicate that adrenalectomy results in a progressive increase in the mean volume of dense-core vesicles to about three times normal. These results suggest that the quantal size and the composition of dense-core vesicles are subject to long-term modulation. The capacity of corticotropin releasing factor-41 neurons to alter dense-core vesicles could enhance or diminish the efficacy of the hypothalamohypophyseal communication underlying physiological adaptation to stress, as well as pathological changes.

Two subpopulations of ganglion cells in hydra are distinguished by an immunogold-double-labeling technique

In the primitive nervous system of Hydra sensory cells and ganglion cells are easily distinguished by their location and distribution, but distinguishing subpopulations within these two groups of neurons is difficult. Both sensory cells and ganglion cells in Hydra and other coelenterates can be immunocytochemically stained with antisera against the molluscan cardio-excitatory neuropeptide FMRFamide as well as other peptides with the general structure &lt; Glu... Arg-X-NH2, where X is an aromatic amino acid. Arg-Phe-amide (RFamide)-like peptides have be e n localized in densecored vesicles of ganglion cells in the peduncle of Hydra. but to date there is no ultrastructural evidence to distinguish between subpopulations of ganglion cells. Therefore, we used an immunogold-double-labeling procedure for electron microscopy with antisera to FMRFamide and RGamide (Arg-Gly-amide) to determine if these two peptides characterized different types of ganglion cells in the peduncle and lower part of the hypostome. In addition, we tested for colocalization of RFamide- and Antho-RFamide-like material in the same neuronal dense-cored vesicles.

Ultrastructural localization of RFamide-like peptides in neuronal dense-cored vesicles in the peduncle of Hydra.

The presence of Arg-Phe-amide (RFamide)-like peptides in dense-cored vesicles in neurons of the peduncle of Hydra was demonstrated by immunogold electron microscopy. Thin sections of Lowicryl-embedded tissue labeled with antisera to RFamide and 5-nm gold-conjugated, secondary antibody and of Epon-Araldite-embedded tissue labeled with 15-nm gold particles revealed a concentration of RFamide-like immunoreactivity over the granular cores of vesicles in epidermal ganglion cells. Gold-labeled, dense-cored vesicles were present in the perikaryon, long thin neurites, and axon terminals of these neurons. The aggregation of labeled dense-cored vesicles in an axon terminal on the myoneme of an epitheliomuscular cell suggests a possible function of RFamide-like peptides in neuromuscular transmission. Gold staining of dense-cored vesicles completely disappeared when the RFamide antiserum was preabsorbed with 10 micrograms/ml RFamide. These results are the first demonstration that the dense-cored vesicles of Hydra neurons contain a neuropeptide.