Vesicular Packaging Research Articles

Real-time imaging of axonal membrane protein life cycles.

The construction of neuronal membranes is a dynamic process involving the biogenesis, vesicular packaging, transport, insertion and recycling of membrane proteins. Optical imaging is well suited for the study of protein spatial organization and transport. However, various shortcomings of existing imaging techniques have prevented the study of specific types of proteins and cellular processes. Here we describe strategies for protein tagging and labeling, cell culture and microscopy that enable the real-time imaging of axonal membrane protein trafficking and subcellular distribution as they progress through some stages of their life cycle. First, we describe a process for engineering membrane proteins with extracellular self-labeling tags (either HaloTag or SNAPTag), which can be labeled with fluorescent ligands of various colors and cell permeability, providing flexibility for investigating the trafficking and spatiotemporal regulation of multiple membrane proteins in neuronal compartments. Next, we detail the dissection, transfection and culture of dorsal root ganglion sensory neurons in microfluidic chambers, which physically compartmentalizes cell bodies and distal axons. Finally, we describe four labeling and imaging procedures that utilize these enzymatically tagged proteins, flexible fluorescent labels and compartmentalized neuronal cultures to study axonal membrane protein anterograde and retrograde transport, the cotransport of multiple proteins, protein subcellular localization, exocytosis and endocytosis. Additionally, we generated open-source software for analyzing the imaging data in a high throughput manner. The experimental and analysis workflows provide an approach for studying the dynamics of neuronal membrane protein homeostasis, addressing longstanding challenges in this area. The protocol requires 5-7 days and expertise in cell culture and microscopy.

Conserved but not critical: Trafficking and function of NaV1.7 are independent of highly conserved polybasic motifs.

Non-addictive treatment of chronic pain represents a major unmet clinical need. Peripheral voltage-gated sodium (NaV) channels are an attractive target for pain therapy because they initiate and propagate action potentials in primary afferents that detect and transduce noxious stimuli. NaV1.7 sets the gain on peripheral pain-signaling neurons and is the best validated peripheral ion channel involved in human pain, and previous work has shown that it is transported in vesicles in sensory axons which also carry Rab6a, a small GTPase known to be involved in vesicular packaging and axonal transport. Understanding the mechanism of the association between Rab6a and NaV1.7 could inform therapeutic modalities to decrease trafficking of NaV1.7 to the distal axonal membrane. Polybasic motifs (PBM) have been shown to regulate Rab-protein interactions in a variety of contexts. In this study, we explored whether two PBMs in the cytoplasmic loop that joins domains I and II of human NaV1.7 were responsible for association with Rab6a and regulate axonal trafficking of the channel. Using site-directed mutagenesis we generated NaV1.7 constructs with alanine substitutions in the two PBMs. Voltage-clamp recordings showed that the constructs retain wild-type like gating properties. Optical Pulse-chase Axonal Long-distance (OPAL) imaging in live sensory axons shows that mutations of these PBMs do not affect co-trafficking of Rab6a and NaV1.7, or the accumulation of the channel at the distal axonal surface. Thus, these polybasic motifs are not required for interaction of NaV1.7 with the Rab6a GTPase, or for trafficking of the channel to the plasma membrane.

ATP citrate lyase links increases in glycolysis to diminished release of vesicular suppressor of cytokine signaling 3 by alveolar macrophages

Extracellular vesicles (EVs) are important vectors for intercellular communication. Lung-resident alveolar macrophages (AMs) tonically secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis in the distal lung via its actions in recipient neighboring epithelial cells. AMs are metabolically distinct and exhibit low levels of glycolysis at steady state. To our knowledge, whether cellular metabolism influences the packaging and release of an EV cargo molecule has never been explored in any cellular context. Here, we report that increases in glycolysis following in vitro exposure of AMs to the growth and activating factor granulocyte-macrophage colony-stimulating factor inhibit the release of vesicular SOCS3 by primary AMs. Glycolytically diminished SOCS3 secretion requires export of citrate from the mitochondria to the cytosol and its subsequent conversion to acetyl-CoA by ATP citrate lyase. Our data for the first time implicate perturbations in intracellular metabolites in the regulation of vesicular cargo packaging and secretion.

Depolarizing NaV and Hyperpolarizing KV Channels Are Co-Trafficked in Sensory Neurons.

Neuronal excitability relies on coordinated action of functionally distinction channels. Voltage-gated sodium (NaV) and potassium (KV) channels have distinct but complementary roles in firing action potentials: NaV channels provide depolarizing current while KV channels provide hyperpolarizing current. Mutations and dysfunction of multiple NaV and KV channels underlie disorders of excitability, including pain and epilepsy. Modulating ion channel trafficking may offer a potential therapeutic strategy for these diseases. A fundamental question, however, is whether these channels with distinct functional roles are transported independently or packaged together in the same vesicles in sensory axons. We have used Optical Pulse-Chase Axonal Long-distance imaging to investigate trafficking of NaV and KV channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to NaV1.7 channels, NaV1.8 and KV7.2 channels are transported in Rab6a-positive vesicles, and that each of the NaV channel isoforms expressed in healthy, mature sensory neurons (NaV1.6, NaV1.7, NaV1.8, and NaV1.9) is cotransported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions (NaV1.7, KV7.2, and TNFR1) are cotransported in the same vesicles. However, vesicular packaging of axonal membrane proteins is not indiscriminate, since another axonal membrane protein (NCX2) is transported in separate vesicles. These results shed new light on the development and organization of sensory neuron membranes, revealing complex sorting of axonal proteins with diverse physiological functions into specific transport vesicles.SIGNIFICANCE STATEMENT Normal neuronal excitability is dependent on precise regulation of membrane proteins, including NaV and KV channels, and imbalance in the level of these channels at the plasma membrane could lead to excitability disorders. Ion channel trafficking could potentially be targeted therapeutically, which would require better understanding of the mechanisms underlying trafficking of functionally diverse channels. Optical Pulse-chase Axonal Long-distance imaging in live neurons permitted examination of the specificity of ion channel trafficking, revealing co-packaging of axonal proteins with opposing physiological functions into the same transport vesicles. This suggests that additional trafficking mechanisms are necessary to regulate levels of surface channels, and reveals an important consideration for therapeutic strategies that target ion channel trafficking for the treatment of excitability disorders.

Reciprocal Packaging of the Main Structural Proteins of Type 1 Fimbriae and Flagella in the Outer Membrane Vesicles of "Wild Type" Escherichia coli Strains.

Fundamental aspects of outer membrane vesicle (OMV) biogenesis and the engineering of producer strains have been major research foci for many in recent years. The focus of this study was OMV production in a variety of Escherichia coli strains including wild type (WT) (K12 and BW25113), mutants (from the Keio collection) and proprietary [BL21 and BL21 (DE3)] strains. The present study investigated the proteome and prospective mechanism that underpinned the key finding that the dominant protein present in E. coli K-12 WT OMVs was fimbrial protein monomer (FimA) (a polymerizable protein which is the key structural monomer from which Type 1 fimbriae are made). However, mutations in genes involved in fimbriae biosynthesis (ΔfimA, B, C, and F) resulted in the packaging of flagella protein monomer (FliC) (the major structural protein of flagella) into OMVs instead of FimA. Other mutations (ΔfimE, G, H, I, and ΔlrhA–a transcriptional regulator of fimbriation and flagella biosynthesis) lead to the packaging of both FimA and Flagellin into the OMVs. In the majority of instances shown within this research, the production of OMVs is considered in K-12 WT strains where structural appendages including fimbriae or flagella are temporally co-expressed throughout the growth curve as shown previously in the literature. The hypothesis, proposed and supported within the present paper, is that the vesicular packaging of the major FimA is reciprocally regulated with the major FliC in E. coli K-12 OMVs but this is abrogated in a range of mutated, non-WT E. coli strains. We also demonstrate, that a protein of interest (GFP) can be targeted to OMVs in an E. coli K-12 strain by protein fusion with FimA and that this causes normal packaging to be disrupted. The findings and underlying implications for host interactions and use in biotechnology are discussed.

Tyramine synthesis, vesicular packaging, and the SNARE complex function coordinately in astrocytes to regulate Drosophila alcohol sedation.

Identifying mechanisms underlying alcohol-related behaviors could provide important insights regarding the etiology of alcohol use disorder. To date, most genetic studies on alcohol-related behavior in model organisms have focused on neurons, leaving the causal roles of glial mechanisms less comprehensively investigated. Here, we report our studies on the role of Tyrosine decarboxylase 2 (Tdc2), which converts tyrosine to the catecholamine tyramine, in glial cells in Drosophila alcohol sedation. Using genetic approaches that drove transgene expression constitutively in all glia, constitutively in astrocytes and conditionally in glia during adulthood, we found that knockdown and overexpression of Tdc2, respectively, increased and decreased the sensitivity to alcohol sedation in flies. Manipulation of the genes tyramine β-hydroxylase and tyrosine hydroxylase, which respectively synthesize octopamine and dopamine from tyramine and tyrosine, had no discernable effect on alcohol sedation, suggesting that Tdc2 affects alcohol sedation by regulating tyramine production. We also found that knockdown of the vesicular monoamine transporter (VMAT) and disruption of the SNARE complex in all glia or selectively in astrocytes increased sensitivity to alcohol sedation and that both VMAT and the SNARE complex functioned downstream of Tdc2. Our studies support a model in which the synthesis of tyramine and vesicle-mediated release of tyramine from adult astrocytes regulates alcohol sedation in Drosophila. Considering that tyramine is functionally orthologous to norepinephrine in mammals, our results raise the possibility that gliotransmitter synthesis release could be a conserved mechanism influencing behavioral responses to alcohol as well as alcohol use disorder.

Assessing Vesicular Monoamine Transport and Toxicity Using Fluorescent False Neurotransmitters.

Impairments in the vesicular packaging of dopamine result in an accumulation of dopamine in the cytosol. Cytosolic dopamine is vulnerable to two metabolic processes-enzymatic catabolism and enzymatic- or auto-oxidation-that form toxic metabolites and generate reactive oxygen species. Alterations in the expression or activity of the vesicular monoamine transporter 2 (VMAT2), which transports monoamines such as dopamine from the cytosol into the synaptic vesicle, result in dysregulated dopamine packaging. Here, we developed a series of assays using the fluorescent false neurotransmitter 206 (FFN206) to visualize VMAT2-mediated vesicular packaging at baseline and following pharmacological and toxicological manipulations. As a proof of principle, we observed a significant reduction in vesicular FFN206 packaging after treatment with the VMAT2 inhibitors reserpine (IC50: 73.1 nM), tetrabenazine (IC50: 30.4 nM), methamphetamine (IC50: 2.4 μM), and methylphenidate (IC50: 94.3 μM). We then applied the assay to investigate the consequences on vesicular packaging by environmental toxicants including the pesticides paraquat, rotenone, and chlorpyrifos, as well as the halogenated compounds unichlor, perfluorooctanesulfonic acid, Paroil, Aroclor 1260, and hexabromocyclododecane. Several of the environmental toxicants showed minor impairment of the vesicular FFN206 loading, suggesting that the toxicants are weak VMAT2 inhibitors at the concentrations tested. The assay presented here can be applied to investigate the effect of additional pharmacological compounds and environmental toxicants on vesicular function, which will provide insight into how exposures to such factors are involved in the pathogenesis of monoaminergic diseases such as Parkinson's disease, and the assay can be used to identify pharmacological agents that influence VMAT2 activity.

Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3.

Extracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis via vesicular transfer to neighboring alveolar epithelial cells. Although changes in the secretion of EV molecules—including but not limited to SOCS3—have been described in response to microenvironmental stimuli, the cellular and molecular machinery that control alterations in vesicular cargo packaging remain poorly understood. Furthermore, the use of quantitative methods to assess the sorting of cytosolic cargo molecules into EVs is lacking. Here, we utilized cigarette smoke extract (CSE) exposure of AMs as an in vitro model of oxidative stress to address these gaps in knowledge. We demonstrate that the accumulation of reactive oxygen species (ROS) in AMs was sufficient to augment vesicular SOCS3 release in this model. Using nanoparticle tracking analysis (NTA) in tandem with a new carboxyfluorescein succinimidyl ester (CFSE)-based intracellular protein packaging assay, we show that the stimulatory effects of CSE were at least in part attributable to elevated amounts of SOCS3 packaged per EV secreted by AMs. Furthermore, the use of a 20S proteasome activity assay alongside treatment of AMs with conventional proteasome inhibitors strongly suggest that ROS stimulated SOCS3 release via inactivation of the proteasome. These data demonstrate that tuning of AM proteasome function by microenvironmental oxidants is a critical determinant of the packaging and secretion of cytosolic SOCS3 protein within EVs.

P2584Extracellular vesicles are relevant transporters of C16 ceramide and induce apoptosis in endothelial vesicle recipient cells

Abstract Introduction Extracellular vesicles (EVs) have recently been identified as important intercellular transporters of biologically active molecules. Besides microRNAs, which have been shown to mediate an important part of the EV function, various sphingolipids have been shown to be exported into EVs. Among these sphingolipids, ceramides have been gained interest in the cardiovascular context, because elevated plasma levels of d18:1–16:0 Ceramide (C16) have been shown to correlate with an increased risk for cardiovascular events. How ceramides are enriched in endothelial cell derived-EVs and if they are transferred to EV recipient cells to exert specific biological functions is currently unknown. Methods and results Endothelial cell derived large EVs were isolated from human coronary artery endothelial cells (HCAECs) via differential centrifugation of the culture supernatant (1 x 1500 g / 15 min + 2 x 20,000 g / 40 min). EVs were characterized by immunoblotting, electron microscopy and nanoparticle tracking analysis (Size: 252±24 nm). Sphingolipids were extracted from HCAECs and EVs (with and without glycemic injury by 30mmol/L glucose for 72 h) by solid phase extraction and analysed via Q-TOF MS/MS mass spectrometry. Lipidomic analysis revealed an enrichment of all sphingolipid classes in EVs, including C16, which was most abundantly present in EVs. As a next step, we tested if ceramides can be transferred between HCAECs by EVs. Therefore, HCAECs were incubated with NBD-labeled ceramide. NBD-Ceramide uptake into HCAECs and vesicular transfer to native HCAECs were confirmed by fluorescence microscopic imaging. Importantly, the transfer of NBD-Ceramide was abrogated if the vesicles were degraded in Triton-X 1% before incubation with EV recipient cells, which confirms that the transfer is vesicle dependent. Additionally, uptake of C16 into HCAECs and release into EVs was confirmed via mass spectrometry. Viability of C16 treated HCAECs was significantly reduced at concentrations above 5 μM of C16 in an MTT-based assay. In order to assess if the increased vesicular packing and transfer of Ceramides also reduces viability of EV recipient cells, native HCAECs were incubated with EVs from HCAECs which were treated with 2 μM and 20 μM C16. Treatment with C16 enriched vesicles lead to a significant reduction of EV recipient cell viability in a dose dependant manner. Conclusion In the present study, we found that C16 is enriched in EVs and that C16 is transferred through EVs among HCAECs. Depending on the amount of transferred C16, this process reduces the viability of native EV recipient HCAECs. Furthermore, vesicular C16 export is increased after glycemic injury. In comparison to the amounts of free C16, which are necessary to induce a similar reduction in HCAEC viability, the amount of EV transferred C16 is negliable. Hence, vesicular packaging results in a high bioavailability of ceramides leading to a relevant regulation of endothelial cell death. Acknowledgement/Funding BONFOR Progamm University Bonn

A novel mechanism strengthens GABAergic inhibition in the hypothalamic PVN in proportion to the level of synaptic L‐glutamate spillover

Perhaps the most robust property of pre‐sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) in unstressed animals is their state of near quiescence. Although discharge quiescence has been strongly linked to the dominance of GABAergic synaptic inhibition, mechanisms that govern the strength of local GABAergic inhibition remain to be fully elucidated. Here, whole cell patch‐clamp recordings were made from identified parvocellular PVN neurons in brain slices and GABAergic inhibitory postsynaptic currents (IPSCs) were evoked by electrical stimuli delivered to the surrounding peri‐nuclear zone. To investigate the impact of GABA synthesis and vesicular packaging on GABAergic IPSC amplitude, we compared eIPSC amplitude before and after bath application of the GABA synthetic substrate L‐glutamate (L‐Glu; 100 μM, 10 min). Within 3–5 minutes of L‐Glu washout, eIPSC amplitude suddenly and significantly increased (+23 ± 6%; P<0.002). This response was unaffected by selective inhibition of glial L‐Glu uptake with dihydrokainate (DHK; 250 μM), but was nearly abolished (−77 ± 8.5%) by DL‐threo‐β‐benzyloxyaspartic acid (TBOA; 100 μM), a pan‐specific inhibitor of L‐glutamate transporters, suggesting that strengthening of GABAergic inhibition by L‐Glu depends on uptake by the neuronal L‐Glu transporter EAAT3. Similar results were obtained when local synaptic activity was increased by bath perfusion with high K+ (10 mM) aCSF. In current‐clamp recordings of spontaneous firing, high bath K+ caused a delayed reduction of discharge (1.58 ± 0.59Hz to 1.18 ± 0.51Hz; P<0.03) that was not observed when depolarizing current was injected to mimic the spiking response to high K+. These findings suggest that GABAergic IPSC strengthening is sufficient to reduce PVN neuronal discharge. High frequency stimulation‐induced GABAergic synaptic depression/vesicular depletion revealed that exposure to bath L‐Glu did not increase quantal content (i.e., the number of GABA vesicles released during each IPSC), but enhance recovery of IPSC amplitude by +36 pA within 10 s. These data indicate that bath L‐Glu selectively increases the number of GABA molecules contained within each GABA vesicle. In anesthetized rats (n=6), acute blockade of GABA‐A receptors in the PVN with picrotoxin (PTX) increased renal sympathetic nerve activity (+23%). Prior PVN injection of L‐Glu greatly amplified the PTX response (+72%), which is consistent with increased functional inhibitory tonus. Collectively, our findings indicate that GABA synaptic vesicles are “over‐filled” when GABA is synthesized from L‐Glu under conditions of heightened L‐Glu availability. This process depends on EAAT3‐mediated L‐Glu uptake, likely into GABA terminals. As over‐filled vesicles transition to the readily releasable pool, the amplitude of IPSCs increases, allowing synaptic inhibitory tonus to increase in proportion to the level of synaptic L‐Glu spillover. This local mechanism is postulated to help prevent neuronal hyper‐activation under conditions of heightened glutamatergic excitation.Support or Funding InformationNIH HL088052 (GMT)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission.

Many neurons influence their targets through co-release of neuropeptides and small-molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small-molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both stimulated emission depletion super-resolution and transmission electron microscopy. Ptp4E also controls the presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content.This article has an associated First Person interview with the first author of the paper.

First person – Juan Tao

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Juan Tao is first author on ‘Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission’, published in JCS. Juan conducted the research described in this article while a postdoc in Edwin Levitan's lab at University of Pittsburgh. She is now a Research Staff in the lab of Michael Parsons at University of California, investigating how neuropeptide and other molecule transmitters are transported long distances in Drosophila synapses.

Blockade and reversal of swimming-induced paralysis in C. elegans by the antipsychotic and D2-type dopamine receptor antagonist azaperone

The catecholamine neurotransmitter dopamine (DA) exerts powerful modulatory control of physiology and behavior across phylogeny. Perturbations of DA signaling in humans are associated with multiple neurodegenerative and behavioral disorders, including Parkinson's disease, attention-deficit/hyperactivity disorder, addiction and schizophrenia. In the nematode C. elegans, DA signaling regulates mating behavior, learning, food seeking and locomotion. Previously, we demonstrated that loss of function mutations in the dat-1 gene that encodes the presynaptic DA transporter (DAT-1) results in a rapid cessation of movement when animals are placed in water, termed Swimming Induced Paralysis (Swip). Loss of function mutations in genes that support DA biosynthesis, DA vesicular packaging and DA action at the extrasynaptic D2-type DA receptor DOP-3 suppress Swip in dat-1 animals, consistent with paralysis as arising from excessive DA signaling. Although animals grown on the vesicular monoamine transporter antagonist reserpine diminish Swip, the drug must be applied chronically, can impact the signaling of multiple biogenic amines, and has been reported to have penetrant, off-target actions. Here, we demonstrate that the antipsychotic drug azaperone potently and rapidly suppresses Swip behavior in either dat-1 mutants, as well as in wildtype animals treated with the DAT-1 antagonist nisoxetine, with genetic experiments consistent with DOP-3 antagonism as the mechanism of Swip suppression. Reversal of Swip in previously paralyzed dat-1 animals by azaperone application demonstrates an otherwise functionally-intact swimming circuit in these mutants. Finally, whereas azaperone suppresses DA-dependent Swip, the drug fails to attenuate the DA-independent paralysis induced by βPEA, aldicarb or genetic disruption of γ-aminobutyric acid (GABA) signaling. We discuss our findings with respect to the use of azaperone as a potent and selective tool in the identification and analysis of presynaptic mechanisms that regulate DA signaling.

Stellate Transcriptomics Reveal Consistent Patterns of Gene Expression Across Species

IntroductionSympathetic hyperactivity is a key feature of human hypertension as well as animal models of hypertension and cardiovascular disease. Sympathetic hyperactivity can result from cell‐autonomous changes in neurons that innervate cardiovascular target tissues. Stellate ganglia are of particular interest because the majority of sympathetic neurons innervating the heart reside there. Transcriptomics provides a powerful tool for identifying genomic changes that contribute to sympathetic dysfunction in disease.AimTo compare the transcriptomes of healthy stellate ganglia from adult rat and mouse.MethodRNA‐sequencing was carried out on individual stellate ganglia from 16‐week male Wistar rats or 18‐week male C57Bl6J mice. Mouse samples were depleted of ribosomal RNA prior to library preparation, while the rat library was prepared used OligoDT to enrich for mRNAs. Sequencing was carried out in an Illumina HiSeq 4000 at the High‐Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (Oxford) or the Massively Parallel Sequencing Shared Resource (OHSU). The rat transcriptome was aligned to the reference genome and transcripts were quantified using the Salmon‐DESeq2 method, which relies on exon matching. The mouse transcriptome was aligned to the reference genome by the OHSU Bioinformatics & Computational Biology Core using STAR, which includes non‐coding RNAs.ResultsA comparison of these independent analyses of gene expression reveals a significant degree of consistency regarding the most highly expressed genes, despite differences in the data processing pipeline. Genes closely associated with sympathetic neuron function were among the most highly expressed genes in both rat and mouse. We identified high levels of genes encoding proteins required for norepinephrine synthesis tyrosine hydroxylase (Th), L‐DOPA decarboxylase (Ddc), and dopamine beta hydroxylase (dbh), as well as genes encoding transporters for vesicular packaging (slc18a2, vesicular monoamine transporter 2) and removal of extracellular catecholamines (slc6a2, norepinephrine transporter, NET). High levels of mRNA encoding the sympathetic co‐transmitter Neuropeptide Y (Npy) were also observed.Genes encoding the Nerve Growth Factor receptors Ngfr (p75 neurotrophin receptor) and Trk1 (TrkA tyrosine kinase receptor) were abundantly expressed in rat and mouse stellate ganglia. Genes encoding structural proteins such as actins and tubulins were abundant in both species, as were genes encoding proteins involved in synaptic transmission including Snap25 (synaptosomal‐associated protein 25), Syt1 (synaptotagmin 1), and several nicotinic acetylcholine receptor subunits. The details of transcript counts differed between the two methods but the overall pattern of gene expression between species was quite consistent.ConclusionThese data will provide a normal baseline for identification of changes that occur in diseases associated with sympathetic over‐activity such as hypertension and cardiovascular disease.Support or Funding InformationNIH OT2TR001984, OHSU School of Medicine Dean's Fund, The Wellcome TrustThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vesicular Glutamate Transporter 1 Knockdown in Infralimbic Prefrontal Cortex Augments Neuroendocrine Responses to Chronic Stress in Male Rats.

Chronic stress-associated pathologies frequently associate with alterations in the structure and activity of the medial prefrontal cortex (mPFC). However, the influence of infralimbic cortex (IL) projection neurons on hypothalamic-pituitary-adrenal (HPA) axis activity is unknown, as is the involvement of these cells in chronic stress-induced endocrine alterations. In the current study, a lentiviral-packaged vector coding for a small interfering RNA (siRNA) targeting vesicular glutamate transporter (vGluT) 1 messenger RNA (mRNA) was microinjected into the IL of male rats. vGluT1 is responsible for presynaptic vesicular glutamate packaging in cortical neurons, and knockdown reduces the amount of glutamate available for synaptic release. After injection, rats were either exposed to chronic variable stress (CVS) or remained in the home cage as unstressed controls. Fifteen days after the initiation of CVS, all animals were exposed to a novel acute stressor (30-minute restraint) with blood collection for the analysis of adrenocorticotropic hormone (ACTH) and corticosterone. Additionally, brains were collected for in situ hybridization of corticotrophin-releasing hormone mRNA. In previously unstressed rats, vGluT1 siRNA significantly enhanced ACTH and corticosterone secretion. Compared with CVS animals receiving the green fluorescent protein control vector, the vGluT1 siRNA further increased basal and stress-induced corticosterone release. Further analysis revealed enhanced adrenal responsiveness in CVS rats treated with vGluT1 siRNA. Collectively, our results suggest that IL glutamate output inhibits HPA responses to acute stress and restrains corticosterone secretion during chronic stress, possibly at the level of the adrenal. Together, these findings pinpoint a neurochemical mechanism linking mPFC dysfunction with aberrant neuroendocrine responses to chronic stress.

Levodopa reverses cytokine-induced reductions in striatal dopamine release.

Background:Studies using neuroimaging and in vivo microdialysis in humans and nonhuman primates indicate that inflammatory cytokines such as interferon-alpha reduce dopamine release in the ventral striatum in association with depressive symptoms including anhedonia and psychomotor slowing.Methods:Herein, we examined whether reduced striatal dopamine release in rhesus monkeys chronically treated with interferon-alpha can be restored by administration of the dopamine precursor levodopa via reverse in vivo microdialysis.Results:Levodopa completely reversed interferon-alpha–induced reductions in striatal dopamine release. No changes were found in the 3,4-dihydroxyphenylacetic acid to dopamine ratio, which increases when unpackaged dopamine is metabolized via monoamine oxidase.Conclusions:These findings suggest that inflammatory cytokines reduce the availability of dopamine precursors without affecting end-product synthesis or vesicular packaging and/or release and provide the foundation for future studies investigating therapeutic strategies that facilitate availability of dopamine precursors to improve depressive symptoms in patient populations with increased inflammation.

Tianeptine modulates amygdalar glutamate neurochemistry and synaptic proteins in rats subjected to repeated stress

Stress is a common environmental factor associated with depressive illness and the amygdala is thought to be integral for this association. For example, repeated stress impairs amygdalar neuroplasticity in rodents and these defects parallel amygdalar deficits in depressive illness patients. Because the excitatory neurotransmitter glutamate is important in neuroplasticity, we hypothesized that alterations in amygdalar glutamatergic systems may serve as key players in depressive illness. Moreover, restoration of amygdalar glutamatergic systems may serve as important therapeutic targets in the successful management of multiple stress-related mood disorders. To address these hypotheses, we measured glutamate efflux in the basolateral and central amygdalar complexes via in vivo microdialysis, as well as the expression of synaptic proteins that regulate vesicular glutamate packaging and release, in rats subjected to repeated stress and treated daily with saline or the antidepressant tianeptine. Glutamate efflux was significantly reduced in the central amygdalar complex of animals subjected to repeated stress. In addition, repeated stress nearly eliminated amygdalar vGLUT2 expression, thereby proving a potential mechanism through which repeated stress impairs amygdalar glutamate neurochemistry. These stress-induced changes in glutamate efflux and vGLUT2 expression were inhibited by daily tianeptine administration. Moreover, tianeptine administration increased the vesicular localization of SNAP-25, which could account for the ability of tianeptine to modify glutamatergic tone in non-stressed control rats. Collectively, these results demonstrate that repeated stress differentially affects amygdalar glutamate systems and further supports our previous studies indicating that tianeptine's antidepressant efficacy may involve targeting amygdalar glutatamatergic systems.

Abstract 3387: Secreted exosomes from cultured bladder cells are enriched for distinct miRNAs detected in circulation of metastatic bladder cancer patients

Abstract Exosomes are small nanovesicles (30-100 nm) secreted from the cell surface of most cell types. The vesicles are secreted at increased levels by cancer cells and can be detected in body fluids of cancer patients. Exosomes function in paracrine delivery of proteins, RNA, and DNA into recipient cells. By this, exosomes are speculated to be involved e.g. in pre-metastatic niche formation at distant sites. Here we present a characterization of exosome vesicles isolated from bladder cancer cells as examined by electron microscopy, dynamic light scattering and by western blotting of common exosome protein markers. The expression of 671 unique human miRNAs by qPCR was analyzed in both exosomes and donor cells from: 1) cultivated normal urothelial cells (NHU), 2) T24 bladder carcinoma cell line (non-metastatic) and its metastatic isogenic derivates FL3 and SLT4 and 3) UMUC3 bladder carcinoma cell line (moderate metastatic) and its highly metastatic isogenic derivative LUL2. Several miRNAs were detected in the vesicles yet absent in their parental donor cell indicating a possible preferential vesicular packaging. miRNAs miR92a, -106a, -20a, -21, -16, and let-7a/b/d/e were detected among the top100 ranked miRNAs in all vesicles. Among the top30 enriched exosomal miRNAs, miR-223 and-383 were detected in all cancer vesicles, and miR-921 in the highly metastatic vesicles (FL3, SLT4, LUL2). The release of miRNAs (miR-21, -16, and let7b) from FL3 cells into the culture media was time-dependent and a fraction of these miRNAs were stable to RNaseOne degradation indicating possible protection inside the vesicles. Examination of miRNAs in human plasma samples also suggested resistance towards endogenous RNAse degradation as well as towards repetitive freeze/thawing. The profiling of miRNA expression by Affymetrix microarray analysis of crude exosome-containing fractions of human plasma samples (10 healthy controls and 10 cases of metastatic bladder cancer) showed significant upregulation in cancer plasma of e.g. miR-92a, -106a, -23a, -20a, -21, some of which were the same as the miRNAs highly expressed in the tumor vesicles. The significant increase was confirmed by q-RT-PCR for miR-92a and -23a and validated on an independent set of plasma samples (8 healthy controls and 8 metastatic cancer cases). Our results suggest that specific miRNAs are encapsulated into exosomes in bladder cancer and that these may be detected in circulation in advanced stages of bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3387. doi:1538-7445.AM2012-3387

Serum albumin disrupts Cryptococcus neoformans and Bacillus anthracis extracellular vesicles

For both pathogenic fungi and bacteria, extracellular vesicles have been shown to contain many microbial components associated with virulence, suggesting a role in pathogenesis. However, there are many unresolved issues regarding vesicle synthesis and stability, including the fact that vesicular packaging for extracellular factors involved in virulence must also have a mechanism for vesicle unloading. Consequently, we studied the kinetics of vesicle production and stability using [1-(14) C] palmitic acid metabolic labelling and dynamic light scattering techniques. Cryptococcus neoformans vesicles were produced throughout all stages of fungal culture growth and they were stable once isolated. Density gradient analysis revealed that only a portion of the vesicle population carried cryptococcal polysaccharide, implying heterogeneity in vesicular cargo. Vesicle incubation with macrophages resulted in rapid vesicle instability, a phenomenon that was ultimately associated with serum albumin. Additionally, albumin, along with mouse serum and murine immunoglobulin destabilized Bacillus anthracis vesicles, but the effect was not observed with ovalbumin or keyhole limpet haemocyanin, demonstrating that this phenomenon is neither host-, microbe- nor protein-specific. Our findings strongly suggest that cryptococcal vesicles are short-lived in vivo and vesicle destabilization is mediated by albumin. The ability of albumin to promote vesicular offload through destabilization indicates a new activity for this abundant serum protein.

Decreased voluntary activity and amygdala levels of serotonin and dopamine in ovariectomized rats

Estrogen is involved in numerous activities in the brain, such as learning, memory, fear, anxiety and mood. However, little is known about the pathways involved in the effects of estrogen in the brain. Therefore, to improve our understanding of the effects of estrogen, we examined the effects of ovariectomy (OVX)—a model of estrogen deficiency and menopause—on psychiatric functions, including voluntary activity. Female Wistar rats underwent OVX or sham operation. Voluntary momentum and circadian activity were monitored at 2 and 6 weeks, respectively. Rats also underwent microdialysis of the amygdala to determine serotonin (5-HT) and dopamine levels. Although the circadian rhythm was unchanged at 2 weeks, voluntary activity at 6 weeks was significantly lower in OVX rats than that in sham rats. This was due to significantly reduced voluntary activity in the 12-h dark phase, while no significant difference was detected in the 12-h light phase. Both 5-HT and dopamine levels in the amygdala were significantly lower in OVX rats than those in sham rats at 6 weeks after the procedure. In conclusion, these results indicate that estrogen is an important mediator of voluntary activity in rats, particularly during the dark phase. These effects of estrogen appear to involve reduced 5-HT and dopamine release in the amygdala. Further studies are needed to determine whether estrogen (and its deficiency) influences the synthesis, vesicular packaging, release, re-uptake and degradation of these transmitters.