Bothrops lanceolatus, commonly named ‘Fer-de-Lance’, is an endemic snake of the French Caribbean Island of Martinique. Envenomations by B. lanceolatus present clinical aspects characterized by systemic thrombotic syndrome and important local inflammation, involving edema and pain but limited hemorrhage. To investigate mechanisms of venom-induced inflammation, B. lanceolatus venom was characterized, its cross-reactivity with bothropic antivenom explored, its cytotoxicity on human keratinocytes and vascular cells, and the production of cytokines and chemokines were analyzed. We used electrophoretic separation, zymography, colorimetric or fluorimetric enzymatic assays, and immunochemical assays. Therapeutic South American bothropic antivenom cross-reacted with B. lanceolatus venom and completely or partially abolished its PLA2, hyaluronidase, and proteolytic activities, as well as its cytotoxicity for keratinocytes. The substrate specificity of B. lanceolatus venom proteases was emphasized. B. lanceolatus venom cytotoxicity was compared to the B. jararaca venom. Both venoms were highly cytotoxic for keratinocytes (HaCaT), whereas B. lanceolatus venom showed particularly low toxicity for endothelial cells (EAhy926). Patterns of cytokine and chemokine production by cells exposed to the venoms were highly pro-inflammatory. Thus, the results presented here show that B. lanceolatus venom toxins share important antigenic similarities with South American Bothrops species toxins, although their proteases have acquired particular substrate specificity. Moreover, the venom displays important cytotoxic and pro-inflammatory action on human cell types such as keratinocytes and endothelial cells, which are important players in the local and systemic compartments affected by the envenomation. Bothrops species account for the majority of venomous ophidian accidents. Envenomation by these snakes has a complex pathophysiology, characterized by prominent local effects  and systemic effects such as coagulation disturbances, hemorrhage, and renal failure [Bothrops lanceolatus, commonly known as ‘Fer-de-lance’, is an endemic species confined to the island of Martinique in the Caribbean, where it is responsible for an average of 30 cases of human envenomation per year [B. lanceolatus venom induces local and systemic effects comparable to bothropic syndrome, but the envenomation is characterized by a predominant prothrombotic profile. Systemic thrombosis development can lead to cerebral, myocardial, or pulmonary infarctions that only rapid treatment with the monospecific commercial antivenom  can prevent [In South and Central America,  failure ,2. Bothr prevent ,5.B. lanceolatus venom contains five major types of enzymes; zinc-dependent snake venom metalloproteases (SVMPs), snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2), L-amino acid oxidases, and a specific C-type lectin-like molecule [Bothrops venoms [Bothrops venoms. They display a wide range of biological effects such as neurotoxicity, cardiotoxicity, myotoxicity, hemolysis, and, anti-coagulating, anti-platelet, and edema-forming activities [B. lanceolatus venom; a hemorrhagic PI-SVMP, an acidic phospholipase, and a potent fibrinogenolytic enzyme [molecule ,7. SVMPss venoms ,9. They s venoms . Class Is venoms ,12. SVMPs venoms . SVSPs hs venoms . PLA2s htivities . Three ec enzyme ,17.B. lanceolatus venom has been described in rat and mouse models [B. lanceolatus venom-induced edema is accompanied by local hemorrhage, involves neutrophil infiltration, mast cell degranulation, and the release of arachidonic acid metabolites, bradykinin, histamine, and serotonin in the first hours after inoculation [Inflammation induced by e models ,19,20,21culation ,19,21. IB. lanceolatus has not yet been elucidated. The venom cleaves purified human fibrinogen but is unable to clot citrated human plasma, and almost normal coagulation profile can be observed in patients developing thrombosis, suggesting that the thrombosis mechanism could involve vascular endothelial cells or platelet activation [The mechanism of thrombosis observed in envenomation by tivation . It is dtivation ,23. The tivation .B. lanceolatus venom, the therapeutic Costa Rican polyvalent antivenom  was shown to recognize venom toxic compounds in vitro and to be fully effective in mice in neutralizing the lethal, hemorrhagic, edema-forming, myotoxic, and indirect hemolytic activities of the venom [Bothrops jararaca (50%), Bothrops alternatus (12.5%), Bothrops jararacussu (12.5%), Bothrops moojeni (12.5%), and Bothrops neuwiedi (12.5%). This antivenom cross reacts with B. lanceolatus venom and its hemorrhagic PI-SVMP, BlaH1, and neutralizes the hemorrhagic activity of this toxin [Like the monospecific antivenom raised against he venom ,22. The is toxin .B. lanceolatus venom activities have been studied [B. lanceolatus venom enzymatic and cytotoxic activities to improve the understanding of the venom-induced local inflammation mechanism. The venom enzymatic activities were confirmed and further investigated. The antivenom cross-reaction and the inhibition of in vitro B. lanceolatus venom enzymatic activities were studied to highlight antigenic similarities between B. lanceolatus and South American Bothrops species venoms. To approach human inflammation mechanisms, we studied B. lanceolatus venom cytotoxicity on human cell lines of keratinocytes and vascular endothelial cells and cytokines/chemokines production upon venom-exposure.The main  studied ,18,22,24B. lanceolatus venom in non-reducing conditions reveals the presence of proteins with molecular masses between 10 and 180 kDa  was submitted to gelatin and fibrinogen zymography. rM > 115 kDa), as well as smaller ones, between 64 and 85 kDa. Several proteins are able to cleave fibrinogen with molecular masses between 20 and 180 kDa ( 180 kDa B. Howeve 180 kDa C. EDTA aB. lanceolatus venom upon FRET peptidic substrates was investigated. In linear kinetics conditions (excess of substrate), the specific activities of B. lanceolatus venom on the peptides Abz-FRSSRQ and Abz-RPPGFSPFRQ were of the values 111.0 ± 4.7 and 78.3 ± 9.6 UF/min/µg, respectively , and, after 72 h, it was 17.6 µg/mL (CI 95: 0–51.2 µg/mL).Incubation of human keratinocytes HaCaT with ing 24 h A. 50% ofing 24 h C. After  EAhy926 B,D. Afteus venom B. The caectively D. After B. lanceolatus venom for the two cell lineages by bothropic antivenom was tested by preincubating venom with antivenom. B. jararaca venom was used as the positive control, and botulinum toxin antiserum was used as the negative control. The concentrations of venom were set so as to obtain about 100% loss of viability within 24 h. Bothrops venoms’ cytotoxicity in keratinocytes by the bothropic antivenom, using 1:20 antivenom dilution. However, no inhibition of the venoms’ cytotoxicity was observed for endothelial vascular cells, using antivenom dilution up to 1:5  activities could be fully abrogated by the antivenom. On the contrary, the inhibition of B. lanceolatus venom proteolytic activity and cytotoxicity for endothelial cells by the antivenom was partial, showing that some, but not all, components from B. lanceolatus venom share antigenic similarities with other Bothrops species from South America. Abz-FRSSRQ substrate is cleaved mainly by serine proteases in B. lanceolatus venom, whereas both serine proteases and metalloproteinases act on Abz-RPPGFSPFRQ. However, in the study by Kuniyoshi et al. [Bothrops venoms. These data suggest that B. lanceolatus proteases present different substrate specificity from their continental bothropic counterparts.The bothropic antivenom produced against Brazilian snakes cross-reacted with i et al. , Abz-RPPBothrops species from South America or the cytotoxicity of proteins purified from these venoms for endothelial cells have been performed [Bothrops venom toxicity for keratinocytes [B. lanceolatus venom. Two human immortalized cell lines were chosen to investigate venom-induced local/systemic inflammation: HaCaT keratinocytes and EAhy926 vascular endothelial cells. In the assay conditions, the venom affected keratinocyte viability from 2 µg/mL concentration and 24 h of exposure. With similar exposure time, B. jararaca venom was as toxic as B. lanceolatus venom, demonstrating a high toxicity of both Bothrops venoms in this cell line. However, both venoms displayed different cytotoxic patterns on EAhy926 endothelial cells. After 24 h incubation, B. lanceolatus venom appeared about 10 times less toxic than B. jararaca venom. Longer times of incubation enhanced B. lanceolatus venom cytotoxicity. Interestingly B. lanceolatus venom results reproduced the in vivo observations of Jiménez et al [Several studies addressing the cytotoxic action of venoms of the erformed ,43,44,45inocytes ,47. To dez et al ,48.Keratinocytes play an important part in the inflammatory process by producing a wide variety of cytokines. Once activated, they also produce chemokines, which attract leucocytes to the inflammation spot . In venoB. lanceolatus venom, we detected four cytokines: IL-6, MCP-1, RANTES, and IL-8. The concentrations of these proteins increased in venom-exposed cell supernatants after 48 or 72 h of venom exposure. In order to complete our study, we analyzed activation markers expression such as adhesion protein ICAM-1 and tissue factors on venom-exposed endothelial cell membranes, using flow cytometry. ICAM-1 expression was significantly augmented by TNF-α exposure, whereas B. lanceolatus venom did not significantly alter its expression. In vivo, B. asper venom stimulates endothelial cells to express adhesion molecules such as ICAM-1 [B. jararaca venom influence the expression of selectin and integrin genes in HUVECs [Endothelial vascular cells also play a key role in inflammation. They control inflammatory cell adhesion and migration from the bloodstream to damaged tissues, as well as fluid exchange between the two compartments. In supernatants of EAhy926 endothelial cells exposed to s ICAM-1 , whereasn HUVECs ,51. The B. lanceolatus venom such as low hyaluronidase activity, protease substrate specificity, and cytotoxicity for keratinocytes and endothelial cells and bring insights into antigenic similarity between B. lanceolatus venom toxins and South American Bothrops venoms toxins. Our in vitro model of venom-induced cytokine and chemokine production reveals the high pro-inflammatory potential of the venom in human cell lines. Further describing B. lanceolatus venom interactions with cytokines and human cell lines that are relevant in inflammation processes could help to improve envenomation management.The data presented here expose new features of N-Cetyl-N,N,N-trimethylammonium bromide (CTAB), ortho-phenylenediamine (OPD), Coomassie Blue R-250, hyaluronic acid, diaminobenzidine (DAB), phenylmethanesulfonyl fluoride (PMSF), 1,10-phenanthroline, bovine serum albumin (BSA), fibrinogen from human plasma, gelatin type A, goat anti-horse (GAH) IgG horseradish peroxidase (IgGHRPO), concanavalin A (Con A), and Wheat Germ Agglutinin (WGA) labelled with peroxidase, were purchased from Sigma-Aldrich . Dithiothreitol (DTT) and iodoacetamide were obtained from GE Healthcare Life Sciences . 3--2,5-di-phenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were from Merck . GAH IgG labelled with alkaline phosphatase (IgG-AP), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), and nitroblue tetrazolium (NBT) were from Promega Corp. . Fluorescent resonance energy transfer (FRET) substrates Abz-FRSSRQ-EDDnp (Abz-FRSSRQ) and Abz-RPPGFSPFRQ-EDDnp (Abz-RPPGFSPFRQ) were obtained from GenOne Biotechnologies .Triton X-100, Tween 20, Brij-35, EDTA, Bothrops lanceolatus was obtained from Latoxan . Venoms from Bothrops jararaca and Crotalus durissus terrificus were supplied by the Herpetology Laboratory of the Butantan Institute. Stock solutions were prepared in sterile saline solution (150 mM NaCl) at 5 mg/mL and stored at −80 °C.Venom from The bothropic antivenom and botulinum toxin antiserum, both containing only purified immunoglobulin F(ab′)2 fraction, were kindly donated by the Hyperimmune Plasma Processing Section of the Butantan Institute .B. lanceolatus venom was solubilized in non-reducing or reducing sample buffer, incubated at 96 °C for 5 min. Then samples were separated by SDS-PAGE on 12% acrylamide gels [2HPO4, 1.5 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.2) containing 5% BSA and incubated with bothropic antivenom  for 1 h at room temperature. Immunoreactive proteins were detected using GAH/IgG-AP (1:7500) in PBS/1% BSA for 1 h at room temperature. After washing three times for 10 min with PBS/0.05% Tween 20, blots were developed using NBT/BCIP according to the manufacturer’s instructions . For the western blot using lectins, after transfer and blocking, the membranes were incubated with Con A or WGA labelled with peroxidase for 1 h at room temperature. The membranes were then washed with PBS containing Tween-20 (0.05%) and developed with DAB (20 mg/mL).ide gels , stainedide gels . After tB. lanceolatus venom for 16 h at room temperature. First dimension separation was carried out using an Ettan IPGphor Isoelectric Focusing System , following th emanufacturer’s instructions. We followed a five-phase electrophoresis program; a step of 500 V for 60 min, a gradient of 1000 V for 60 min, a gradient of 8000 V for 2 h 30 min, a step of 1000 V for 12 h (so as to run overnight), and finally a step of 8000 V for 30 min. Right at the end of the separation, the IPG strips were sequentially incubated in equilibration buffer  containing 10 mg/mL DTT for 15 min and then in equilibration buffer containing 25 mg/mL iodoacetamide for 15 min. After that, they were briefly washed in electrophoresis buffer  and applied to 12% SDS-polyacrylamide gels (10 × 10 cm) for second dimension electrophoresis, using the Hoefer Mini VE apparatus . The gels were finally silver-stained.Precast IPG strips  were rehydrated with 250 µL of DeStreak rehydration solution  containing 1% IPG buffer  and 100 µg of B. lanceolatus venom  and an ELISA assay was performed with bothropic antivenom, as previously described [Microtitre plates were coated with escribed . BotulinB. lanceolatus venom was investigated using an EnzChek® Phospholipase A2 Assay Kit , following the manufacturer’s instructions. Venoms from B. jararaca and C. durissus terrificus were used as the positive controls. The venom concentrations were chosen according to preliminary dose-response curves. Fluorescence measurements were performed at 37 °C using the spectrometer FLUOstar Omega  at the wavelength λEM = 515 nm, with an excitation at λEX = 485 nm every 30 s for 5 min. Specific activity was expressed as the units of free fluorescence of cleaved substrate/min/µg of venom.The PLA2 activity of The hyaluronidase activity of the venom was measured following the procedures of Pukrittayakamee et al. . The venB. lanceolatus venom’s fibrinogenolytic activity, samples of fibrinogen (30 µg) were incubated with the venom for 30 min at 37 °C and submitted to electrophoretic separation in reducing conditions.The venom samples were pre-incubated or not with 20 mM of inhibitors . To evaluate B. lanceolatus venom’s gelatinolytic and fibrinogenolytic activities were analyzed by zymography. Venom samples (25 µg) were solubilized in normal sample buffer and separated on gels containing 10% of polyacrylamide and 1 mg/mL of gelatin type A or fibrinogen from human plasma. The separation was realized under constant tension (80 V) and temperature (4 °C) in the Mighty Small system . After separation, the gelatin gels were washed once for 30 min in Triton X-100 (2.5%) at room temperature, whereas the fibrinogen-containing gels were submitted to this wash three times. The gels were incubated overnight at 37 °C in substrate buffer . They were finally stained in Coomassie Brilliant Blue R-250 (0.2%) for 30 min under constant agitation in order to reveal the bands where proteolytic digestion occurred.B. lanceolatus venom’s proteolytic activity in the presence or absence of inhibitors and bothropic antivenom, following the procedures described by Queiroz et al. [Bothrops venom SVSPs [® 96-well plates. The reactions were monitored at 37 °C (λEM = 420 nm and λEX = 320 nm) in a fluorescence spectrophotometer . The specific activity was expressed as units of free fluorescence (UF) of cleaved substrate/min/µg of venom.The fluorescence resonance energy transfer (FRET) substrates, Abz-FRSSRQ and Abz-RPPGFSPFRQ, were used to study z et al. . Abz-RPPom SVSPs . Based oB. lanceolatus venom was investigated using pre-incubation protocols. Venom samples and antivenom were incubated at room temperature for 30 min, and the residual enzymatic activity was measured as described above. Initially, the amount of antivenom that inhibited 95% of the enzymatic activity of interest in B. jararaca venom (IC95) was determined. This amount of antivenom was then tested against B. lanceolatus venom. If complete inhibition was not achieved, then we determined the IC95 for the inhibition of this activity. It was not possible to determine the IC95 in the proteolytic assay because of interference by the antivenom. In this case, venom samples (5 µg or 2.5 µg depending on the substrate) were incubated with the maximum volume of bothropic antivenom (20 µL) that would not interfere with the measurements.The ability of bothropic antivenom to neutralize the PLA2, hyaluronidase, and proteolytic activities of v/v) heat-inactivated fetal bovine serum  and the following antibiotics; penicillin and streptomycin (100 IU/mL) . Cells were grown at 37 °C in humidified air with 5% CO2. To assess the cell viability upon venom exposure, the colorimetric MTT assay adapted from Mosmann [4 cells/well for HaCaT cells; 104 cells/well for EAhy926; 200 µL/well) and maintained in FBS-containing medium for 24 h and in medium without FBS for an additional 24 h, the cells were then exposed to the venom for 24, 48, and 72 h. DMEM without FBS was used as the control. At the end of the exposure, the supernatant was collected. Endothelial cells were incubated with MTT (0.5 mg/mL in 200 µL DMEM) for 4 h at 37 °C (5% CO2), whereas keratinocytes, which metabolize MTT more rapidly, were incubated with 0.83 mg/mL of MTT solution (60 µL/well) for 30 min. After removing the MTT solution, 100 µL of DMSO was added to each well. The absorbance was measured in a spectrophotometer  at 540 and 620 nm. The relative cell viability was calculated as follows:The human keratinocyte HaCaT cell line was obtained from the ‘Banco de Células do Rio de Janeiro’ . The cells were kept in Dulbecco’s modified eagle medium  containing 10%  to calculate the concentration able to impair cell viability by 50% (IC50).B. lanceolatus venom cytotoxicity for human cells was assessed by MTT assay, as described above. The venom samples were previously incubated with the antivenom for 30 min at room temperature and incubated with the cells for 24 h. B. jararaca venom was used as the positive control, and botulinum toxin antiserum was used as the negative control. Keratinocytes were incubated with a venom concentration of 4 µg/mL. Due to the difference in cytotoxic potential for endothelial cells between the two venoms, these cells were incubated with final concentrations of 125 µg/mL of B. lanceolatus venom and 10 µg/mL of B. jararaca venom.The inhibition of g and stored at −80 °C until use. The presence of cytokines and chemokines in the samples was assessed using ‘Cytometric Bead Arrays’ (CBA) with the ‘Human Chemokine Kit’, ‘Human Inflammatory Cytokine Kit’, and ‘Human Th1/Th2/Th17 Kit’ , following the manufacturer’s instructions.The cell culture supernatants collected from the cell viability assay were centrifuged at 400× B. lanceolatus venom, which could interfere with the analysis of cells supernatants, samples of cytokines and chemokines standard mixtures were incubated with the venom for 30 min at 37 °C in the presence or absence of proteases inhibitors . The samples were subsequently submitted to ‘Cytometric Bead Arrays’, following the manufacturer’s instructions. Concentrations of cytokines and chemokines were calculated in pg/mL of supernatant from the standard linear regression curves, using FCAP Array 3.0 software .In order to evaluate a potential direct cleavage of these cytokines and chemokines by 6 cells/mL) were incubated with 100 µg/mL of B. lanceolatus venom for 2 h at 37 °C under constant agitation. TNF-α  and saline solution were used as the positive and negative controls, respectively. The cells were centrifuged at 400× g and resuspended in FACS buffer (PBS containing 1% BSA and 0.01% NaN3). Then, cells  were incubated for 1 h at 4 °C, with PE-conjugated murine anti-tissue factor  antibodies . Alternatively, the cells were incubated  for 1 h at 4 °C with the following antibodies: murine monoclonal antibodies anti-intercellular adhesion molecule 1 , monoclonal murine antibodies donated by Dr Vaclav Horeji  against vascular cell adhesion molecule 1 (VCAM-1), and E-selectin (CD62E), diluted 1:50. After centrifugation  and removal of the supernatant, cells were resuspended in FACS buffer and incubated with secondary antibodies, diluted in the ratio 1:100 for 1 h at 4 °C, and protected from light . The samples incubated with isotype-matched, non-specific antibodies , or only with secondary antibodies were assayed in parallel as controls. All antibodies used were previously titrated. After washing, cells were resuspended in 200 µL of FACS buffer and analyzed by flow cytometry  using FACSDiva software . After gate definition, 5000 events per sample were analyzed and the fluorescence median was calculated.Endothelial cells . A


Sleep is thought to be involved in the regulation of synaptic plasticity in two ways: by enhancing local plastic processes underlying the consolidation of specific memories and by supporting global synaptic homeostasis. Here, we briefly summarize recent structural and functional studies examining sleep-associated changes in synaptic morphology and neural excitability. These studies point to a global down-scaling of synaptic strength across sleep while a subset of synapses increases in strength. Similarly, neuronal excitability on average decreases across sleep, whereas subsets of neurons increase firing rates across sleep. Whether synapse formation and excitability is down or upregulated across sleep appears to partly depend on the cell’s activity level during wakefulness. Processes of memory-specific upregulation of synapse formation and excitability are observed during slow wave sleep (SWS), whereas global downregulation resulting in elimination of synapses and decreased neural firing is linked to rapid eye movement sleep (REM sleep). Studies of the excitation/inhibition balance in cortical circuits suggest that both processes are connected to a specific inhibitory regulation of cortical principal neurons, characterized by an enhanced perisomatic inhibition via parvalbumin positive (PV+) cells, together with a release from dendritic inhibition by somatostatin positive (SOM+) cells. Such shift towards increased perisomatic inhibition of principal cells appears to be a general motif which underlies the plastic synaptic changes observed during sleep, regardless of whether towards up or downregulation. Research of the last decades has identified sleep as a key contributor to the continuing changes the brain undergoes while adapting to its environment. Basically, two different functions of sleep have been revealed in this context: on the one hand, sleep was shown to serve the consolidation of memories and, thus, to support synaptic plasticity in memory-specific circuitry mediating these consolidation processes  and rapid eye movement (REM) sleep in the regulation of synapse morphology. In mice, training of a motor task was followed by the reactivation of layer 5 pyramidal cells in motor cortex during SWS, which was accompanied by branch-specific spine formation on the apical dendrites of these cells  and through the balance of excitatory (glutamatergic) and inhibitory (GABAergic) synaptic inputs. Inhibitory interneurons do not only enable the homeostatic regulation of neuronal excitability, but are likewise involved in regulating information processing and neuronal plasticity underlying the encoding and formation of specific memories  cells in cortical layers 2 and 3. Although there is a great variety of interneurons in the cortex, these cells represent the great majority of such cells, with PV+ interneurons constituting ~40% and SOM+ interneurons constituting ~30% of all cortical interneurons  and concomitantly low levels of dendritic inhibition (mediated by SOM+ interneurons) which is similarly observed during REM sleep and slow oscillation-spindle events in SWS. During slow oscillation-spindle events such inhibitory dynamics occur in the presence of maximum pyramidal cell activity, which might effectively support the upregulation of select synapse formation through local dendritic calcium influx and, thereby, the consolidation of memories. During REM sleep, the same inhibitory constellation of pyramidal cells accompanied by lowest activity of pyramidal cells might support global synaptic down-scaling. In fact, a shift towards predominant PV+ cell mediated perisomatic inhibition of pyramidal cells is also observed during learning: in mice, after learning a motor task, new spines grew locally on the apical dendrites of layer 2/3 pyramidal cells while axonal boutons of SOM+ interneurons were eliminated. At the same time, PV+ interneurons showed increased numbers of axonal boutons  and simultaneously diminished dendritic inhibition of the cells (via SOM+ interneurons). This constellation of pyramidal cell inhibition might favor plasticity in both directions, towards synaptic growth and potentiation during SWS as well as towards synaptic pruning and diminished neural excitability during REM sleep.The underlying plastic processes during SWS and REM sleep, respectively, might share some basic mechanisms. Slow oscillation-spindle events represent the EEG signatures of memory related plasticity during SWS, whereas during REM sleep plasticity has been mainly linked to the pervasive EEG theta activity during this state. CaThe specific brain oscillations might actively cooperate to favor plasticity. PV+ interneurons are not only involved in the generation of spindles and theta oscillations, but the perisomatic inhibition these cells impose on pyramidal cells is highly modulated by these rhythms (e.g., Wulff et al., NN: conception and draft of the article; figure panels. AB: revision of the article. JB: conception and revision of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although trehalose profoundly attenuated mTORC1 signaling, trehalose-induced mTORC1 suppression was insufficient to activate autophagy in the absence of AMPK or GLUT8. Strikingly, transient, heterologous Tret1 overexpression reconstituted autophagic flux and AMPK signaling defects in GLUT8-deficient hepatocyte cultures. Together, these data suggest that cytoplasmic trehalose access is carrier-mediated, and that GLUT8 is a mammalian trehalose transporter required for hepatocyte trehalose-induced autophagy and signal transduction.Trehalose is a disaccharide demonstrated to mitigate disease burden in multiple murine neurodegenerative models. We recently revealed that trehalose rapidly induces hepatic autophagy and abrogates hepatic steatosis by inhibiting hexose transport via the SLC2A family of facilitative transporters. Prior studies, however, postulate that intracellular trehalose is sufficient to induce cellular autophagy. The objective of the current study was to identify the means by which trehalose accesses the hepatocyte cytoplasm, and define the distal signaling mechanisms by which trehalose induces autophagy. We provide gas chromatographic/mass spectrometric, fluorescence microscopic and radiolabeled uptake evidence that trehalose traverses the plasma membrane via SLC2A8 (GLUT8), a homolog of the trehalose transporter-1 (Tret1). Moreover, GLUT8-deficient hepatocytes and GLUT8-deficient mice exposed to trehalose resisted trehalose-induced AMP-activated protein kinase (AMPK) phosphorylation and autophagic induction  The role of cellular macroautophagy  in manifold disease processes is a target of intense investigation. Autophagy is a tightly regulated process that ensues as the end-manifestation of cellular stressors such as starvation, protein and fat accumulation1bona fide human therapeutic for metabolic disease is gaining tractionTrehalose is a disaccharide previously demonstrated to induce autophagy and mitigate multiple disease models through incompletely defined mechanisms345678slc2a genes) family of transmembrane-spanning facilitative carbohydrate transporters are ubiquitously expressed carriers that mediate transport of carbohydrates and other substrates down their concentration gradients across lipid bilayers11121314151617103The glucose transporter  with FITC-labeled trehalose. Standard immunofluorescence microscopy further demonstrated minimal fluorescence preceding labeling, with scattered trehalose uptake of variegated intensity 5-minutes post-FITC labeling .5 cells−1*min−1 was observed in trehalose-treated cultures (P < 0.001 versus untreated cultures), suggesting intact trehalose accumulation within the hepatocyte in the absence of mass changes,  prior to extensive washing, lysis and GC-MS analysis of soluble methanol extracts. An uptake rate of 10–12 nmol trehalose*5 × 1014C]-trehalose uptake is competitively inhibited by unlabeled trehalose 2114C]-trehalose uptake in glucose-starved HepG2 cells stably expressing scrambled (“control”) shRNA or GLUT8-directed (“GLUT8′) shRNA. qRT-PCR analysis of GLUT isoforms 1–12 revealed that shGLUT8 cells exhibit 85% knockdown of GLUT8 mRNA relative to control cultures without significant alterations in any other GLUT (3H]-2-deoxyglucose (2DG) in shGLUT8 cells was attenuated by 76% relative to shScramble controls  phosphorylation, which in turn activates ULK1 via phosphorylation at the AMP-activated protein kinase (AMPK) substrate site, Ser317 within 30 minutes post-trehalose stimulation in vitro and in vivo757)Prior work demonstrated that trehalose activates AMPK (ThrWT), from which GFP is cleaved upon autophagic activation29To test the role of ULK1 in trehalose-stimulated autophagy in a primary hepatocyte culture system, primary murine hepatocytes were isolated from mice constitutively expressing a transgene encoding a GFP-LC3 fusion protein (GFP-LC3172) and ULK1 (Ser317) phosphorylation and LC3B-II accumulation, concomitant with attenuated phosphorylation of mTORC1 targets, ribosomal S6 protein kinase (S6K phospho-Thr389) and 4E-binding protein-1  , followed by liver dissection and immunoblotting for mTORC1 targets  concomitant with decreased S6K (Thr389) and 4E-BP1 (Thr37/46) phosphorylation . Lysis and immunoblot analysis following trehalose stimulation demonstrated enhanced AMPK (Thr757), including at TSC2 (Ser1387 792 172) phosphorylation, and decreased S6K (Thr389) phosphorylation during the trehalose stimulation timecourse (1387) and raptor (Ser792) phosphorylation at AMPK crosstalk-specific nodes within the mTORC1 pathway were observed in response to trehalose.The above data suggested that ULK1 is required for trehalose-induced autophagic flux. Observations that AMPK and mTORC1 pathways were coordinately modulated by trehalose activation prompted us to assess whether AMPK→ mTORC1 crosstalk occurred at signaling nodes other than ULK1  was enhanced in KD-transfected cell cultures, and S6K (Thr389) phosphorylation remained relatively elevated throughout the 30-minute timecourse post-trehalose stimulation. Nevertheless, trehalose reduced mTORC1 signaling through S6K  mutant was sufficient to prevent trehalose-induced autophagic fluxough S6K .317), TSC2 (Ser1387) and raptor (Ser792) phosphorylation), and with diminished mTORC1 signaling (e.g. diminished ULK1 (Ser757), S6K (Thr389) and 4E-BP1 (Thr37/46) phosphorylation, 317), TSC2 (Ser1387) and raptor (Ser792) phosphorylation in response to trehalose were abrogated. However, despite AMPK knockdown and abrogation of distal AMPK signal transduction and AMPK-mTORC1 crosstalk, diminution of S6K (Thr389) and 4E-BP1 (Thr37/46) phosphorylation remained intact  virus, or with scrambled shRNA-encoding adenovirus (shScramble) prior to 100 mM trehalose stimulation in regular growth media. LC3B-II increased during the 30-minute trehalose stimulation timecourse in shScramble cells. This was associated with enhanced AMPK signal transduction (e.g increased ULK1 (Serd intact .37/46) and S6K (Thr389)) was similarly suppressed in both trehalose-treated control and GLUT8-deficient cultures, AMPK (Thr172) and the AMPK substrate TSC2 (Ser1387) phosphorylation were stimulated only in trehalose-treated shScramble cultures. Indeed, trehalose-induced AMPK signaling was blunted in trehalose-treated GLUT8-deficient cells  AMPK signaling is required for trehalose-induced hepatic autophagic flux independent of mTORC1 cross-talk, 2) GLUT8 is required for trehalose-induced AMPK activation and trehalose transport, and 3) intracellular trehalose is sufficient to induce cellular autophagy33Consistent with biochemical data in hepatocytes, treatment-blinded analysis of electron micrographs obtained from bafA1-treated shScramble and shGLUT8 hepatocytes treated with or without trehalose (1 hr) revealed an overabundance of lysosomal and multivesicular bodies . Overallin vivo GFP-LC3WT and GFP-LC38KO models. We first quantified trehalose-induced GFP cleavage from GFP-LC3 fusion protein in primary GFP-LC3WT and GFP-LC38KO hepatocytes incubated with or without trehalose  or with the mTORC1 inhibitor, Torin-1  as a positive control. GFP-specific immunoblotting revealed that both Torin-1 and trehalose stimulated GFP cleavage in GFP-LC3WT hepatocytes, whereas no GFP cleavage was detectable in GFP-LC3G8KO hepatocytes , followed by liver dissection, lysis and whole liver lysate immunoblotting. GFP-specific immunoblotting revealed that trehalose induced GFP cleavage from GFP-LC3 fusion protein in vivo, and this GFP cleavage response was attenuated in GFP-LC3G8KO mice (The function of GLUT8 in hepatic autophagic flux 8KO mice .in vitro triglyceride (TG) accumulation model. 500 μM FFA (48 hr) induced robust TG accumulation in shScramble hepatocytes, which was significantly mitigated by concomitant trehalose treatment  or Tret1-1, stimulated in the presence or absence of 100 mM trehalose and subjected to immunoblot analysis. GFP-LC3WT cultures responded to trehalose by increasing GFP-LC3 cleavage in a time-dependent manner, whereas GFP cleavage was attenuated in trehalose-treated GFP-LC38KO hepatocytes (WT and GFP-LC38KO hepatocyte cultures (172) and ULK1 (Ser317) phosphorylation in GFP-LC38KO hepatocyte cultures after 15 minutes trehalose incubation  in wild-type primary hepatocyte cultures . Primaryatocytes . In contcultures . Accordicubation . To quanatocytes . ImportaKnown for its capacity to mitigate neurodegenerative disease and non-alcoholic fatty liver disease by inducing autophagic flux, the precise mechanisms by which trehalose stimulates autophagy have been elusive. Data from our group and others310Kinetic data quantifying outside-in 2DG uptake are consistent with trehalose binding either outside the glucose binding pocket or within the glucose binding pocket in the inward open conformation2326Intracytoplasmic glucose transport inhibition of both GLUT family and non-GLUT homolog hexose transporters is well documented. The HIV protease inhibitors23383922sufficient to induce autophagic flux101146bona fide substrate sugars (e.g. glucose and fructose) suggesting a role as an influx amplitude modulator (e.g. a “sensor”). Moreover, acknowledging incomplete abrogation of acute trehalose transport in contexts of GLUT8 deletion, GLUT8 knockdown and various GLUT inhibitors, there are likely to be additional means by which trehalose enters the hepatocyte. Regardless, GLUT8 seems to mediate an important signal and/or delivery mechanism in the trehalose-induced cascade. Analogously, GLUT2 is considered to be a signaling “transceptor” via binding and docking of signaling molecules such as L-pyruvate kinase to its large intracytoplasmic loop  and 4E-BP1 . GFP-LC3Methods used to generate the HEK 293 cell lines selectively overexpressing human GLUT8 with concomitant knockdown of endogenous GLUT1 via shRNA overexpression were recently described1919HepG2 cultures were obtained from the American Type Culture Collection . Cultures were maintained in DMEM containing 4.5 g/L glucose and 5% FBS (e.g. “regular growth media”) through passage 20. To generate GLUT8-deficient HepG2 cell lines, HepG2 were transfected with lentivirus carrying the puromycin-resistance transgene and either scrambled shRNA-encoding vector  or GLUT8-directed shRNA-encoding vector . All stably transfected cell lines were maintained under constant 2 μg/mL puromycin selection. Relative GLUT8 mRNA and protein knockdown is demonstrated in WT and GFP-LC3G8KO were isolated as describedPrimary murine hepatocytes obtained from GFP-LC3cDNA and adenoviral constructs used for transient transfections were obtained as follows: AMPKα-shRNA, tret1-1 and tret1-2 were purchased from Vector Biolabs . Wild-type ULK1, was purchased from Applied Biological Materials .Accession numbers for sequences aligned using Clustal Omega were Tret1-1 (NP_725068.1), Tret1-2 (AAM68715.1), GLUT1 (EDL30474.1), GLUT2 (NP_112474.2), GLUT3 (NP_008862.1), GLUT4 (NP_001033.1), GLUT5 (AAA52570.1), GLUT6 (NP_060055.2), GLUT7 (NP_997303.2), GLUT8 (NP_062361.1), GLUT9 (NP_064425.2), GLUT10 (NP_110404.1), GLUT11 (NP_110434.3), GLUT12 (NP_660159.1), HMIT (NP_443117.3). The following program-default parameters were used: Output format – Clustal without numbers; no input sequence dealignment; MBED-like clustering iteration and MBED-like clustering guide-tree; 0 combined guide-tree iterations; “default” maximum guide tree and “default” maximum HMM iterations were used; aligned order output.HepG2 cells were plated on 35 mM glass-bottom dishes . All images were collected using a Zeiss  LSM510 META NLO multiphoton imaging system with 8000 nm excitation and a 40X lens. Preliminary experiments demonstrated closely related emission spectra when comparing cellular autofluorescence and tracer. To separate the tracer’s spectral signature we resorted to emission fingerprinting using tracer and unstained cells as reference. DMEM was replaced by 1 × PBS immediately before imaging to reduce background fluorescence. Emission spectra were acquired using beam splitter HFT-KP650, with an excitation wavelength of 800 nm.13C12 trehalose  was used as a sample extraction and derivatization internal standard. 5 nmol internal standard were added to the samples which were then extracted into 200 μl isopropanol:CH3CN:H2O. Samples were centrifuged and the supernatant was taken to dryness under nitrogen. Samples were derivatized using 100 ul N-Methyl-N-(Trimethysilyl) trifluoroacetamide (MSTFA): 10% pyridine in CH3CN (1:3) at room temperature overnight. Derivatized samples were analyzed using an Agilent 7890 A gas chromatograph interfaced to an Agilent 5975 C mass spectrometer. The GC column was a HP-5MS . A linear temperature gradient was used. The initial temperature (80 °C) was held for 2 min and increased to 300 °C at 10 °C/min. The temperature was held at 300 °C for 2 min. Samples were run by electron ionization (EI) mode and the source temperature, electron energy and emission current were 200 °C, 70 eV and 300 μA, respectively. The injector and transfer line temperatures were 250 °C. The ions monitored were m/z 361 and 13-Trehalose at m/z 367.Trehalose uptake quantification by gas chromatography and mass spectrometry was performed precisely as reported14C]-trehalose  uptake was determined at 37 °C. To adhere the cells to tissue culture plates, dishes were first pretreated with 25 μg/ml polyethyleneimine  in 150 mM NaCl for 20 min followed by the removal of the solution by vacuum. Cells were then plated at 2*105 cells/ml MEM in regular growth medium in 12-well plates and incubated overnight at 37 °C. Following a 30-minute incubation at 37 °C in glucose-free HEPES, pH 7.3, 1 μCi/mL [3H]-2-deoxy-D-glucose (2DG) uptake was measured at 37 °C in glucose-free HEPES, pH 7.3 for 180 min at 37 °C. Cultures were washed extensively in cold phosphate-buffered saline and lysed in 1% triton X-100.buffer and 80% of each lysate was subjected to liquid scintillation counting . Protein concentration was determined in the remaining lysate and total protein per well was used to normalize total radiolabeled trehalose uptake in each well.GLUT8OE and control cell lines were grown in MEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (regular growth medium). [5 cells per well in 12-well plates and maintained at 37 °C overnight in regular growth media until radiolabeled trehalose uptake was assayed. On the day of assay, cultures were incubated (15 minutes at 37 °C) in glucose-free HEPES, pH 7.3, in the presence or absence of unlabeled trehalose (50 mM), quercetin  in glucose-free HEPES, pH 7.3, at 37 °C  (#2535); phospho-ULK1 (Ser317) (#2535); phospho-ULK1 (Ser757) (#6888); ULK1 (#8054); phospho-p70 S6K (Thr389) (#9234), p70 S6K ; pTSC2 (Ser1387) (#5584); pRAPTOR (Ser792) (#2083); phospho-4E-BP1 (Thr37/46) (#2855); 4E-BP1 (#9644); HA-tag 6E2 (#2367); GFP (#2956); Vinculin (#13901); Actin (#4970); GAPDH (#5174); LC3B antiserum was obtained from Novus Biologicals  (#NB-100-2200).All immunoblot experiments depicted represent a minimum of three independent experiments with n = 2–3 per experiment. Immunoblotting was performed as describedFor autophagic flux assays, primary hepatocytes and HepG2 cell lines were maintained in regular growth media prior to experiment. Cultures were then incubated in the presence or absence of dimethyl sulfoxide (DMSO) or 100 nM Bafilomycin A1  with or without 100 mM trehalose for 1 hour. Cultures were lysed and subjected to immunoblot analysis as above.in vivo autophagic flux assays, 8-week old GFP-LC3WT and GFP-LC3G8KO mice were gavaged with saline or with 6 g/kg trehalose dissolved in 0.9% NaCl. Mice were killed two hours after orogastric gavage, and livers were rapidly dissected and snap-frozen in liquid nitrogen prior to homogenization in ice-cold RIPA lysis buffer and immunoblot analysis.For in vivo signaling assays, 8-week old wild-type mice were subjected to orogastric gavage with 6 g/kg trehalose dissolved in saline. Mice were killed 0.5 hours after gavage, and livers were rapidly dissected and snap frozen in liquid nitrogen prior to homogenization in ice-cold RIPA lysis buffer and immunoblot analysis. Control mice were gavaged with saline alone and killed 0.5 hours post-gavage.For 20 prior to en bloc staining with 1% aqueous uranyl acetate  for 1 hr. Following several rinses in ultrapure H20, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT7 ultramicrotome , stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope  equipped with an AMT 8 megapixel digital camera and AMT Image Capture Engine V602 software .Ultrastructural analysis was as previously performedA genotype- and treatment-blind expert electron microscopist calculated quantity and organelle-to-cytosolic area ratios by NIH imageJ 1.47 v analysis software. A lysosome/MVB was defined as any circumscribed, electron-dense intracytosolic structure containing variegated intraorganellar densities.in vitro biochemical analysis, cultured hepatocytes were grown in 6-well plates and equilibrated 24 hr prior to treatment and assay as previously describedFor qPCR was performed with primer sequences and method precisely as described19post hoc correction where multiple comparisons were made. Data shown are as mean ± SEM. Specific statistical tests applied are noted in the Figure Legends.Data were analyzed using GraphPad Prism version 6.0. P < 0.05 was defined as statistically significant after How to cite this article: Mayer, A. L. et al. SLC2A8 (GLUT8) is a mammalian trehalose transporter required for trehalose-induced autophagy. Sci. Rep.6, 38586; doi: 10.1038/srep38586 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Approximately 60% of perioperative anaphylactic reactions are thought to be immunoglobulin IgE mediated, whereas 40% are thought to be non-IgE mediated hypersensitivity reactions (both considered non-dose-related type B adverse drug reactions). In both cases, symptoms are elicited by mast cell degranulation. Also, pharmacological reactions to drugs  may sometimes mimic symptoms triggered by mast cell degranulation. In case of hypotension, bronchospasm, or urticarial rash due to mast cell degranulation, identification of the responsible mechanism is complicated. However, determination of the type of the underlying adverse drug reaction is of paramount interest for the decision of whether the culprit drug may be re-administered. Neuromuscular blocking agents (NMBA) are among the most frequent cause of perioperative anaphylaxis. Recently, it has been shown that NMBA may activate mast cells independently from IgE antibodies via the human Mas-related G-protein-coupled receptor member X2 (MRGPRX2). In light of this new insight into the patho-mechanism of pseudo-allergic adverse drug reactions, in which as drug-receptor interaction results in anaphylaxis like symptoms, we critically reviewed the literature on NMBA-induced perioperative anaphylaxis. We challenge the dogma that NMBA mainly cause IgE-mediated anaphylaxis via an IgE-mediated mechanism, which is based on studies that consider positive skin test to be specific for IgE-mediated hypersensitivity. Finally, we discuss the question whether MRGPRX2 mediated pseudo-allergic reactions should be re-classified as type A adverse reactions.  The term “anaphylaxis” was previously used for IgE-mediated reactions only, whereas the term pseudo-allergic (or anaphylactoid) was used for similar clinical reactions, which occur via a non-IgE-dependent mechanism ,2. Both The incidence of anaphylactic reactions during general anesthesia has been estimated at 1/4000 to 1/25,000, and about 1/5000 for Neuromuscular blocking agents (NMBA) . The incSkin tests have so far been considered to be specific for IgE-mediated hypersensitivity, whereas pseudo-allergic reactions have been considered to yield negative results. However, the term “pseudo-allergy” has been too frequently used to describe any kind of immediate-type allergic-like reaction that is not IgE-mediated . This isThe recent identification of the Mas-related G-protein-coupled receptor member X2 (MRGPRX2), now allows for better classification of different types of non-IgE mediated allergic reactions . AccordiA better understanding of the underlying mechanism is of clinical relevance for deciding whether the culprit drug may be re-administered or has to be avoided. According to the still generally accepted and widely used classification of adverse drug reactions (ADR) that was developed in the 1970s , ADR canType A ADR are pharmacological adverse reactions, which typically do not involve the immune system, and are therefore predictable. This means that the drug may be re-administered with a lower dose or reduced speed without re-eliciting the same adverse reaction. Examples of type A ADR are diarrhea after antibiotics, or gastric ulcers following prolonged NSAID treatment. Also, toxic reactions are typical type A reactions. The reason why some individuals suffer from type A ADR, whereas others do not, is often unknown. Identifying type A ADR is important, since this will have implications for the future management of the patient. An incorrect labeling as “allergic” may result in withholding optimal treatment for a subsequent illness, which can be deleterious in infectious disease and in anesthetic procedures .Type B reactions are ADR that are not predictable. They mainly include hypersensitivity reactions, which are mediated by the immune system and occur in a susceptible subgroup of patients. This susceptibility is classically considered to be due to environmental factors, in particular previous exposure during which the adaptive immune system develops hypersensitivity. However, it has been increasingly noted that some type B reactions that are highly predictable were not due to environmental factors, but to the genetic profile of the patient, linked to particular human leukocyte antigens (HLA). The reason for this is that the antigen presentation to T-cells is dependent on specific HLA haplotypes, for example in abacavir, carbamazepine and allopurinol ADR, which seem to be dose-dependent ,19.Mast cells can release preformed mediators , newly formed lipid mediators  and cytokines  during anaphylactic reactions. The clinical picture of IgE-mediated hypersensitivity, including hypotension, bronchospasm and urticaria, is mainly caused by these mediators, in particular histamine. However, mast cell degranulation can be also elicited by other mechanisms than IgE crosslinking, as these cells carry a variety of other receptors on their surface that can induce degranulation . Mast cePatients with chronic spontaneous urticaria often suffer from flares caused rather by a non-IgE mediated mechanism than by IgE-mediated allergy . DiffereThe combination of different co-factors is thought to have a cumulative effect. This means that a single co-factor alone does not necessarily lead to a clinically apparent mast cell degranulation, but the simultaneous presence of several co-factors have the potential to trigger symptoms mediated by histamine release in these patients . NeverthTherapeutically used monoclonal antibodies, as well as NMBA, have been described to induce anaphylactic reactions more frequently upon first exposure than subsequent re-exposure. The mechanism of these reactions remains unclear because previous sensitization to the drug is unlikely . For decAccording to the current concept, a possible explanation for an IgE-mediated reaction upon first exposure could be cross-reactivity among different drugs, as shown in patients reacting to NMBA who have been previously exposed to pholcodine. Pholcodine is a non-prescription antitussive drug that contains a substituted ammonium ion . Epidemiological studies have shown a correlation between the intake of pholcodine and the incidence of NMBA anaphylaxis. This is considered to be due to common substituted ammonium ions (tertiary and/or quaternary ammonium (QA) structures) which are found in a wide variety of chemical structures, including NMBA and pholcodine . The maiMRGPRX2 gene have already been reported [MRGPRX2 gene may be associated with an increased risk for pseudo-allergic ADR.Whereas severe cutaneous ADR associated with particular HLA-types are mainly delayed ADR, the recent identification of the MRGPRX2 receptor has introduced the concept of a genetical, possibly innate, predisposition to develop immediate-type pseudo-allergic ADR. Although environmental influence by epigenetic modification is certainly possible, genetic variants in the reported ,35. Thisreported . Three oNMBA are considered to be responsible for the majority of IgE-mediated reactions occurring during general anesthesia, followed by latex, antibiotics, hypnotics and opioids ,38,39. OSkin tests have been so far considered to have a high specificity for IgE-mediated reactions and to be negative in non-IgE mediated, immediate type reactions . In NMBAp < 0.001) [Moreover, patients with immediate type hypersensitivity against antibiotics, confirmed by positive skin test, have an increased risk for positive allergy skin tests for NMBA, independently from their atopic status . This su< 0.001) . This su< 0.001) ,52. Howe< 0.001) . Also, i< 0.001) .Tryptase and histamine release are not specific for an IgE-mediated reaction ,61. TrypAs to other in vitro diagnostic tools, the value of specific IgE measurements for rocuronium, QA and morphine remains a matter of debate ,43. AlthBasophil activation test (BAT) appears to be more reliable than measurement of specific IgE ,44. If tAtracurium ADR represents an exception among NMBA as the responsible epitope seems to be different than the ammonium ion in IgE-mediated reactions ,70. MoreThe recent discovery of the MRGPRX2 receptor gave important new insights into the patho-mechanism of pseudo-allergic ADR and questions a few old dogmas, in particular that skin tests are supposed to be specific for IgE-mediated reactions. Therefore, data in the literature reporting ADR based on positive skin tests have to be critically reviewed. Skin testing is not a formal proof for IgE-mediated reactions from the adaptive immune system. A positive skin test can mirror alternative activation pathways of cutaneous mast cell activation. Especially the assumption that anaphylaxis due to NMBA could be mainly IgE-mediated has to be questioned. On one hand the reported decreased incidence of anaphylaxis due to NMBA in Norway after pholcodine withdrawal and the often overlapping results of skin test and BAT, might argue in favor of an IgE-mediated reaction. On the other hand, as discussed in the previous chapters, these studies have several limitations and possible biases, and there are several facts that argue in favor of a pseudo-allergic mechanism: (1) most reactions occur upon first exposure, (2) there is a high rate of cross-sensitization to several NMBA, (3) specific IgE is present in non-allergic individuals without a clear causal link to the anaphylactic reaction, and (4) there is an increased risk of a second anaphylactic reactions to NMBA in skin test-negative patients with previous reaction to another NMBA compared to patients without previous reaction . There is little doubt that IgE against the substituted ammonium ion might be induced by exposure to different chemical substances, i.e., pholcodine, that in turn puts the patient at risk for IgE-mediated anaphylaxis to succinylcholine due to cross-reactivity. However, the recent identification of the MRGPRX2 receptor indicates that other NMBA might induce mainly pseudo-allergic reactions. As mast cell degranulation mediated by MRGPRX2 was found to be dose-dependent and highly predictable , we propThe hypothesis that most NMBA may cause pseudo-allergic rather than IgE-mediated reactions raises several new questions. (1) Supposing that NMBA reactions are due to non-IgE-mediated mast cell degranulation, why do some individuals react more easily than others? (2) Does the expression of MRGPRX2 vary in a single individual over time? Studies addressing the genetic variants and epigenetic modifications of MRGPRX2 are urgently needed to provide answers to these questions and gain more insight in the field of pseudo-allergic reactions.


Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that interact trans-synaptically with neurexins to mediate synapse development and function. NLGN2 is only at inhibitory synapses while NLGN3 is at both excitatory and inhibitory synapses. We found that NLGN3 function at inhibitory synapses in rat CA1 depends on the presence of NLGN2 and identified a domain in the extracellular region that accounted for this functional difference between NLGN2 and 3 specifically at inhibitory synapses. We further show that the presence of a cytoplasmic tail (c-tail) is indispensible, and identified two domains in the c-tail that are necessary for NLGN function at inhibitory synapses. These domains point to a gephyrin-dependent mechanism that is disrupted by an autism-associated mutation at R705 and a gephyrin-independent mechanism reliant on a putative phosphorylation site at S714. Our work highlights unique and separate roles for the extracellular and intracellular regions in specifying and carrying out NLGN function respectively.DOI:http://dx.doi.org/10.7554/eLife.19236.001 Proper balance between inhibitory and excitatory connections is important for proper functioning of neuronal circuits . Cell adNeuroligins are a family of postsynaptic cell adhesion molecules that interact trans-synaptically with their corresponding presynaptic neurexin binding partners to mediate proper synaptic function . Rats exA receptors at inhibitory synapses  background to study the effects of our constructs in isolation . ExpressWe constructed further chimeric constructs to determine where in the extracellular region this difference between NLGN2 and 3 resides and if we could effectively confer NLGN2 function onto NLGN3 by transplanting a critical domain. There are various components located in the extracellular region which have previously been suggested to be important for NLGN function . One keyStarting from the proximal region closest to the transmembrane region, we progressively added back regions of NLGN2 onto the NLGN3 extracellular region while keeping the NLGN2 c-tail intact. We first inserted a region of NLGN2 up to the 623rd amino acid that contains substantial differences between it and NLGN3 . NeitherSince the c-tails of the NLGNs share many similar domains , and ourTo determine what domain(s) in the c-tail are necessary for NLGN function at inhibitory synapses, we made a series of truncations to the c-tail of NLGN2, deleting previously identified domains proposed to be important for its function  and found that deletion of a 35 amino acid stretch from that point (NLGN2Δ98–133) resulted in the loss of enhancement of inhibitory responses . FurtherWe next sought to identify any candidate residues that could be important within this domain. We were informed by our previous results that any NLGN c-tail would be able to function at inhibitory synapses and thus looked for residues that were shared among all the NLGNs. We focused on two sites, a residue at R705 linked to an autism mutation previously found in NLGN4 that inhibited its function at excitatory synapses , and a pIndividual mutations at these sites, either by expressing the phospho-null (NLGN2-S714A) , phosphoOur finding that there are two distinct requirements in the c-tail for NLGN function at inhibitory synapses could explain why we were unable to see an effect earlier with our gephyrin mutants if there are both gephyrin-dependent and gephyrin-independent mechanisms for NLGN function at inhibitory synapses. To determine whether this was the case, we made two more double mutants, combining either the autism mutation or phospho-null mutation with the point mutation in the gephyrin-binding domain that inhibits gephyrin binding (NLGN2-R705C-Y770A or NLGN2-S714A-Y770A). Since the Y770A mutation inherently blocks the gephyrin-dependent pathway, we wanted to know which of our mutations when combined with this manipulation would reveal the presence of a gephyrin-independent pathway. We expect that if either the R705C or S714A point mutations were in a gephyrin-independent pathway, we would observe reduced enhancement of inhibitory currents when combined with the Y770A point mutation that blocks gephyrin binding.The combined autism mutation and gephyrin-binding double point mutant (NLGN2-R705C-Y770A) exhibited enhancement comparable to full-length NLGN2 , while tOur results have uncovered the critical features of NLGN2 for proper inhibitory synaptic function and reveal domains in both the extracellular and intracellular regions of the NLGNs that are necessary for their function specifically at inhibitory synapses. Our systematic dissection of the relative contributions of NLGN2 and 3 to inhibitory synaptic function through targeted knockdown of one or the other revealed that while NLGN3 appears to be a dispensable component, NLGN2 is not and that, in fact, NLGN3 function depends on the presence of NLGN2. Further analysis via expression of chimeric NLGN2/3 constructs on a reduced endogenous NLGN background revealed that this difference between NLGN2 and 3 function at inhibitory synapses is due to differences within a domain in the extracellular region corresponding to a site important for neurexin binding, as well as a proximal uncharacterized region.While this result seemed to suggest that the intracellular region is not important, our results clearly show that, although the intracellular region of any NLGN is sufficient for function at inhibitory synapses, if paired with the extracellular region of NLGN2, a c-tail is very much required. Thus, a NLGN2 truncation lacking most of the intracellular region failed to enhance inhibitory responses while maintaining a normal ability to traffic to the surface. Further refinement identified two distinct regions of the NLGN c-tail corresponding to gephyrin-dependent and gephyrin-independent mechanisms that enable proper function at inhibitory synapses. These results suggest that while the extracellular region is important for specifying function at an inhibitory or excitatory synapse, the intracellular region is critical for carrying out that function at both an inhibitory or excitatory synapse.Due to the heterogeneous nature of inhibitory synapses, one question that arises is whether our observations are relevant only for particular subsets of inhibitory synapses. Previous work using simultaneous recording from connected neurons showed that NLGN2 knockout animals specifically displayed a deficit in PV but not SOM inputs onto hippocampal pyramidal neurons . A similOne limitation of our study is that the results we observe may not occur universally among all brain regions. Indeed, recent work has shown that within the cerebellum the basket/stellate cell synapses onto Purkinje cells exhibit a decrease in mIPSC frequency but not amplitude with knock-out of just NLGN2 while the double knockout of NLGN2 and 3 exhibit a much more dramatic impairment in both mIPSC frequency and amplitude . In addiWhat might be the role of NLGN3 at inhibitory synapses since our results show that its absence does not affect inhibitory transmission, and that NLGN3 requires the presence of NLGN2 to function at inhibitory synapses? One possible explanation is that NLGN3 functions as an available mechanism to quickly enhance existing inhibitory synapses. Previous work looking at overexpression of NLGN2 or 3 in cerebral cortical neurons noticed that the majority of NLGN3 was not localized synaptically while the majority of NLGN2 was synaptically located . From thThis hypothesis is further strengthened by our result showing that transplanting a domain of the NLGN2 extracellular region corresponding to a key area associated with neurexin binding fully enabled NLGN3 to function at inhibitory synapses by itself. It suggests that NLGN3 is unable to bind to the corresponding neurexin partner at inhibitory synapses alone and that it needs to heterodimerize with NLGN2 in order to function. Previous work has already shown that different neurexin isoforms are expressed at different types of synapses, and that the neurexin isoform expressed at inhibitory synapses can bind to NLGN2 but not NLGN1 . Thus, iOur results showing that a chimeric NLGN3 extracellular region with a NLGN2 intracellular region was unable to potentiate inhibitory responses but could still enhance excitatory responses indicates that there are differential requirements for NLGN function at excitatory versus inhibitory synapses and that the extracellular region is critical for mediating this specificity. This is not surprising given that neurexin binding occurs at the extracellular region and that different neurexin isoforms are present preferentially at particular synapses. However, there could be other possible interactors that help define this functional specificity. Previous work has shown that NMDA receptors are able to bind to the extracellular region of NLGN1 but not NLGN2 or 3 . In addiOur results with chimeric constructs show that any NLGN c-tail is able to function at inhibitory synapses if linked with a NLGN2 extracellular region, and that the NLGN2 c-tail is able to function at excitatory synapses when linked with the NLGN3 extracellular region. This suggests that there is no specificity within the c-tail itself to determine NLGN function at either an excitatory or inhibitory synapse. These results are consistent with previous work showing that overexpression on a wild-type background of chimeric constructs containing NLGN1 extracellular linked to NLGN2 intracellular, or NLGN2 extracellular linked to NLGN1 intracellular, is fully capable of enhancing excitatory and inhibitory currents respectively . Our worWhile the c-tail is the toolbox that contains all the functional residues necessary to perform at either an excitatory or inhibitory synapse, it is important to note that the particular type of synapse determines which residues will be utilized. Indeed, prior work has shown that phosphorylation at the gephyrin binding domain enables preferential recruitment of PSD-95 versus gephyrin to the intracellular region of NLGN1 . In addiA receptors but no change in gephyrin clustering, suggesting there might be dystrophin-dependent and gephyrin-independent mechanisms for clustering of specific GABAA receptor subtypes . Supernatant was collected 40 hr later, filtered, and concentrated using PEG-it Virus Precipitation Solution . Resulting pellet was resuspended in Opti-mem, flash-frozen, and stored at –80°C.Primary rat hippocampal dissociated neurons were prepared at E18.5 and infected with lentivirus expressing a Gephyrin-miR, NLGN2miR, or control GFP construct at DIV 4–7. Neurons were harvested at DIV17-18 by lysis and reverse transcribed to synthesize cDNA using a Power SYBR Green Cells-to-CT kit . Amplification of cDNA by real-time PCR was quantified using SYBR Green with the following sequence specific primers: Neuroligin 2, forward-CATTCAGAAGGGCTGTTCCA, reverse- GTCTTCCCGGGAGCTAGTAG; Gephryin, forward- GGGAATGAGCTACTAAATCCTG, reverse- TGATACCCTCATTCAAGGCA.Primary rat hippocampal dissociated neurons were prepared at E18.5 and infected with lentivirus expressing Gephyrin-miR construct at DIV 4–7. Neurons were harvested at DIV17-18 in Tris-buffered saline  plus 0.5% Triton-X and protease inhibitor mix . After incubation at 4°C for 30 min, cell lysates were centrifuged for 15 min at 12000 g. Proteins were resolved by SDS-PAGE and analyzed by western blot using antibodies against gephyrin  and actin .2, 1.5 mM CaCl2, adjusted to pH 7.3 with NaOH) then incubated with anti-HA antibody  for 20 mins in the cold. Cells were then washed with HEPES external buffer and fixed in 4% paraformaldehyde in 4% PBS for 15 min room temperature. Cells were then washed with PBS and blocked in 10% goat serum in PBS with 0.1% Triton X-100. Cells were then washed with PBS with 0.1% Triton X-100 and labeled with Alexa 546- conjugated anti-rabbit secondary antibody  in PBS with 0.1% Triton X-100 with 2.5% goat serum and mounted with SlowFade Gold Antifade Mountant . Neurons were imaged with a 100x objective on a Zeiss LSM 510 confocal microscope. For analysis, images were collected and quantified by normalizing the fluorescence intensity of surface-expressed NLGN2 (or NLGN2Δ133) with the fluorescence intensity of GFP in each cell using ImageJ . All data analysis was done blinded to experimental conditions.Primary rat hippocampal dissociated neurons were prepared at E18.5 and transfected using Lipofectamine 2000 with pCaggs NLmiRs-GFP and either pCaggs-NLGN2 or pCaggs-NLGN2Δ133. 7 days later cells were prepped for immunostaining for surface and intracellular markers. Cells were first washed with cold HEPES external buffer . When biolistically expressing two plasmids, gold particles were coated with equal amounts of each plasmid and plasmids always expressed different fluorescent markers. Observed frequency of coexpression was nearly 100%. Slices were maintained at 34°C with media changes every other day.Sparse biolistic transfections of organotypic slice cultures were performed 1 day after culturing as previously described . Brieflyn) reported in the figure legends refer to the number of pairs. Pyramidal neurons were identified by morphology and location. To ensure stable recording, membrane holding current, input resistance, and pipette series resistance were monitored throughout recording. All recordings were made at 20–25°C using glass patch electrodes filled with an internal solution consisting of 135 mM CsMeSO4, 8 mM NaCl, 10 mM HEPES, 0.3 mM EGTA, 4 mM Mg-ATP, 0.3 mM Na-GTP, 5 mM QX-314, and 0.1 mM spermine and an external solution containing 119 mM NaCl, 2.5 mM KCl, 4 mM MgSO4, 4 mM CaCl2, 1 mM NaH2PO4, 26.2 mM NaHCO3 and 11 mM glucose bubbled continuously with 95% O2 and 5% CO2. Recordings of IPSCs were made in the presence of D-APV  and NBQX  to block NMDA and AMPA-mediated currents respectively. Recordings of excitatory current were made in the presence of picrotoxin  to block inhibitory currents and a small (50 nM) amount of NBQX to reduce epileptiform activity at −70 mV (AMPA). AMPAR-mediated currents were measured at the peak of the current.Recordings were performed at 7–10 DIV after 6–9 days of expression. Dual whole-cell recordings of CA1 pyramidal neurons were done by simultaneously recording responses from a fluorescent transfected neuron and a neighboring untransfected control neuron. Synaptic responses were evoked by stimulating with a monopolar glass electrode filled with aCSF in stratum radiatum of CA1. Typically each pair of neurons is from a separate slice, whereas on rare occasions two pairs may come from one slice. For all paired recordings, the number of experiments , GraphPad Prism  and Excel . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Gary Westbrook (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your article "Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses" for consideration by Summary:eLife. The findings provide novel insight into the relevance and function of previously unidentified regions of Nlgn at inhibitory synapses.Nguyen et al. report the roles of distinct extracellular and intracellular domains of neuroligins (NLGNs) at inhibitory synapses. The authors first find that NLGN2 but not NLGN3 is required for inhibitory synaptic transmission in cultured neurons. In follow-up experiments, the authors mainly use molecular replacement approaches to map out critical regions associated with NLGN2-dependent regulation of inhibitory transmission. The authors identify two domains in the extracellular region of NLGN2 (splice A site domain and an uncharacterized adjacent domain) that confer NLGN function at inhibitory synapses. Intracellularly, the authors find that a c-tail from NLGN1/2/3 is required for NLGN function at inhibitory synapses. Furthermore, they find that previously identified cytoplasmic domains including the PDZ-binding motif and gephyrin-binding domain are not required for NLGN2 function, but that an upstream region (δ 117-133) and two residues within this region, one autism-related (R705) and the other phosphorylatable (S714), are important for NLGN2 function. Interestingly, the authors find that these residues together with a residue in the gephyrin-binding domain (Y770) are collectively important for NLGN2 function; combinations of residues both in the gephyrin-dependent and -independent pathways are important to fully support NLGN2 function. This is an interesting, well-performed and thorough study, and a well-written manuscript that is appropriate for Essential revisions:1) This study used many constructs carrying deletions and mutations of NLGN2 and NLGN3. Ideally, their total and surface expression levels should be tested using biochemical (i.e. biotinylation) or cell biological methods, although some were tested by surface staining . The ext2) The authors identify the proximal part of the extracellular region of Nlgn2 as required for the functional differences between Nlgn2 and Nlgn3 . The stu3) A challenge in dissecting the contribution of the Nlgn c-tail to inhibitory synaptic function is that the downstream signaling pathways are largely unknown. Although their experiments suggest that the R705C mutation acts via a gephyrin-dependent pathway, the molecular basis of this is less clear. The authors previously showed that in Nlgn4, this mutation impairs phosphorylation of neighboring T707 by PKC . T707 is absent in Nlgn1-3, suggesting an alternative mechanism. Biochemical analysis of gephryin binding in R705C mutants would help to clarify how this mutation might affect the gephyrin-dependent pathway. In these experiments, the R705C mutant can be compared to S714A mutant, which would not be expected to affect gephyrin binding. In addition, is it possible to check that the phosphorylation of neighboring S714 is not affected by the R705C mutation?4) The authors propose that S714 modulates a gephyrin-independent pathway, based on their findings that the S714A single mutation does not impair inhibitory responses, whereas the S714A-Y770A double mutant does. The authors could functionally test the effect of the single S714A mutation by expressing the single Nlgn2-S714A mutant in an Nlgn1-3 miR+/gephyrin-miR+ background, which would be expected to fail to enhance inhibitory responses.5) The main approach employed in the current study is electrophysiological recordings of rat organotypic slice cultured neurons. To corroborate the key findings, some of the phenotypes  should be confirmed by independent immunocytochemical approaches. […] Essential revisions:1) This study used many constructs carrying deletions and mutations of NLGN2 and NLGN3. Ideally, their total and surface expression levels should be tested using biochemical (i.e. biotinylation) or cell biological methods, although some were tested by surface staining . The extWe have done surface staining for the HA-tag construct in two of our most critical double point mutations which exhibited impaired inhibitory synaptic transmission . As show2) The authors identify the proximal part of the extracellular region of Nlgn2 as required for the functional differences between Nlgn2 and Nlgn3 . The stuThere are multiple hurdles underlying the suggested experiment to express different variants of neurexins to test binding of NLGN3. First, there is no readily available library of all the neurexin variants expressed at inhibitory synapses from which we can obtain constructs to overexpress. Even if there were, there are a plethora of neurexin variants and combinations, including splice isoforms, which could be possibly expressed, making it unwieldy to perform this experiment.However, we have attempted to characterize our critical extracellular domain further by looking at the crystal structure of NLGN2 which has previously been resolved [1]. We aligned the structure of NLGN2 (3BL8) to the crystal structure of the NLGN1/Neurexin1β complex [2] (3BIW) , to apprWe were unable to characterize and align NLGN3 to our structural analysis due to the lack of a crystal structure and the inability of structure prediction software to fully predict the structure of our protein due to the highly disordered region around splice site A.We do not think that our identified critical domain in the proximal region is required to allow NLGN3 to associate with NLGN2 in heteromers since wild-type NLGN3 already associates with NLGN2, as shown in the blot . In this3) A challenge in dissecting the contribution of the Nlgn c-tail to inhibitory synaptic function is that the downstream signaling pathways are largely unknown. Although their experiments suggest that the R705C mutation acts via a gephyrin-dependent pathway, the molecular basis of this is less clear. The authors previously showed that in Nlgn4, this mutation impairs phosphorylation of neighboring T707 by PKC . T707 is absent in Nlgn1-3, suggesting an alternative mechanism. Biochemical analysis of gephryin binding in R705C mutants would help to clarify how this mutation might affect the gephyrin-dependent pathway. In these experiments, the R705C mutant can be compared to S714A mutant, which would not be expected to affect gephyrin binding. In addition, is it possible to check that the phosphorylation of neighboring S714 is not affected by the R705C mutation?We transfected HEK cells with gephyrin and either NLGN2, NLGN2-R705C, or NLGN2-S714A. We then immunoprecipitated for the HA-tagged NLGN2 and blotted for gephyrin to see if we could detect any differences in gephyrin binding to the different point mutants. As shown , we coulUnfortunately, we are not able to check whether phosphorylation of neighboring S714 is affected by the R705C mutation due to lack of a phospho-specific antibody for S714.4) The authors propose that S714 modulates a gephyrin-independent pathway, based on their findings that the S714A single mutation does not impair inhibitory responses, whereas the S714A-Y770A double mutant does. The authors could functionally test the effect of the single S714A mutation by expressing the single Nlgn2-S714A mutant in an Nlgn1-3 miR+/gephyrin-miR+ background, which would be expected to fail to enhance inhibitory responses.We thank the reviewer for this suggestion. We have performed this experiment and indeed expression of NLGN2-S714A along with NLGN1-3miR + Gephyrin-miR fails to enhance inhibitory responses.5) The main approach employed in the current study is electrophysiological recordings of rat organotypic slice cultured neurons. To corroborate the key findings, some of the phenotypes  should be confirmed by independent immunocytochemical approaches.With due respect, for multiple reasons we would argue that immunocytochemical approaches would not significantly enhance the impact of our study. First, previous work in our lab has shown that neurons expressing point mutants which affect the function of a NLGN, as determined by electrophysiological recordings, could display normal presynaptic specializations [4]. In addition, it is difficult to determine the effects our mutants have on postsynaptic specializations since there is no structural approximation of inhibitory synapse number equivalent to the spine density used for excitatory synapses. Indeed, the postsynaptic marker that is used most often for inhibitory synapses is gephyrin, which would not be ideal to use for our purposes.References:1) Koehnke, J. et al. Crystal structure of the extracellular cholinesterase-like domain from neuroligin-2. Proc Natl Acad Sci U S A 105, 1873-1878, doi:10.1073/pnas.0711701105 (2008).2) Arac, D. et al. Structures of neuroligin-1 and the neuroligin-1/neurexin-1 beta complex reveal specific protein-protein and protein-Ca2+ interactions. Neuron 56, 992-1003, doi:10.1016/j.neuron.2007.12.002 (2007).3) Kang, Y. et al. A combined transgenic proteomic analysis and regulated trafficking of neuroligin-2. J Biol Chem 289, 29350-29364, doi:10.1074/jbc.M114.549279 (2014).4) Shipman, S. L. & Nicoll, R. A. A subtype-specific function for the extracellular domain of neuroligin 1 in hippocampal LTP. Neuron 76, 309-316, doi:10.1016/j.neuron.2012.07.024 (2012).