The transient receptor potential canonical 1 (TRPC1) channel has been shown to contribute for agonist-stimulated Ca2+ influx in some types of cells. However, the relationships among IL-4, TRPC1 and mucus overproduction in bronchial epithelial cells (BECs) in asthma are poorly understood.Mucus overproduction is an important feature of asthma. Interleukin (IL)-4 is required for allergen-induced airway inflammation and mucus production. MUC5AC gene expression is regulated by transcript factors NF-κB. The intracellular Ca2+]i was measured after loading with Fura-2. NF-κB activities were measured by an ELISA-based assay. MUC5AC mRNA and protein levels were detected by real-time quantitative RT-PCR, ELISA analysis and immunofluorescence staining respectively.BECs were isolated from large bronchial airway of rats and used as cell model. To present changes of lipid raft, caveolin-1 and TRPC1, immunofluorescence staining and sucrose gradient centrifugation were performed. [Ca2+ influx in BECs, and this was blocked by a Ca2+ influx inhibitor (2-APB). 2-APB also prevented MUC5AC protein synthesis induced by IL-4. Depletion of extracellular Ca2+ resulted in partial decrease in expression of MUC5AC in IL-4 treated cells. NF-κB rather than STAT6 activation mediated IL-4-induced MUC5AC protein synthesis. Then the mechanism of Ca2+ influx was investigated. Immunofluorescence staining and sucrose gradient centrifugation revealed that caveolin-1-containing lipid rafts aggregation was involved in TRPC1 activation and Ca2+ influx in BECs. Lastly, the data revealed that blocking lipid rafts aggregation exactly prevented Ca2+ influx, NF-κB activation and MUC5AC synthesis induced by IL-4.IL-4 induced CaOur results indicate that IL-4-induced caveolin-1-containing lipid rafts aggregation at least partly contributes to MUC5AC synthesis in BECs.The online version of this article (10.1186/s12931-017-0657-z) contains supplementary material, which is available to authorized users. Mucus overproduction is an important feature of asthma . ExcessiAllergic pulmonary inflammation and airway hyper-reactivity in most cases of asthma is closely related in T helper type 2 cells (Th2) responses . Th2 celMUC5AC gene expression is regulated by transcript factors NF-κB and STAT6 i) signal is required for activation of NF-κB i were measured after loading with 10 μM of the acetoxymethyl ester form of Fura-2 for 30 min at room temperature. Then, the fluorescence of Fura-2 was recorded with a stimulation of 20 ng/ml IL-4, in the presence or absence of 10 mM M-βCD or 50 μM 2-APB after excitation at 340 ± 10 and 380 ± 10 nm using a xenon short-arc lamp (Ushio). Bandpass interference filters  selected wavelength bands of emitted fluorescence at 510 ± 10 nm. In the process of [Ca2+]i measurements, we firstly got stable [Ca2+]i signal, 250 s later, we treated cells with 2-APB or IL-4. M-βCD was used before [Ca2+]i measurements. In some experiments, we firstly treated cells with 2-APB or M-βCD, then added IL-4 to the cells at 1800s. Emitted Fura-2 fluorescence was collected and measured using a spectrofluorometer .BECs [Ca2+]i with the ratios from Fura-2 fluorescence, we had detected the minimum and maximum Fura-2 fluorescence ratio (Rmin and Rmax) with Ca2+-free buffer without calcium  and Ca2+-high buffer with high concentration of Ca2+ . The Rmin was determined using Ca2+-free buffer and stimulated by 3 μM ionomycin (Sigma). The same to Rmin, the Rmax was measured with Ca2+-high buffer and stimulated by 3 μM ionomycin. The final [Ca2+]i was calculated with Rmin and Rmax follow this equation: [Ca2+]i = Kd*β*(R – Rmin)/(Rmax – R), Kd is a constant which is 224 for BECs, and the β values is the fluorescence intensity emitted by 380 ± 10 nm within Ca2+-free buffer. Ca2+ influx duration was calculated from the beginning of Ca2+ level rise to the time of Ca2+ level return to the base.To acquire the final i in BECs, we treated BECs with IL-4 in the presence or absence of 2-APB, an inhibitor of Ca2+ release. As shown in Fig. 2+]i, but this was abolished by 2-APB. After careful analysis of Ca2+ influx duration and amplitude, we found both Ca2+ influx duration and amplitude were increased with the treatment of IL-4 in BECs (Fig. 2+ influx could be prevented by 2-APB in BECs.To study the effect of IL-4 on i levels. BECs were exposed to IL-4 in the presence or absence of M-βCD. As shown in Fig. 2+]i including amplitude and duration, but after disruption of lipid raft clustering with M-βCD, IL-4 did not induce any change in [Ca2+]i signal. These data suggested that IL-4 induced intracellular calcium response is mediated by lipid rafts aggregation.To further confirm the colocalization of TRPC1 in caveolin-1-containing lipid rafts aggregation, we detected [CaTo study the role of IL-4-induced caveolin-1-containing lipid rafts aggregation in MUC5AC synthesis in BECs, we used M-βCD to disrupt lipid rafts aggregation, and then detected changes in NF-κB activity and MUC5AC levels. As shown in Fig. In this study, we provided substantial evidence linking asthmatic cytokine IL-4 and airway epithelial cells MUC5AC overproduction. Our additional data revealed that MUC5AC proteins were over-expressed in asthmatic rat airway epithelial cells in vivo (Additional file 2+ influx and MUC5AC overproduction in BECs which prevented by 2-APB. Because 2-ABP is nonspecific and blocks both TRP channels and IP3R, to distinguish between extracellular and intracellular Ca2+ source, we depleted extracellular Ca2+ using EGTA and found extracellular Ca2+ influx played a role in IL-4 induced MUC5AC expression. We next found NF-κB rather than STAT6 activation mediated IL-4-induced MUC5AC protein synthesis.In the current study, firstly we found IL-4 induced CaFor the first time, we found that IL-4 induced caveolin-1-containing lipid rafts aggregation in BECs. Membrane lipid rafts are highly ordered membrane domains that are enriched in cholesterol, sphingolipids and gangliosides, and function as platforms for the recruitment of signaling proteins to facilitate protein-protein interaction and signal transduction. An increasing number of proteins involved in signal transduction have been found to locate in these ordered membrane domains. It is well known that lipid rafts organize receptors, ion channels and their downstream acting molecules to regulate intracellular signaling pathways –30.2+ signaling microdomains. TRPC1 channelosome responsible for SOCE is located in caveolar lipid raft domains [2+ influx in BECs.Recent studies have demonstrated that lipids rafts also contribute to the organization and function of Ca domains , 31–33.  domains , 34. Cav domains . In the 2+ oscillation frequency regulated NF-κB transcriptional activity via increased cumulated Ca2+ spike duration [Calcium signal was observed to be related with NF-κB activation. Sequence analyses of MUC5AC promoter revealed the presence of NF-κB response elements within it . NF-κB aduration , 39. OurIn the current study, we found IL-4 caused aggregation of caveolin-1-containing lipid rafts which activated TPRC1, subsequently activated calcium signal and NF-κB, and finally increased MUC5AC synthesis in BECs. This study elucidates a new mechanism underlying asthmatic cytokines induce mucus overproduction, which may be beneficial in developing novel strategies for the treatment of asthma.


We tested the hypothesis that excitatory neurotransmitters regulate Ca2+ transients in ICC-DMP as a means of regulating intestinal muscles. High-resolution confocal microscopy was used to image Ca2+ transients in ICC-DMP within murine small intestinal muscles with cell-specific expression of GCaMP3. Intrinsic nerves were stimulated by electrical field stimulation (EFS). ICC-DMP exhibited ongoing Ca2+ transients before stimuli were applied. EFS caused initial suppression of Ca2+ transients, followed by escape during sustained stimulation, and large increases in Ca2+ transients after cessation of stimulation. Basal Ca2+ activity and the excitatory phases of Ca2+ responses to EFS were inhibited by atropine and neurokinin 1 receptor (NK1) antagonists, but not by NK2 receptor antagonists. Exogenous ACh and substance P (SP) increased Ca2+ transients, atropine and NK1 antagonists decreased Ca2+ transients. Neurokinins appear to be released spontaneously (tonic excitation) in small intestinal muscles and are the dominant excitatory neurotransmitters. Subcellular regulation of Ca2+ release events in ICC-DMP may be a means by which excitatory neurotransmission organizes intestinal motility patterns.Interstitial cells of Cajal (ICC) regulate smooth muscle excitability and motility in the gastrointestinal (GI) tract. ICC in the deep muscular plexus (ICC-DMP) of the small intestine are aligned closely with varicosities of enteric motor neurons and thought to transduce neural responses. ICC-DMP generate Ca Neural responses in ICC in the deep muscular plexus (ICC-DMP) of the small intestine were studied by confocal imaging of Ca2+ transients in these cells. Excitatory neural input was due to cholinergic and peptidergic neurotransmitters [acetylcholine (ACh) and neurokinins], as excitatory effects on Ca2+ transients were blocked by atropine and neurokinin receptor antagonists. Neurokinins are the dominant excitatory regulators of Ca2+ transients in ICC-DMP. ICC-DMP are innervated by enteric motor neurons and mediate significant excitatory responses in intestinal muscles.Interstitial cells of Cajal (ICC) are innervated by enteric motor neurons and thought to transduce neural responses in GI muscles. Ca2+ sensitization mechanisms intrinsic to these cells but also by interstitial cells that are electrically coupled to SMCs. Together SMCs and interstitial cells, i.e., interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor α-immunopositive (PDGFRα+) cells  tract are innervated by both excitatory and inhibitory enteric motor neurons , and mot+) cells , form an+) cells . It is t+) cells .In the small intestine, a network of ICC in the myenteric region (ICC-MY) serves as the pacemaker cells that generate and actively propagate electrical slow waves and organize contractile activity into a phasic pattern that underlies segmental contractions . Another2+ release from intracellular stores (2+ release is important because it activates Ca2+-activated Cl- channels (CaCCs), encoded by Ano1, that are strongly expressed in ICC  is Car stores . Ca2+ red in ICC . We have muscles .Excitatory neurotransmission in the gut is mediated predominantly via cholinergic neurotransmitters and neurokinins. The tachykinin (TKs) family of peptides  is expressed throughout the GI tract . SP, NKA2+ release events. To test this hypothesis, we explored whether excitatory neural inputs to ICC-DMP are coupled to Ca2+ release and characterized the nature of the Ca2+ responses that constitute this transduction pathway for postjunctional excitatory transmission.In the present study, we investigated the hypothesis that a major mechanism by which enteric motor neurotransmitters regulate ICC is through modulation of Catm38(CAG-GCaMP3)HzeGt(ROSA)26Sor/J) and their wild-type siblings (C57BL/6) were acquired from The Jackson Laboratory and crossed with Kit-Cre mice (+/Cre-ERT2c-Kit), provided by Dr. Dieter Saur . Kit-Cre-GCaMP3 mice (both sexes) were injected with tamoxifen at six to eight weeks of age (2 mg for three consecutive days), as previously described (GCaMP3-floxed mice (B6.129S-escribed  to activSegments of jejunum (2 cm in length) were removed from mice and bathed in Krebs-Ringer bicarbonate solution (KRB). Jejunal segments were opened along the mesenteric border and luminal contents were washed away with KRB. The mucosa and sub-mucosa layers were removed by sharp dissection, and the remaining tunica muscularis was pinned flat within a Sylgard coated dish.3, 1.2 mmol/l NaH2PO4, 1.2 mmol/l MgCl2, 2.5 mmol/l CaCl2, and 11.5 mmol/l glucose. The KRB was bubbled with a mixture of 97% O2-3% CO2 and warmed to 37 ± 0.2°C.Tissues were maintained and perfused with KRB containing 120.35 mmol/l NaCl, 5.9 mmol/l KCl, 15.5 mmol/l NaHCOAll drugs were purchased from Tocris Bioscience and dissolved in the solvents recommended by the manufacturer to obtain stock solutions. Final concentrations used in experiments were obtained by dilution into KRB.+/copGFPKit mice as previously described . The PCR primers used and their GenBank accession numbers are provided in 2+ imaging data were acquired with an Eclipse E600FN microscope (Nikon Inc.) equipped with a 60× 1.0 CFI Fluor lens (Nikon instruments Inc). In this system, GCaMP3 was excited at 488 nm (T.I.L.L. Polychrome IV), as previously described (2+ imaging experiments were performed in the presence of nicardipine (100 nM) to minimize contractile movements.Jejunal muscle sheets (5.0 × 10.0 mm) were pinned to the base of a 5-ml, 60-mm diameter Sylgard-coated dish. The muscles were perfused with warmed KRB solution at 37°C for an equilibration period of 1 h. Fluorescence imaging was performed with a spinning-disk confocal microscope  mounted to an upright Nikon Eclipse FN1 microscope equipped with a 60× 1.0 NA CFI Fluor lens (Nikon Instruments Inc). GCaMP3, expressed solely in ICC, was excited at 488 nm using a laser coupled to a Borealis system (ANDOR Technology). Emitted fluorescence (>515 nm) was captured using a high-speed EMCCD Camera . Image sequences were collected at 33 fps using MetaMorph software (Molecular Devices Inc). Additional Caescribed . All Ca22+ activity in ICC-DMP was performed, as described previously (2+ activity in ICC-DMP were converted to a stack of TIFF (tagged image file format) images and imported into custom software  for analysis. Tissue movement was stabilized to ensure accurate measurement of Ca2+ transients from ICC-DMP. Whole cell ROIs were used to generate spatio-temporal (ST) maps of Ca2+ activity in individual ICC-DMP. ST maps were then imported as TIFF files into ImageJ  for post hoc quantification analysis of Ca2+ events.Analysis of Caeviously . Briefly2+ event frequency in ICC-DMP was expressed as the number of events fired per cell per second (s−1). Ca2+ event amplitude was expressed as ΔF/F0, the duration of Ca2+ events was expressed as full duration at half maximum amplitude (FDHM), and Ca2+ event spatial spread was expressed as μm of cell propagated per Ca2+ event. Unless otherwise stated, data are represented as mean ± SEM. Statistical analysis was performed using either a Student’s t test or with an ANOVA with a Dunnett post hoc test where appropriate. In all statistical analyses, p < 0.05 was taken as significant; p < 0.05 are represented by a single asterisk (*), p < 0.01 are represented by two asterisks (**), and p < 0.001 are represented by three asterisks (***). When describing data throughout the text, n refers to the number of animals used in that dataset while c refers to the numbers of cells used in that same dataset.Ca2+ transients that fired in a stochastic manner  an initial inhibitory phase; (2) an excitatory response that occurred largely after cessation of EFS  caused two distinct Can of EFS . The iniP ceased A–C. In tstimulus A. These e to EFS B. These Movie 1.2+ transient responses to enteric neuronal stimulation. Movie of intracellular Ca2+ transients in ICC-DMP labeled with the genetically encoded Ca2+ indicator GCaMP3 in response to EFS . The top left FOV shows typical elongated ICC-DMP using a 60× objective . Note that Ca2+ transients fired in stochastic fashion the blue bit-masked cell. The right window shows Ca2+ transient particles thresholded  after differentiation (Δt = 0.5 s) and smoothing  as shown in the middle window. Scale bar in top left window is 15 μm and pertains to all windows. The blue overlay of ICC-DMP in the FOV (blue bit-masked cell) was used to construct an ST map of Ca2+-induced fluorescence intensity across the diameter of the cell, which better displays the firing and propagation of Ca2+ transients along the length of the cell in response to EFS . The bottom panel shows active area of Ca2+ transients across the FOV (area of active particles). Note the caseation of Ca2+ transients in response to EFS and enhanced Ca2+ firing during post stimulus period. Scale bar in the lower ST map and bottom active area map: 50 μm.ICC-DMP Ca10.1523/ENEURO.0080-18.2018.video.12+ response to EFS was quantified during the final 3 s of EFS . The frequency of Ca2+ transients in the post-EFS period was also significantly increased from control, firing on average at 2.1 ± 0.1 events s−1 . There was a significant increase in Ca2+ transient amplitude in the final 3 s of EFS from 0.8 ± 0.06-1.1 ± 0.05 ΔF/F0 , although there was no significant increase in amplitude in the post-EFS period compared to control . Ca2+ transient duration increased in the final 3 s of EFS from 193 ± 3.7 to 219.6 ± 7.9 ms  and was also significantly increased in the post-EFS period, increasing to 222 ± 6.5 ms . Ca2+ transient propagation spread was also increased in the final 3 s of EFS from 11 ± 0.6 to 15.4 ± 0.9 μm  and was also significantly increased, as compared to control, in the post-EFS period, with Ca2+ transients propagating an average of 12.9 ± 0.6 μm . The number of Ca2+ firing sites in ICC-DMP was decreased significantly during the final 3 s of EFS (p < 0.001) and during the post-EFS period . This is likely a result of the increased propagation spread of Ca2+ transients during these periods, as shown in 2+ transients increased and they propagated over longer distances, individual firing sites may summate to create the increase in propagation distances observed during the final seconds of EFS and post-EFS. This could lead to an apparent reduction in firing sites, as the underlying sites were masked by propagating Ca2+ waves. A small increase in Ca2+ transient propagation velocity, that did not reach significance, was also observed during the final 3 s of EFS and during the post-EFS period .The excitatory Ca2+ responses in ICC-DMP were dependent on the frequency of EFS. EFS was applied to muscles from 1 to 20 Hz . No changes in Ca2+ transient parameters were resolved during 1-Hz stimulation , although a significant increase in the frequency of Ca2+ transients occurred in the post-EFS period . Higher EFS frequencies  increased Ca2+ transients significantly during EFS  and during the post stimulus period . EFS 5 Hz also increased Ca2+ transient frequency during the post-EFS period to 2.5 ± 0.4 events s−1, as compared to 1.6 ± 0.4 events s−1 in control . During EFS, the amplitude and duration of Ca2+ transients were not significantly changed at all frequencies tested (p > 0.05). However, Ca2+ transient duration increased during the post-EFS period at 5 Hz from 198 ± 10 to 228 ± 13.1 ms . The spread of Ca2+ transients was not significantly affected by 1-Hz EFS but increased significantly at 5 Hz during EFS . At 20 Hz, the spatial spread increased from 8.5 ± 0.6 to 11.9 ± 1.2 μm during EFS . The change in firing frequency (% change) for each stimulus 1, 5 10, and 20 Hz was calculated and plotted in 2+ transients was dependent on the stimulus frequency during both periods  from small intestinal muscles of Kit+/copGFP mice by FACS from, as previously described . Chrm3 transcripts were also higher in ICC relative to unsorted cells . Tacr1 was also highly expressed in ICC , and expression of Tacr2 was not resolved in ICC. Thus, the dominant receptor transcripts in ICC were Chrm3 and Tacr1.Excitatory neurotransmitters mediate responses by binding to specific post junctional receptors. In the case of excitatory enteric neurotransmission, responses have been attributed to muscarinic (M2 and M3) receptors and neurokinin (NK1 and NK2) receptors expressed in small intestinal muscles . In thisescribed , and cha2+ transients from 1.9 ± 0.31 to 1.2 ± 0.2 events s−1 . No significant effects on the other parameters of Ca2+ transients were noted: amplitude (p = 0.39), duration (p = 0.83) or spatial spread . When cholinergic stimulation was initiated by exogenous acetylcholine , Ca2+ transients increased markedly. ACh increased the frequency of Ca2+ transients from 0.85 ± 0.2 to 1.85 ± 0.4 events s−1 , and Ca2+ transient amplitude increased from 0.3 ± 0.04 to 0.6 ± 0.1 ΔF/F0 . ACh increased the duration of Ca2+ transients from 240 ± 16.1 to 296 ± 15.3 ms . The spatial spread of Ca2+ transients also increased in response to ACh, sometimes leading to propagating Ca2+ waves in contrast to more spatially limited events. Spatial spread increased from 7.1 ± 0.7 to 11 ± 1.3 μm .Atropine (1 μM) decreased the frequency of basal Ca2+ transients in ICC-DMP. EFS  in the presence of atropine (1 μM) resulted in a decrease in the frequency of Ca2+ transients during stimulation , from 1.7 ± 0.4 to 0.9 ± 0.2 events s−1 . Atropine also decreased the Ca2+ transient frequency during the post stimulus period, from 2.4 ± 0.3 to 1.6 ± 0.2 events s−1 . This suggests that cholinergic neurotransmission can also affect the poststimulus excitatory period in ICC-DMP. There were no significant changes in Ca2+ transient amplitude , duration , or spatial spread  during either the final 3 s of EFS or during the poststimulus period in the presence of atropine .Next, we investigated the extent of regulation exerted by cholinergic neurotransmission on Ca2+ transients in ICC-DMP from 1.2 ± 0.2 to 0.5 ± 0.1 events s−1 . The amplitude (p = 0.0039), duration (p = 0.002), and spatial spread (p = 0.005) of Ca2+ transients were also significantly depressed by RP 67580 . Amplitudes (p = 0.042) and spatial spread (p = 0.003) of Ca2+ transients were also significantly decreased by SR 140333 . A selective NK2 receptor antagonist, MEN 10376 (1 μM), had no effect on Ca2+ transients in ICC-DMP .NK1 receptors are the major neurokinin receptors expressed in ICC, and results from this study confirmed previous reports . Therefo2+ transients with RP 67580 . The duration of Ca2+ transients was increased by CCh from 183.4 ± 37.4 to 308.3 ± 18.9 ms  and the spatial spread of Ca2+ transients was also increased from 6.2 ± 1.3 to 12.1 ± 1.3 μm . CCh also increased Ca2+ transients after treatment with SR 140333 (data not shown). These results show that the effects of the NK1 antagonists were not due to off-target effects, such as inhibition of IP3-dependent signaling or Ca2+ release from intracellular stores.After inhibition of CaRP 67580 A,B, carb2+ signaling in ICC-DMP can be modulated by neurokinins via NK1, but not NK2 receptors. Regulation by neurokinins was further tested by application of NK1 agonists. SP  increased Ca2+ transients significantly , duration increased from 207 ± 15.9 to 342 ± 21.8 ms  and spatial spread increased from 7 ± 0.6 to 10.9 ± 1.2 μm . A more selective NK1 agonist, GR 73632  also increased the frequency of Ca2+ transients from 0.3 ± 0.03 to 1.2 ± 0.2 events s−1 , but effects on amplitude (p = 0.92), duration (p = 0.78), or spatial spread (p = 0.42) were not changed significantly . These data suggest that neurokinins are released tonically in small intestinal muscles, and responses of ICC-DMP to neurokinins are mediated largely by NK1 receptors.The observations above suggest that Caficantly A–E; freq2+ responses in ICC-DMP evoked by EFS. RP 67580 . During the post-EFS period, the frequency of Ca2+ transients was also significantly reduced from 1.8 ± 0.3 to 0.1 ± 0.05 events s−1 . Ca2+ transient amplitude, duration and spatial spread during the final 3 s EFS and post-EFS periods were also inhibited .We also tested whether NK1 receptors mediate Ca80 1 μM; A,B causes to EFS C–F. The 2+ transients persisted during the final 3 s of EFS and during the post stimulus period, as shown in n = 4, c = 20).When cholinergic and NK1 receptors were both antagonized by adding both atropine (1 μM) and RP 67580 (1 μM), pronounced inhibition of Cal-arginine  and MRS 2500 (1 μM) were used to examine excitatory neural regulation of Ca2+ transients in ICC-DMP after blocking major inhibitory pathways of neurotransmission. In the presence of L-NNA, MRS 2500, and atropine (1 μM), Ca2+ transient frequency , amplitude , and spatial spread  in the final 3-s period were not significantly affected. Ca2+ transient duration decreased significantly from 266 ± 14.15 to 199 ± 18.9 ms  in the final 3-s period. In the presence of L-NNA, MRS 2500, and atropine, Ca2+ transients in the poststimulus period were not reduced in amplitude  or spatial spread . However, the frequency of Ca2+ transients was reduced during the poststimulus period from 2.6 ± 0.25 to 1.9 ± 0.2 events s−1  and duration decreased from 237 ± 11.6 to 177 ± 11.9 ms .Nitrergic and purinergic antagonists N-ω-nitro-2+ transients during the final 3 s of EFS . The amplitude decreased from 1.4 ± 0.2 to 0.42 ± 0.8 ΔF/F0 , the duration decreased from 214.4 ± 16.9 to 122 ± 22 ms , and the spatial spread decreased from 15.4 ± 3.2 to 5.8 ± 1 μm . Overall the frequency of Ca2+ transients in the final 3 s of the EFS period was not significantly affected . In the presence of L-NNA, MRS 2500, and RP 67580, the frequency of Ca2+ transients in the post-EFS period was significantly reduced from 1.8 ± 0.3 to 0.4 ± 0.1 events s−1 . The amplitude of Ca2+ transients during this period was not significantly affected , but the duration of Ca2+ transients was reduced from 237 ± 23.9 to 121 ± 19.7 ms , and spatial spread decreased from 12.61 ± 1.6 to 6.8 ±1.2 μm .We next examined the neurokinin input into EFS-mediated excitatory responses in ICC-DMP in the presence of blockers of nitrergic and purinergic neurotransmission. With L-NNA, MRS 2500, and RP 67580 present, responses to EFS were significantly reduced in amplitude, duration, and spatial spread of Ca2+ transients were significantly diminished across all parameters tabulated, as shown in n = 5, c = 32).Next, we inhibited cholinergic and neurokinin transmission with atropine and RP 67580 in the presence of L-NNA and MRS 2500. Under these conditions all Ca2+ transients that occur in the absence of extrinsic stimuli in these cells , a period of escape from inhibition during sustained EFS, and a period of strong excitation after cessation of the stimuli (poststimulus or “rebound” excitation). The complexity of these responses is likely due to the fact that the enteric nervous system contains both inhibitory and excitatory motor neurons  and NK1 (Tacr1) were enriched in ICC-DMP versus unsorted cells. However, transcripts of Chrm2 were also present, suggesting these receptors and coupling to effectors via Gi/Go may also have a role in transduction or modulation of excitatory neurotransmission. Our findings are consistent with previous studies showing muscarinic receptors and NK1 receptor expression in ICC with immunohistochemical techniques  which activates Ca2+ release from the endoplasmic reticulum (ER). All of the molecular components of this pathway are expressed in ICC, as shown by transcriptome analyses , mediated, in part, by IP3R , and enhance the amplitude of phasic contractions , similar in concept to the tonic inhibition phenomena observed in many GI muscles . As discussed above, much of the excitatory response was mediated through NK1 receptors that are expressed largely by ICC-DMP . Response events , suggest by SMCs , were ap


As a Ca2+-induced Ca2+ release (CICR) channel that mediates endoplasmic reticulum (ER) Ca2+ release, the role of ryanodine receptors (RyRs) in oligodendroglial development is unexplored. In the present study, we observed that among the three mammalian isoforms, oligodendroglial lineage cells selectively expressed RyR3. Strong RyR3-positive signal was distributed all over the cytoplasm and processes in OPCs and/or immature OLs (imOLs), whereas it gradually decreased and was located mainly around the perinuclear region in mature oligodendrocytes (OLs). In addition, RyR3-mediated intracellular Ca2+ waves following caffeine stimulation were correlated with the expression pattern of RyR3, in which high flat Ca2+ fluctuations and oscillatory Ca2+ waves were more frequently recorded in OPCs and/or imOLs than in OLs. Through further functional exploration, we demonstrated that pretreatment with the RyR antagonist ryanodine could neutralize the increase in intracellular Ca2+ induced by OPC differentiation and reduce the number of mature OLs. Moreover, gene-level knockdown of RyR3 by lentivirus in OPCs resulted in inhibition of OPC differentiation. Taken together, our results provide new insight into the crucial role of RyR3-mediated ER Ca2+ release in the regulation of OPC differentiation and/or myelination.Increased intracellular Ca Moreover, inhibiting the function of RyR3 either pharmacologically or by gene knockdown suppressed the differentiation of OPCs. Our results revealed a critical role for RyR3-mediated Ca2+ signaling in oligodendroglial differentiation that may provide new insight into therapeutic approaches for demyelinating diseases.In the present study, we sought to characterize the expression and function of RyRs during OPC differentiation. Our results showed that RyR3 was selectively expressed and widely distributed in the soma and processes of the oligodendroglial lineage cells and that its expression level was downregulated following OPC differentiation. Using confocal CaCortical OPCs were purified as previously described , N2 supplement , fetal bovine serum , insulin , N-acetyl-l-cysteine , PDGFAA . The SD rats related procedures were performed in accordance with the guidelines approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University  for 20 min at 37°C in a modified imaging buffer containing (in mM): NaCl, 135; KCl, 3; MgCl2, 2; Glucose, 8; HEPES, 10 and CaCl2, 2 (pH adjusted to 7.4 with NaOH). The dye-loaded cells were washed twice and maintained for at least 20 min at RT in fresh imaging buffer to allow complete dye de-esterification. Ca2+ wave and fluorescence images were real-time recorded for at least 15 min in all experiments, using a confocal laser-scanning microscope  with the UplanFl40× objective (N.A. 0.95). The image acquisition frequency is 100–180 ms/image. Cell morphology was detected using differential interference contrast (DIC) under confocal microscopy. Ca2+ concentrations were measured by exciting Fluo-3 AM at 488 nm.OPCs were grown and differentiated in glass-bottom dish and loaded with the fluorescent Ca2+ responses in OPCs, imOLs and mature OLs were recorded with caffeine  stimulation in a Ca2+ free imaging buffer. For spontaneous Ca2+ recordings, OPCs were grown in proliferation medium or differentiation medium for 6 h with or without ryanodine treatment . Fluo-3 loading and cell washing was followed by applying normal proliferating medium (Pro-medium) or differentiating medium (Diff-medium) for the following recording.Intracellular CaCells were grown on coverslips, fixed in cold 4% paraformaldehyde for 20 min, rinsed with 0.01 M PBS, blocked with 1% bovine serum albumin (BSA) and 0.2% Triton-X100 for 30 min and then incubated with primary antibodies diluted in 1% BSA overnight at 4°C and then by fluorophore-conjugated secondary antibodies at room temperature (RT) for 2 h. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole  for 10 min.In this study, the following antibodies were used: mouse polyclonal anti-Olig2 , rabbit polyclonal anti-RyR3 , and goat anti-myelin basic protein . The secondary antibodies were as follows: Alexa 568-labeled donkey anti-mouse , Alexa 488-labeled donkey anti-goat , Alexa 568-labeled donkey anti-goat , Alexa 568-labeled donkey anti-rabbit  and Cy5-labeled rabbit anti-mouse .Fluorescent images were captured using an Axio Imager M2 fluorescence microscope  or a confocal laser-scanning microscope  with excitation wavelengths appropriate for Alexa Fluor 488 (488 nm), 596 (568 nm), 647 (628 nm) or DAPI (380 nm). Digital images of the oligodendroglial lineage cells in the supplemental figure were acquired with an Olympus IX51 microscope with an Olympus C-7070 camera (Tokyo). For the statistical analysis, randomly selected images in at least three representative fields were acquired from each sample. Detection and quantification were performed using ImageJ software .The cells were lysed using RIPA lysis buffer  with freshly added 1% phenylmethylsulfonyl fluoride  solution. Protein concentration was determined using Coomassie brilliant Blue G-250. SDS-PAGE and Western blotting were carried out as reported previously . The amplification procedure and melt curve analysis were performed using three independent replicates for each sample.Total ribonucleic acid (RNA) was isolated from different stages of OPC cultures . Real-time polymerase chain reaction (RT-PCR) was performed with the C1000 Touch™ Real-time PCR Detection System (Bio-Rad) and GoTaqThe oligonucleotide primers used were as follows:8 transducing units (TU)/ml for the interference group (shRyR3) and 6.71*108 TU/ml for the control group (shCTL). Lentivirus was removed after transfection for 16 h. OPC were further differentiated for another 32 h and fixed.The shRNA lentivirus was purchased from Obio Technology, Shanghai. Targeted sequence in rat RyR3 gene is: AGATGCTAATTGCATCTC. The primary lentivirus solution was diluted in several gradient concentrations  to check the best work concentration . The lentivirus was diluted 1:300 in the differentiation medium with a primary concentration of 8.67*10Phosphodiester ODNs protected by terminal phosphorothioate double substitution (capped ODNs) against possible exonuclease-mediated degradation were purchased from Tib-Molbiol (Sigma). The sequences are as follows: anti-RyR3: 5′-A*G*ATGCTAATTGCATC*T*C-3′ (*indicates the phosphorothioate residues) and an 18-mer fully degenerated ODN (dODN), 5′-N*N*NNNNNNNNNNNNNN*N*N-3′ , which was used as a control ODN. ODNs were transported using an artificial cationic lipid  to enhance both uptake and stability. Antisense ODNs (aODNs) or dODNs were pre-incubated at 37°C for 30 min. Differentiated OLs were collected on day 3.post hoc test. Comparisons between two experimental groups were made using Student’s t-test (GraphPad Prism 6). A probability of p < 0.05 was considered significant.All experiments were repeated at least three times. Data are shown as the means ± SEM. Statistical analyses of three groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s Similar to our previous studies  are relatively quiescent in comparison to those in neurons  and imOLs . Likely, the oscillatory Ca2+ transients were present at a higher ratio in OPCs  and imOLs  than in mature OLs . The low plateaus Ca2+ response was the dominant reaction of mature OLs  but was barely found in OPCs and imOLs -induced Ca2+ transient is likely due to release of Ca2+ from ER mediated mainly by RyR3 channel. We assume that this stage-specific response is probably correlated to the downregulated expression of RyR3 during OL development.Although OPCs are reported to have spontaneous oscillatory-like Cas Figure . More im Figures . The higs Figure . Notablys Figure , cerebels Figure , B lymphs Figure . In our 2+ response. By transforming the time-lapse Ca2+ imaging into pseudocolor changes and 3D surface plots with Image J software, we found that OPCs respond rapidly and strongly in both the soma and processes , which was characterized by a gradual increase in the Ca2+ concentration to a peak and a subsequently decreasing Ca2+ signal . Those Ca2+ activities appeared at higher frequency; moreover, the elevation of Ca2+ signaling was more persistent in certain individual cells  and measured the number of mature OLs by immunofluorescence staining after 3 days of differentiation. The number of MBP-positive OLs was significantly decreased in the ryanodine-treated group  have the ability to selectively reduce the mRNA level of RyR3  or a control lentivirus (shCTL). After differentiation for 48 h, we observed efficient infection of both lentiviruses, visualized as the GFP expression in the cytoplasm Figure . Immunof2+ signaling is regulated and how it contributes to oligodendroglial differentiation remains unclear. In our present study, we systematically analyzed the expression and function of the ER Ca2+ release channel—RyR3. We found that RyR3 is the only RyR expressed in oligodendroglial cells and dynamically regulated during OPC differentiation. Importantly, inhibition of RyR3 resulted in blockage of OPC differentiation  are major Ca2+ responses to depolarization in OPCs were more active than those in mature OLs, and this phenomenon was mainly explained by the downregulation of L-type voltage-operated Ca2+ channels (VOCCs) following OPC maturation  which could trigger Ca2+ influx in oligodendroglial cells and possibly evoke the following CICR through RyR3 (Butt, + are sufficient to depolarize VOCCs in nearby oligodendroglial cells to produce a significant rise in intracellular Ca2+ which also need CICR to form the final Ca2+ signal (Cheli et al., 2+ signaling (Haak et al., 2+ channel in several cell types (Brown et al., 2+ signaling formation in oligodendroglial differentiation. Channel-specific antagonists or a genetic approach may be needed in our future studies.Even if it has not been studied in this work, we speculate that in physiological R3 Butt, . Moreove2+ release is dynamically regulated and plays an essential role in initiating OPC differentiation (Figure 2+ channels in oligodendroglia and may provide valuable insights into therapeutic strategies for demyelinating diseases.In summary, we provide direct evidence showing that RyR3-mediated ER Can Figure . Our resLX, HL and TL designed experiments and wrote the manuscript. TL, LW, TM and SW conducted the experiments. TL, LW and JN collected and analyzed the data.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.


We systematically examined influence on SCOs of an array of ion channel modifiers by recording intracellular Ca2+ dynamics in rat ventricular cardiomyocytes using Ca2+ specific fluorescence dye, Fluo-8/AM. Voltage-gated sodium channels (VGSCs) activation elongates SCO duration and reduces SCO frequency while inhibition of VGSCs decreases SCO frequency without affecting amplitude and duration. Inhibition of voltage-gated potassium channel increases SCO duration. Direct activation of L-type Ca2+ channels (LTCCs) induces SCO bursts while suppressing LTCCs decreases SCO amplitude and slightly increases SCO frequency. Activation of ryanodine receptors (RyRs) increases SCO duration and decreases both SCO amplitude and frequency while inhibiting RyRs decreases SCO frequency without affecting amplitude and duration. The potencies of these ion channel modifiers on SCO responses are generally consistent with their affinities in respective targets demonstrating that modification of distinct targets produces different SCO profiles. We further demonstrate that clinically-used drugs that produce Long-QT syndrome including cisapride, dofetilide, sotalol, and quinidine all induce SCO bursts while verapamil has no effect. Therefore, occurrence of SCO bursts may have a translational value to predict cardiotoxicants causing Long-QT syndrome.Primary cultured cardiomyocytes show spontaneous Ca Ether-à-go-go Related Gene) are the major contributors to rapid delayed rectifier potassium currents (IKr) which are involved in AP repolarizationThe orchestrated mechanical activity of the heart is controlled by electrical pulses initiating from the sino-atrial node and finally conveys to the ventricles leading to rapid depolarization of all ventricular myocytes and coordinated contraction of the heart1362+ concentration 2+ homeostasis in ventricular cardiomyocytes are associated with the ventricular tachycardia. Re-opening of L-type Ca2+ channels (LTCCs) or other depolarizing currents before normal repolarization completes contributes to the early afterdepolarization (EAD). Gain-of-function mutations on Cav1.2  produces Timothy syndrome which characterized by a heart condition similar to LQT syndrome132+ leakage from the sarcoplasmic reticulum due to point mutation on type 2 ryanodine receptors (RyR2) contributes to formation of delayed after-depolarization (DAD) which leads to heart failure and catecholaminergic polymorphic ventricular tachycardia (CPVT)2+ also undergoes derangements that promote arrhythmogenesis through Ca2+ -dependent and coupled electrophysiological effects. Aberrant Ca2+ signals can modulate CaMKII activity which in turn regulates the activity of a variety of ion channels and transporters, for examples Nav1.517Primary cultured cardiomyocytes show spontaneous transient increase in intracellular Ca2+ dynamics in primary cultured rat ventricular cardiomyocytes using Fluorescence Imaging Plate Reader (FLIPR) in 96-well format. We demonstrate that modification of distinct ion channels differentially affects SCO patterns. In addition, we demonstrate that clinically-used drugs including cisapride, dofetilide, sotalol, and quinidine which cause LQT syndrome all produce characteristic SCO bursts therefore prolong the SCO/burst duration. Our results demonstrate that occurrence of SCO bursts may have a translational value to predict cardiotoxicants causing LQT syndrome.In this study, we systematically examined the influence of an array of ion channel modulators on SCO patterns by detecting the intracellular Cadata not shown). It has been well established that the spontaneous beating was correlated with SCO generation®TETRA. FLIPR®TETRA can simultaneous record the fluorescence intensity from the center area of each well in 96- or 384- well formats therefore representing an overall Ca2+ signals from a population of cells. At a plating density of 1.5 × 105 cells/well and after cultured for 60 h, cardiomyocytes displayed rhythmic SCOs, with minimum variability in the frequency, amplitude as well as full width at half maximum (FWHM)  . The frem (FWHM) . It shoum (FWHM) . To dimi2+ influx or intracellular Ca2+ store release in our culture system, the cells were bathed in normal extracellular Ca2+ (2.3 mM), decreased extracellular Ca2+ (1.15 mM) as well as 0 Ca2+  and SCOs were recorded. Reducing extracellular Ca2+ concentration from 2.3 mM to 1.15 mM significantly decreased SCO mean amplitude with slightly enhanced SCO frequency without changing SCO FWHM. Further decreasing the extracellular cellular Ca2+ from 1.15 mM to 0 mM (no EGTA) produced an additional increase in SCO frequency and an additional decrease in SCO amplitude without effect on SCO duration ] and 0.86 nM , respectively  . Acetylcholine also moderately increased the SCO amplitude with EC50 value of 3.77 nM . Acetylcholine was without effect on the SCO duration (Acetylcholine and epinephrine can modulate heart beating through activation of β-adrenergic receptors and muscarinic acetylcholine receptors (subtype 2 in cardiomyocytes), respectivelyectively . Epineph 95% CI) . In contduration .2+ dynamics and are also responsible for the repolarization of the action potentials (APs)50 values for Bay K 8644 stimulating SCO amplitude and mean SCO/bursts duration were 4.63 nM  and 45.7 nM , respectively. Nifedipine, an LTCC inhibitor, concentration-dependently suppressed the SCO amplitude  while slightly increased the SCO frequency  without significant effect on the SCO duration (LTCCs are directly pertinent to the intracellular Caduration .2+ channel located in the endoplasmic/sarcoplasmic reticulum and is responsible for Ca2+ -induced Ca2+ release (CICR). RyR2 is primarily expressed in cardiac tissues. Upon activation following LTCC mediated Ca2+ influx, RyR2 releases Ca2+ from the sarcoplasmic reticulum into the cytosol, enabling cardiac muscle contraction50 = 0.93 μM, 0.81–1.06 μM, 95% CI) without affecting the SCO duration. Higher concentration of FLA-365 ( ≥3 μM) eliminated SCOs (The ryanodine receptor (RyR) is one of the store operated Cated SCOs . Low conted SCOs  with inc2+ overloading (Data not shown). TTX concentration-dependently suppressed the SCO frequency with IC50 value of 0.86 μM  without changing SCO amplitude and duration  without affecting SCO amplitude and duration  and 88.7 μM , respectively. 4-AP had no effect on SCO frequency (Ether-à-go-go Related Gene) mediates the repolarizing IKr current in the cardiac APs. Loss of hERG channel function by pharmacological inhibition or genetic mutation has been shown to delay the AP repolarization and widen the ventricular AP, which is the underlying physiological abnormality in LQT syndrome50 values for E-4031 increasing SCO burst duration and decreasing SCO frequency were 38.4 nM  and 40.6 nM , respectively , 0.63 μM , 9.38 μM , and 27.5 μM , respectively , 0.36 μM  and 3.24 μM , respectively. However, sotalol was without effect on the SCO frequency. Cisapride and quinidine also moderately suppressed the SCO amplitude with IC50 values of 3.34 μM  and 11.3 μM , respectively. Verapamil, a dual blocker of hERG channel and LTCC, suppressed the SCO frequency and amplitude with IC50 values of 0.79 μM  and 1.47 μM , respectively. Verapamil was without effect on the SCO duration  dynamics in primary cultured rat ventricular cardiomyocytes. We demonstrate that altering the activity of Ca2+ channels which contribute to [Ca2+]i dynamics or ion channels which are involved in AP generation both affect SCO patterns. The potencies of tested reference compounds are generally consistent with their affinities on their respective targets (2+ response in cultured rat ventricular cardiomyocytes. The [Ca2+]i not only governs contractile events (including systolic and diastolic function), but also undergoes derangements that promote arrhythmogenesis through Ca2+ -dependent and coupled electrophysiological effects®TETRA system may serve as a rapid throughput method capable of identifying potential cardiotoxicants from environment or early stage of drug development.In this study, using rapid throughput imaging system, we systematically investigated the influence of an array of ion channel modifiers on intracellular Ca targets  suggestiWe demonstrate that acetylcholine dramatically decreases the SCO frequency consisting with previous study where acetylcholine decreases cardiomyocytes contraction resulting from activation of subtype 2 muscarinic acetylcholine receptors2+]i dynamics or shape APs affect SCO pattern differently. Activation of LTCC by Bay K 8644An important finding is that compounds which contribute to [Ca50 and IC50 values of E-4031 on SCO/burst duration and frequency are 38.3 nM and 40.6 nM, respectively, which were consistent with its affinity on hERG channels3850 values for cisapride and quinidine are significantly deviated from their affinities to hERG channels40+ and Ca2+ currents44Another important finding is that E-4031, a specific Kv11.1 (hERG) blocker, produces SCO bursts and decreases the SCO/burst frequency. The ECIn addition to clinically-used drugs, we also demonstrate that sodium channel agonist, veratridine and LTCC activator, Bay K 8644 as well as the universal potassium channel blocker, 4-AP produce SCO bursts. It has been demonstrated that veratridine prolongs AP duration in rabbit ventricular myocytes62+ handling of iPSC-CM were reportedA variety of compounds-induced SCO patterns have been investigated in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs). The hERG channel inhibitors cisapride, terfenadine, astemizole, and pimozide, all characteristically delay SCO recoveryIn summary, we demonstrate that modification of distinct targets differentially affects the SCO pattern in primary rat cardiomyocyte cultures. Although a large set of LQT positive and negative compounds were required to be investigated to fully establish the relationship between LQT and SCO bursts, we demonstrate that clinically-used drugs which produce LQT all produce SCO bursts while negative drugs do not. Therefore, this method may serve as a primary screening for detecting the cardiotoxicants in the early stage of drug development. In addition, mapping the profiles induced by cardiotoxicants may predict their molecular target(s). More importantly, this method may have translational value for detecting the compounds which have the potential to cause LQT syndrome.® 488-conjugated goat anti-rabbit were from ThermoFisher Scientific . The Ca2+ dye Fluo-8/AM was from TEFLab .Bay K 8644, veratridine, terfenadine, cisapride, anti-cTnT (1:400) antibody, E-4031 and verapamil were purchased from Abcam . Quinidine was from Tocris Bioscience . Nifedipine, 4-aminopyridine, fetal bovine serum (FBS), cytosine β-D-arabinofuranoside (ARA-C), poly-D-lysine and epinephrine, FLA-365, ryanodine, lamotrigine, carbamazepine, phenytoin and all inorganic salts were purchased from Sigma-Aldrich . Bovine serum albumin, DMEM/F12, Triton™ X-100, Alexa Fluor4 0.81, dextrose 5.55, KH2PO4 0.44, Na2HPO4 0.34, HEPES 20, pH 7.4). After centrifugation at 200 g for 5 min, the cells were resuspended in the culture medium . Dissociated myocardial cells were seeded in 60 mm petri-dishes for 45 min to remove the fibroblast cells. The unattached cells (enriched cardiomyocytes) were then plated onto poly-D-lysine (20 μg/mL) coated clear-bottom, black wall, 96-well plate  at a density of 1.5 × 105 cells/well in the culture medium. The medium was changed after 24 h culture by replacing half volume of the medium by fresh culture medium. The cells were maintained at 37 °C with 5% CO2 and 95% humidity until used.Animal experiments were conducted in accordance with the guidelines of Animal Use and Care of the National Institutes of Health approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University. Sprague-Dawley rat were supplied by Qing-Long-Shan animal center . Cardiomyocytes were dissociated from ventricles dissected from Sprague-Dawley rat pups at postnatal day 3 and maintained in cold dissection buffer  were used to investigate the basal characteristics of intracellular calcium dynamics and how ion channel ligands or arrhythmogenic drugs alter these signals. Briefly, the growth medium was removed and replaced with dye loading buffer (50 μL/well) containing 4 μM Fluo-8/AM, 0.5% bovine serum albumin in pre-warmed Locke’s buffer . After 45 min incubation in dye loading buffer, cells were washed four times by adding and removing 150 μL fresh Locke’s buffer repeatedly. The final volume of each well was adjusted to be 175 μL. After loading, the plate was then transferred to a Fluorescence Imaging Plate Reader  chamber which was pre-warmed to 37 °C. Cells were excited at 488 nm, and Ca2+ -bound Fluo-8 emitted fluorescence at 515–575 nm range was recorded. The basal fluorescence was recorded for approximately 300 s at a sampling rate of 8 Hz. After recording the basal fluorescence, the drugs  were added to different wells by an automated, programmable pipetting system yielding a final volume of 200 μL/well. The fluorescence was recorded for additional 500 s at a sampling rate of 8 Hz. The fluorescence data were presented as ΔF/F0, where F is the fluorescence signal at different time point minus background fluorescence whereas the F0 is the basal fluorescence minus the background fluorescence. The background fluorescence was from the sister wells which were not loaded with Fluo-8/AM. To quantify the drug influence on the SCOs, we analyzed SCO frequency, peak amplitude, as well as SCO duration (FWHM). Many drugs induced SCO bursts and in this case, the SCO duration was calculated as SCO burst duration. A burst was defined to be a cluster of SCOs (at least two) where the previous SCO can’t recover to below 20% of the previous SCO amplitude when followed SCO occurred. When drugs induced SCO bursts, a burst was count as an SCO.Cardiomyocyte cultures at 2 days ® 488-conjugated goat anti-rabbit (1:500) secondary antibody for 2 h at RT. After aspirating secondary antibody, a concentration of 2 μg/mL of Hoechst 33342 was added to stain the nuclei. Images were taken using a Nikon eclipse fluorescence microscope using FITC filter and DAPI filter.Cardiomyocytes at 2 DIV were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.25% Triton™ X-100 for 15 min under gentle shaking. After blocking with 5% goat serum in PBS for 30 min, cells were incubated with anti-cTnT (1:400) antibody overnight at 4 °C. Cells were then incubated with Alexa Fluor50 values and the 95% confidence intervals were determined by non-linear regression using GraphPad Prism . Statistical significance between different groups was calculated using an ANOVA and, where appropriate, a Dunnett’s multiple comparison test; p values below 0.05 were considered to be statistically significant.Graphing and statistical analysis were performed using GraphPad Prism software  and Origin Pro 8 . Peaks were fit by Origin Pro 8 . The ECHow to cite this article: Li, X. et al. Modification of distinct ion channels differentially modulates Ca2+ dynamics in primary cultured rat ventricular cardiomyocytes. Sci. Rep.7, 40952; doi: 10.1038/srep40952 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


MLL-rearranged acute myeloid leukemia (AML) remains a fatal disease with a high rate of relapse and therapeutic failure due to chemotherapy resistance. In analysis of our Affymetrix microarray profiling and chromatin immunoprecipitation (ChIP) assays, we found that ALOX5 is especially down-regulated in MLL-rearranged AML, via transcription repression mediated by Polycomb repressive complex 2 (PRC2). Colony forming/replating and bone marrow transplantation (BMT) assays showed that Alox5 exhibited a moderate anti-tumor effect both in vitro and in vivo. Strikingly, leukemic cells with Alox5 overexpression showed a significantly higher sensitivity to the standard chemotherapeutic agents, i.e., doxorubicin (DOX) and cytarabine (Ara-C). The drug-sensitizing role of Alox5 was further confirmed in human and murine MLL-rearranged AML cell models in vitro, as well as in the in vivo MLL-rearranged AML BMT model coupled with treatment of “5 + 3” (i.e. DOX plus Ara-C) regimen. Stat and K-Ras signaling pathways were negatively correlated with Alox5 overexpression in MLL-AF9-leukemic blast cells; inhibition of the above signaling pathways mimicked the drug-sensitizing effect of ALOX5 in AML cells. Collectively, our work shows that ALOX5 plays a moderate anti-tumor role and functions as a drug sensitizer, with a therapeutic potential, in MLL-rearranged AML. The mixed lineage leukemia (MLL) gene on chromosome 11q23 is the frequent target of chromosomal translocations and rearrangements in infant and adult leukemias. AML bearing MLL fusion often associates with poor prognosis and is resistant to most of the current clinical chemotherapies4. Though immense efforts have been exerted in developing novel therapeutic strategies in the past decades, the standard chemotherapy with intensive administration of chemotherapeutic drugs, e.g. the “7 + 3” regimen (i.e. combined treatment of anthracycline (daunorubicin or doxorubicin) and cytarabine (Ara-C)), remains the main therapeutic approach for treating AML, including MLL-rearranged AML8. Resistance to the standard chemotherapy remains the major cause of relapse and therapeutic failure9. Despite intensive chemotherapy, the overall survival of AML patients is still less than 30% for adults and 60% for children, and most of the patients with MLL-rearranged AML fail to survive longer than 5 years11. Therefore, it is crucial to understand the mechanisms underlying the pathogenesis and chemotherapy resistance of AML, such as MLL-rearranged AML, and identify potential new therapeutic targets that can suppress AML or improve the response to the currently existing chemotherapies.Acute myeloid leukemia (AML), the most common type of acute leukemia attacking adults, is a heterogeneous disease characterized by the malignant expansion of dysfunctional myeloid progenitors and the suppression of normal hematopoiesis9. Among these factors, the JAK/STAT signaling is known to be closely associated with myeloid malignancies. Activation of JAK/STAT pathway is a hallmark of the myeloproliferative neoplasms, and is found in many AML cases13. JAK/STAT pathway inhibitors, e.g. pacritinib and ruxolitinib, have already been studied in pre-clinical and clinical trials15. The proto-oncogene K-RAS is another factor involved in both leukemogenesis and therapeutic response16. K-RAS mutations have been reported in many AML cases and are associated with prognosis and chemotherapy resistance18. It holds great potential to cure AML by either targeting these factors directly or targeting a common upstream regulator that controls all these factors in AML.The mechanism of AML drug response remains obscure. A number of key factors including transcriptional factors, oncogenes and tumor suppressors, as well as cell metabolism regulators, have been identified to play important roles in determining drug sensitivity or resistanceALOX5) encodes a non-heme iron-containing enzyme of the lipoxygenase family. It catalyzes the production of leukotrienes (LTs) and reactive oxygen species (ROS) from arachidonic acid20. ALOX5 is known to be involved in various physiological and pathological processes, including oxidative stress response, inflammation and cancer25. It was reported previously that loss of Alox5 impairs the function of leukemic stem cells (LSCs) in BCR-ABL-induced chronic myelogenous leukemia (CML)26. To our surprise, our gene profiling reveals that ALOX5 expression is particularly down-regulated in MLL-rearranged AML. Both in vitro and in vivo studies were carried out to investigate the effects and underlying mechanism of ALOX5 in AML pathogenesis and drug response.The arachidonate 5-lipoxygenase gene (MLL-rearrangements/t(11q23) (n = 12), inv(16) (n = 27), t (n = 30) or t (n = 31), along with 9 normal bone marrow (BM) control ) samples29, we found that ALOX5 was expressed at a significantly lower level in MLL-rearranged AML, but not in other AML subtypes, relative to the normal controls   Fig. . The dowPCR Fig.  and WestPCR Fig.  in MLL-AALOX5 in MLL-rearranged AML, we conducted chromatin immunoprecipitation (ChIP) assays. We found no significant enrichment of MLL fusion proteins at the promoter region32 of the ALOX5 locus , a marker for repressive transcription associated with EZH235, also exhibited high enrichement at these genomic locus  with MSCV-PIG-Alox5 (Alox5) or MSCV-PIG (Ctrl) into mouse BM progenitor cells for in vitro colony-forming/replating assays. We showed that along with the increased number of passages, Alox5 overexpression showed a more significant degree of repression on MLL-AF9-induced colony forming  cells  assay first and found that forced expression of Alox5 showed no significant influence on overall survival   vs. 37 days (MA9_Ctrl); P = 0.018, log-rank test)  Fig. . We thenst) Fig. . While aunt Fig.  and spleunt Fig. . Moreoveter Fig. . Tissue ere Fig. . Thus, oAlox5 implies a therapeutic potential in treating AML. Since restoration of Alox5 expression/function alone showed only moderate inhibitory effect on AML progression, we sought to investigate whether Alox5 restoration could facilitate chemotherapeutic response and thus yield a more effective therapeutic effect. We first used lentivirus to overexpress human ALOX5 in MONOMAC-6/t AML cells, and treated the transduced cells with doxorubicin (DOX) or cytarabine (Ara-C). Our results showed that overexpression of ALOX5 significantly sensitized MONOMAC-6 cells to DOX and especially Ara-C treatment  or without  into secondary recipient mice, and after the onset of leukemia, the recipient mice were treated with or without DOX + Ara-C (i.e. “5 + 3” regimen5). As shown in Fig. MLL-AF9 AML, forced expression of Alox5 dramatically improved the response of MLL-AF9 AML to the DOX + Ara-C treatment 43, those bearing K-RAS mutations (n = 5) are associated with a lower ALOX5 level  of two pairs of mouse BM leukemic blast cells collected from the MA9_Ctrl and MA9_Alox5 mice in secondary BMT assays shown in Fig. lls Fig. . Stat5 aMYC Fig. . In BM lg40 Fig. . In ordeapy Fig. . In a daALOX5 reported previously in CML26, here we show that ALOX5, suppressed by PRC2 at the transcription level, exhibits a moderate anti-tumor effect both in vitro and in vivo in MLL-rearranged AML, in which ALOX5 expression is especially repressed. More interestingly, we show that restoration of Alox5 expression can substantially increase the sensitivity of MLL-rearranged AML cells to standard chemotherapeutic agents such as DOX and Ara-C both in vitro and in vivo, as well as the underlying mechanism through suppressing the STAT and K-RAS oncogenic signaling pathways.In contrast to the oncogenic role of ALOX5 was largely known as an oncogene in solid tumors, e.g. prostate cancer and pancreas cancer25. However, its role remains vague in leukemia. It was reported that Alox5 deficiency resulted in a significant reduction of LSCs in BM, and thus largely prolonged survival of BCR-ABL-induced CML mice26. Nonetheless, there was no significant influence of Alox5 deficiency on normal hematopoietic stem cells (HSCs) or on the induction of lymphoid leukemia by BCR-ABL26. Another study showed that Alox5 deficiency inhibited in vitro colony-forming capacity of BM progenitor cells induced by some AML oncogenic fusion genes such as AML1-ETO9a, a potent oncogenic isoform of AML1-ETO resulting from t44, but had no significant effects on AML1-ETO9a-induced leukemogenesis in vivo32. Those findings together with our present results suggest that Alox5 likely plays distinct roles at different lineages (myeloid and lymphoid) or different stages (i.e. induction and maintenance) of various hematopoietic malignancies.Interestingly, 7. Thus far, a variety of factors, such as JAK/STAT signaling and RAS pathway, have been identified to be closely related with chemotherapy response16. Abnormal activation of JAK/STAT signaling is found in most de novo AML13. K-RAS is a proto-oncogene essential for many solid tumors and hematopoietic malignancies16. Although RAS (i.e. N-RAS or K-RAS) gene mutations were only found in no more than 10% of AML cases, activation of RAS pathway by mutations in upstream receptors, e.g. FLT3 and c-KIT, or downstream effectors, broadly exist in AML45. A set of inhibitors have been developed to target JAK/STAT signaling (e.g. OPB-31121 and pacritinib) or RAS pathway , but their efficacy in clinical trials to treat hematopoietic malignancies was still limited48. Our data suggest that restoration of ALOX5 expression/function could suppress both JAK/STAT and RAS signaling pathways simultaneously, and thus represents an alternative strategy, other than individual small-molecule inhibitors of JAK/STAT and RAS signaling pathways, to target these two critical oncogenic pathways in treating AML. Especially, restoration of ALOX5 expression/function in combination with the standard chemotherapy represents a potentially more effective therapeutic strategy for curing AML, at least MLL-rearranged AML. Thus, it would be interesting to identify small-molecule compound(s), such as natural product(s), that can specifically induce endogenous expression of ALOX5 expression in AML cells, and such compound(s) can be applied together with standard chemotherapy to treat MLL-rearranged AML.One of the major challenges in AML therapy is the high frequency of occurrence of therapeutic resistance and the subsequent relapse29, a total of 100 human AML , 27 inv(16), 31 t and 12 MLL-rearranged) and 9 normal BM samples  samples) were analyzed by use of Affymetrix GeneChip Human Exon 1.0 ST arrays . Robust Multi-array Average (RMA)49 was used for the data normalization with Partek Genomics Suite . The complete microarray data set has been deposited into the GEO database under the accession code GSE34184 and GSE3028543.As described previouslyALOX5, encoding the human ALOX5 gene, was amplified through PCR using primers 5′-ATAACCGGTCCACCATGGATTACAAGGATGACGATGACAAGCCCTCCTACACGGTC-3′ and 5′-AGCGAATTCTCAGATGGGCACACTGTTCGGA-3′, and then cloned into pLJM1-EGFP lentiviral vector . The CDS of mouse Alox5 gene was amplified through PCR using primers 5′-AATCTCGAGCCACCATGGATTAC AAGGATGACGATGACAAGCCCTCCTACACG and 5′-ATTGAATTCTTAGATGGCTACGCTGTTGGGAAT-3, and then ligated into a retroviral vector, namely MSCV-PIG , a kind gift from Drs. Gregory Hannon, Scott Hammond, and Lin He .The CDS of MONOMAC-6 cells were maintained in RPMI 1640 supplemented with 10% FBS, 1% HEPES, 2 mM L-Glutamine, 100 × Non-Essential Amino Acid, 1 mM sodium pyruvate, 9 μg/ml insulin and 1% penicillin-streptomycin. Mouse leukemic cells were kept in RPMI 1640 supplemented with interleukin 3 (IL-3) and IL-6, 10 ng/ml; SCF 100 ng/ml; 10% FBS and 1% penicillin-streptomycin. siRNAs were transfected with Cell Line Nucleofector Kit V following program T-037, using the Amaxa Nucleofector Technology . Experiments were performed 48 h after transfection.ALOX5 or pLJM1-EGFP control were co-transfected into HEK-293T cells in 60 mm cell culture dish with Effectene Transfection Reagent . Lentiviral particles were harvested at 48 and 72 hours after transfection and concentrated with PEG-itTM Virus Precipitation Solution (SBI). The lentivirus particles were directly added into leukemic cells and the cells were washed with PBS 24 hours after infection.pMD2.G, pMDLg/pRRE, pRSV-Rev and pLJM1-EGFP plasmids were purchased from Addgene . 0.5 μg pMD2.G, 0.3 μg pMDLg/pRRE, 0.7 μg pRSV-Rev and 1.5 μg pLJM1-EGFP constructs, i.e. pLJM1-Cells were seeded into 96-well plates at the concentration of 10,000 cells/ well in triplicates and MTT  was used to assess cell proliferation and viability following the manufacturer’s instructions.50, with SABiosciences Corporation’s ChampionChIP One-Day kit  following the manufacturer’s protocol. Chromatin from THP-1 cells were cross-linked, sonicated into an average size of ~500 bp, and then immunoprecipitated with antibodies against the N’-terminal of MLL (MLL-N), the C’-terminal of MLL (MLL-C), EZH2, SIN3A, H3K27Me3 or IgG . Purified DNA was amplified by real-time qPCR using primers targeting the promoter of ALOX5 as described before32.ChIP assay was conducted as described previouslyCells were washed twice with ice-cold phosphate-buffered saline (PBS) and ruptured with RIPA buffer  containing 5 mM EDTA, PMSF, cocktail inhibitor, and phosphatase inhibitor cocktail. Cell extracts were microcentrifuged for 20 min at 10,000 g and supernatants were collected. Cell lysates (20 μl) were resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked for 1 hour with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 and incubated overnight at 4 °C with anti-ALOX5 antibody  or anti-ACTIN antibody . Membranes were washed 30 min with Tris-buffered saline containing 0.1% Tween-20, incubated for 1 hour with appropriate secondary antibodies conjugated to horseradish peroxidase, and developed using chemiluminescence substrates.51. Briefly, retrovirus vectors were co-transfected with pCL-Eco packaging vector  into HEK293T cells using Effectene Transfection Reagent  to produce retrovirus. BM cells were harvested from a cohort of 4- to 6-week-old B6.SJL (CD45.1) donor mice after five days of 5- fluorouracil (5-FU) treatment, and primitive hematopoietic progenitor cells were enriched with Mouse Lineage Cell Depletion Kit . An aliquot of enriched hematopoietic progenitor cells was added to retroviral supernatant together with polybrene in cell culture plates, which were centrifuged at 2,000 g for 2 hours at 32 °C  and then the medium was replaced with fresh media and incubated for 20 hours at 37 °C. Next day, the same procedure was repeated once.CFAs were conducted as described previously with some modifications4 cells were plated into a 35 mm Petri dish in 1.5 ml of Methocult M3230 methylcellulose medium  containing 10 ng/ml each of murine recombinant IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and 30 ng/ml of murine recombinant SCF , along with 1.0 mg/ml of G418 and 2 μg/ml of puromycin. For each transduction, there were two duplicate dishes. Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. The colonies were replated every 7 days under the same conditions. The colony-forming/replating assays were repeated 3 times.Then, on the day following the second spinoculation, an equivalent of 2.0 × 1054.C57BL/6 (CD45.2), B6.SJL (CD45.1) mice were purchased from the Jackson Lab  or Harlan Laboratories, Inc  and maintained in house. Both male and female mice were used for the experiments. All laboratory mice were maintained in the animal facility at University of Cincinnati and the University of Chicago. All experiments on mice in our research protocol were approved by Institutional Animal Care and Use Committee (IACUC) of University of Cincinnati and the University of Chicago. All methods were performed in accordance with the relevant guidelines and regulations. The maintenance, monitoring, and end-point treatment of mice were conducted as described previouslyMLL-AF9 + MSCV-PIG  or MSCVneo-MLL-AF9 + MSCV-PIG-Alox5 , through two rounds of spinoculation, as described previously54. Then, the transduced donor cells were injected by tail vein into lethally irradiated (960 rads) 8- to 10-week-old C57BL/6 (CD45.2) recipient mice with 3 × 105 donor cells plus a radioprotective dose of whole BM cells  per recipient mouse.For primary bone marrow transplantation (BMT) assays, normal BM cells of B6.SJL (CD45.1) mice were retrovirally co-transduced with MSCVneo-+ percentage >90%) were isolated from primary leukemic mice (CD45.2) that received BMT of wild-type BM progenitor donor cells (CD45.1) infected with MSCVneo-MLL-AF9 retrovirus alone. The leukemic cells from the primary recipients were retrovirally transduced with MSCV-PIG (MA9_Ctrl) or MSCV-PIG-Alox5 (MA9_Alox5), and then transplanted into sublethally irradiated (480 rads) 8- to 10-week-old C57BL/6 (CD45.2) secondary recipient mice via tail vein injection. 1.5 × 105 donor cells were transplanted into each secondary recipient mouse.For secondary BMT assays, leukemic BM cells (with CD45.1via tail bleeding and at any time that the mice showed signs of systemic illness to obtain the complete blood counts with white blood cell (WBC) differentials and the blood smears for the presence of immature or abnormal hematopoietic cells. The engraftment was assessed by flow cytometry analysis of CD45.1 in PB. Leukemic mice were euthanized by CO2 inhalation if they showed signs of systemic illness to collect specimens as controls for further analyses. The spleen, liver and thymus were weighed. Cells were obtained from PB, BM, spleen, and liver for flow cytometric analysis. Blood smear and BM cytospin slides were stained with Wright-Giemsa. For histological analysis, portions of the spleen and liver were fixed in formalin, sectioned, embedded in paraffin, and stained with hematoxylin and eosin (H&E).Peripheral blood (PB) cells were collected from the transplanted recipient mice monthly 36 was used to analyze the signal pathway enrichment in different groups of samples. “Hallmark gene sets” obtained from MsigDB (The Molecular Signatures Database) were used as the “gene sets database” input. Vertebrate homology resource from the Mouse Genome Database (MGD)55 was extracted to convert between homologous human and mouse gene symbols.Gene Set Enrichment Analysis (GSEA)56, and/or Bioconductor R packages. The t-test, Kaplan-Meier method, and log-rank test, etc. were performed with WinSTAT (R. Fitch Software), GraphPad Prism version 5.0 , and/or Partek Genomics Suite . The P-values less than 0.05 were considered as statistically significant. Significance analysis of microarrays (SAM)57, embedded in the TMeV package , was used to identify the genes that are significantly  dysregulated in human AML samples relative to the normal controls.The gene/exon array data analyses and qPCR data analyses were conducted by use of Partek Genomics Suite , TIGR Mutiple Array Viewer software package ALOX5 levels and K-RAS mutations is under accession code GSE62190.Data referenced in this study are available in The Gene Expression Omnibus. The Affymetrix exon array data and the microarray data are available under accession codes code GSE34184 and GSE30285. The mouse RNA sequencing data is available under accession code GSE94840. The RNA sequencing results of the AML sample cohort analyzed for the correlation of Supplementary Information


ZBTB7A mutations -AML) were shown to be a target of copy-neutral losses of heterozygosity (CN-LOH) at chromosome 19p. FOXP1 focal deletions were identified in 5% of inv(16)-AML while sequence analysis revealed that 2% carried FOXP1 truncating mutations. Finally, CCDC26 disruption was found in both subtypes (4.5% of the whole cohort) and possibly highlighted a new lesion associated with aberrant tyrosine kinase signaling in this particular subtype of leukemia.Acute myeloid leukemia (AML) with t and inv(16), together referred as core binding factor (CBF)-AML, are recognized as unique entities. Both rearrangements share a common pathophysiology, the disruption of the CBF, and a relatively good prognosis. Experiments have demonstrated that CBF rearrangements were insufficient to induce leukemia, implying the existence of cooperating events. To explore these aberrations, we performed single nucleotide polymorphism (SNP)-array in a well-annotated cohort of 198 patients with CBF-AML. Excluding breakpoint-associated lesions, the most frequent events included loss of a sex chromosome (53%), deletions at 9q21 (12%) and 7q36 (9%) in patients with t compared with trisomy 22 (13%), trisomy 8 (10%) and 7q36 deletions (12%) in patients with inv(16). SNP-array revealed novel recurrent genetic alterations likely to be involved in CBF-AML leukemogenesis.  Compared to other AML subsets, CBF-AML is considered to have a good prognosis. Both alterations result in disruption of genes encoding subunits of the CBF (i.e. RUNX1 and CBFB), a heterodimeric transcription factor complex required for normal hematopoiesis . Measurements were performed on a Navios flow cytometer and analyzed with Kaluza software (Beckman-Coulter).Diagnostic blast cells were obtained from thawed cryopreserved BM samples after red blood cell lysis. Fixation, permeabilization and staining (with both intracellular and cell surface markers) were performed using the PerFix-no centrifuge assay kit (Beckman Coulter) according to the manufacturer’s instructions. The antibody panel contained: anti-AKTpS473-Vio515 , anti-CD33-PC5.5 , anti-CD34-PC7 , anti-CD117/KIT-APC , anti-CD3-AA750 , anti-CD4-PB  and anti-CD45-KO . Blast cells were gated as CD45Failure time data were analyzed and compared after censoring at transplant for patients who received allogeneic stem cell transplantation in first complete remission (CR). RFS and OS were estimated by the Kaplan-Meier method. RFS was estimated taking into account death in first CR for competing risk. Comparisons between patient subgroups were performed by the Mann-Whitney test for continuous variables and by Fisher’s exact test for categorical variables. All statistical tests were performed with the SPSS Statistics software (IBM).


Consequently, overexpression of Otub1 markedly stabilized MDMX and increased its levels, whereas knockdown of Otub1 reduced the levels of MDMX. Interestingly, MDMX induced by Otub1 can localize to mitochondria in addition to the cytosol, enhance p53 phosphorylation at S46 (p53S46P) and promote mitochondria-mediated apoptotic pathway. Knockdown of MDMX reduced Otub1-induced p53S46P, which was shown to be critical for p53's mitochondrial function and apoptotic activity. Furthermore, Otub1 promotes UV-irradiation-induced p53S46P and apoptosis, which can be significantly inhibited by MDMX depletion. Together, these results suggest that Otub1 stabilizes MDMX and promotes p53S46P and mitochondria-mediated apoptosis, providing an alternative mechanism of Otub1's role in apoptosis.Otub1 regulates p53 stability and activity via non-canonical inhibition of UbcH5, the MDM2 cognate ubiquitin-conjugating enzyme (E2). However, whether Otub1 regulates MDMX stability and activity is not clear. Here we report that Otub1 also suppresses MDM2-mediated MDMX ubiquitination in cells and  The tumor suppressor protein p53 inhibits cell growth and proliferation in response to stress by transcriptionally activating or suppressing myriad target genes, whose protein products induce cell cycle arrest, cell death, senescence and other outcomes –3. p53 cUnder physiological conditions, p53 is tightly controlled at low levels mainly by MDM2, a RING-finger domain-containing ubiquitin (Ub) ligase (E3) , 11 thatDeubiquitination catalyzed by the deubiquitinating enzymes (Dubs) has recently emerged as another key regulation of the p53-MDM2-MDMX axis . p53 is in vitro. Interestingly, the stabilized MDMX can localize into mitochondria and mediate mitochondrial apoptotic pathway. Knockdown of MDMX reduced Otub1-induced p53 phosphorylation at S46, which was shown to be critical for p53's mitochondria function, and apoptotic activity. Also, Otub1 promotes UV-irradiation-induced apoptosis, which can be inhibited by MDMX depletion. Together, these results suggest that Otub1 also induces mitochondrial-mediated apoptosis by stabilizing MDMX and relocalizing it to mitochondria to induce mitochondria-mediated apoptosis.Here we report that Otub1 suppresses MDM2-mediated MDMX ubiquitination and degradation. Overexpression of Otub1 markedly stabilized MDMX and induced its levels, whereas knockdown of Otub1 reduced the levels of MDMX. Similar to its regulation of p53, Otub1 stabilizes MDMX independently of its Dub activity. We show that both wild-type Otub1 and its catalytically-inactive mutant (C91S), suppresses MDM2-mediated MDMX ubiquitination in cells and To test whether Otub1 regulates MDMX levels, we first performed transient transfection followed by IB analysis in H1299 cells. As shown in Figure in vivo ubiquitination assays in H1299 cells using Ni2+-NTA purification method. As shown in Figure in vitro reactions. As shown in Figure in vitro (compare lane 4 to lane 3). Also, the recombinant His-Otub1C91S (lane 5), but not the His-Otub1D88A (lane 6), mutant also suppressed the MDM2-mediated MDMX ubiquitination. Thus, Otub1 directly suppresses MDM2-mediated MDMX ubiquitination independently of its canonical Dub catalytic activity.To understand how Otub1 stabilizes MDMX, we tested whether Otub1 suppresses MDM2-mediated MDMX ubiquitination, similar to the suppression of p53 ubiquitination , 40. We As MDMX assists MDM2 to degrade p53, thus inhibiting p53 function –31 whileAs described in our previous work, Otub1 stabilizes and activates p53 and can induce apoptosis . It alsoWe have previously shown that Otub1 plays a critical role in p53 induction in response to DNA damage such as UV irradiation . ConsistIn this study, we show that Otub1 inhibits MDM2-mediated MDMX ubiquitination, leading to the stabilization of MDMX, its accumulation in mitochondria and the cytosol, p53 S46 phosphorylation, and mitochondria-mediated apoptosis. Knockdown of MDMX impaired both the p53-S46P and the induction of apoptosis induced by Otub1 expression as well as in response to UV irradiation, revealing an important role for MDMX in Otub1-induced and mitochondria-mediated apoptosis  supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C in a 5% COescribed , 56. T-Rescribed . Myc-MDMescribed . Anti-Flescribed .®-LT1 reagents following the manufacturer's protocol (Mirus Bio Corporation). Cells were harvested at 36–48 hours posttransfection and lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 0.5% Nonidet P-40, 1 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, 1 μg/ml pepstatin A, and 1 mM leupeptin. For detection of phosphorylated p53, the phosphatase inhibitor cocktails (Sigma) were added in above lysis buffer. Equal amounts of clear cell lysate were used for IB analysis.Cells were transfected with plasmids using TransITIn vivo ubiquitination assay under denaturing conditions was conducted in H1299 cells as previously described [2+-NTA pulldown. After washing, the bead-bound proteins were analyzed by IB.escribed , 56. BriE. coli and purified using Ni2+-NTA purification method as previously described [in vitro ubiquitination reactions were assembled in a total of 20 μl reaction mixture containing recombinant UbE1 , UbcH5 , Ub , 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 2 mM ATP and 1 mM DTT in the absence or presence of 3.2 μM purified His-Otub1 at 37°C for 2 hours, followed by IB as described previously [Recombinant His-Otub1  and His-MDMX proteins were expressed in escribed , 40. GSTeviously , 40.The 21-nucleotide siRNA duplexes with a 3′ dTdT overhang were synthesized by Dharmacon Inc . The target sequences for Otub1 are 5′-GACCAGGCCTGACGGCAAC-3′ (siRNA-1), 5′-GCAGACCTCTGTCGCCGAC-3′ (siRNA-2). The control scramble RNA sequence was described . MDMX kn® reagent following manufacturer's protocol (Invitrogen). Reverse transcriptions were performed as described [™ Universal SYBR Green Supermix (Bio-Rad) as described previously [p21, mdm2 and GAPDH were described [Total RNA was isolated from cells using Trizolescribed . Quantiteviously , 58. Allescribed . The pricells-1 buffer . After hypotonic swelling on ice, the cells were homogenized in dounce tissue grinder until 80–90% of cell damage was attained. The homogenate was centrifuged twice at 600 g for 5 min at 4°C. The supernatant was collected and centrifuged at 7000 g for 10 min at 4°C. The supernatant was saved as the cytoplasmic fraction, and the mitochondrial pellet was resuspended in IBcells-2 buffer , followed by a 10 min centrifugation at 10,000 g. The mitochondrial pellet was lysed in mitochondria lysis buffer  and assayed by IB.T-Rex-U2OS-Flag-Otub1 cells cultured in the presence or absence of doxycycline (2 μg/ml) were resuspended in a hypotonic buffer A  and homogenized. After centrifugation, the supernatant was collected as the cytoplasmic fraction. The pellets were resuspended in buffer C  and sonicated. The nuclear fraction (supernatant) was collected by centrifugation. For isolating mitochondria, cells were trypsinized and washed twice in phosphate-buffered saline (PBS) and resuspended in ice-cold IB


We find that presynaptic Ca2+ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long‐term activity changes, dependent on the Ca2+‐sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1‐mediated activity‐dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca2+ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity.Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca Moreover, we demonstrate that mitochondrial localisation at presynaptic terminals is tuned by long‐term changes in network activity dependent on Miro1‐mediated mitochondrial trafficking. Further, we show that baseline Ca2+ responses and homeostatic changes in the presynaptic response are altered in the absence of Miro1‐mediated Ca2+‐dependent positioning of mitochondria. This provides evidence for a novel mechanism by which mitochondria can alter presynaptic transmission and play a role in the tuning of synaptic signals during homeostatic plasticity.Here, by imaging presynaptic Ca2+ signals evoked in presynaptic terminals containing mitochondria compared to terminals without mitochondria, we co‐transfected hippocampal cultures with the mitochondrial marker MtDsRed and the presynaptically targeted version of the genetically encoded Ca2+ indicator GCaMP5  was significantly greater (ΔF/F0 = 3.5 ± 0.4) than in terminals containing mitochondria ; see Materials and Methods), we compared the average presynaptic Ca2+ response in the presence of a mitochondrion. We found that a train of 10 stimuli (10 APs) was insufficient to generate a difference in Ca2+ response  and thus used 30 min Ru360 treatment to block its activity. In the control cultures, presynaptic terminals containing a mitochondrion had a decreased mean Ca2+ signal (ΔF/F0 = 3.5 ± 0.4) compared to terminals without a mitochondrion . When MCU activity was blocked, the presence of mitochondria no longer affected the mean stimulation Ca2+ signal ΔF/F0 . Thus, mitochondria play a key role in the regulation of the size of presynaptic Ca2+ signals during trains of action potentials, contingent on their ability to buffer Ca2+ in the terminal via MCU.We then asked whether the observed difference in Ca2+ buffering can significantly affect presynaptic Ca2+ signals at individual synapses, we determined whether the presence of mitochondria impacts on transmitter release. To image vesicular release, we used the superecliptic pHluorin‐based probe, VGlut1pHluorin, while imaging mitochondrial positioning with a mitochondrially targeted variant of LSS‐mKate2   P < 0.01), whereas conversely increasing neuronal activity, driven by PTX treatment, increased the fraction of SYN‐GFP synapses containing mitochondria compared to control  and SYN‐GFP clusters  throughout the axon was unaltered following both TTX and PTX treatments  following 48 h of PTX treatment. When stimulating neurons for 20 s at 10 Hz, we again detected a significant increase in VGlut1pHluorin signal in terminals without a mitochondrion compared to terminals occupied by a mitochondrion within the same axon  and a Ca2+‐dependent mitochondrial stopping and recruitment to areas of high Ca2+ load 2+ as Miro1 overexpression increases mitochondrial trafficking 2+ responses during TTX treatment might be sufficient to allow mitochondrial arrest when Miro1 is overexpressed, and therefore, a higher percentage of terminals are always occupied by a mitochondrion. PTX on the other hand has the same effect whether Miro1 is overexpressed or not, because during periods of high activity mitochondria are eventually repositioned to presynaptic terminals in response to the frequent Ca2+ signals. We therefore propose that mitochondria are arrested at terminals in a Miro1‐mediated activity‐dependent manner, followed by tethering of the mitochondria by a protein such as syntaphilin Several studies have shown that Miro1 and its two EF‐hand domains contribute to Ca2+ responses 2+ signals and transmitter release  resulted in the expected increase in presynaptic Ca2+ signals, likely due to the fact that less terminals contain a mitochondrion  and determined whether this interferes with the presynaptic rescaling of Ca2+ signals. To facilitate live cell imaging in ΔEF‐Miro1‐transfected neurons, we co‐transfected SyGCaMP5 with ΔEF‐Miro1‐IRES‐MtDsRed, which allows bicistronic expression of both transgenes thus permitting us to readily identify ΔEF‐Miro1 expression in MtDsRed‐positive neurons co‐expressing SyGCaMP5  is known to vary even in terminals from the same axon 2+ dynamics and fusion probability of vesicles 2+ response upon a train of APs. This is in agreement with a recent report that presynaptic mitochondria in cortical axons attenuate neurotransmitter release by enhancing Ca2+ clearance in an LKB1‐dependent manner 2+ buffering by mitochondria may be the more important mediator of local mitochondrial influence at presynaptic terminals during synaptic transmission.Our findings suggest that mitochondrial occupancy of a presynaptic terminal can tune local CaHomeostatic changes are important for regulating overall levels of activity in neuronal networks by avoiding extreme states of excitation or inhibition in the brain 2+ sensing. This can increase mitochondrial occupancy when terminals are particularly active, thus enabling the mitochondria to provide energy and buffer Ca2+ in those demanding conditions. Further, repositioning of mitochondria when network activity is altered on a longer timescale can contribute to the rescaling of presynaptic Ca2+ signals during homeostatic plasticity.Thus, we put forward a model whereby mitochondrial localisation at presynaptic terminals is tuned by neuronal activity and Miro1‐mediated Ca2+‐phosphate precipitation or by lipofection with Lipofectamine 2000 at DIV 7 and then imaged at DIV 10–12.Primary hippocampal cultures were prepared as previously described from E16 mice MtDsRed, synaptophysin–GFP, myc‐Miro1, myc‐ΔEF‐Miro1, VGlut1pHluorin and Mito‐LSSmKate2 have been previously described After fixation using 4% PFA for 5 min, cells were washed twice and blocked in PBS solution containing 10% horse serum, 0.5% BSA and 0.2% Triton. Cells were stained with primary antibody for 1 h: anti‐myc antibody obtained from 9E10 hybridoma lines and used as supernatant at 1:100, anti‐SV2 (Neuromab) used at 1:200, anti‐Piccolo (Synaptic Systems) used at 1:500, anti‐tau (Millipore) used at 1:1,000 and anti‐MAP2 (Synaptic Systems) used at 1:1,000. Cells were washed and stained with secondary antibody for 1 h: anti‐mouse 405 (Jackson Dylight) antibody was used at 1:500, anti‐mouse Alexa 568 (Invitrogen) was used at 1:1,000, anti‐rabbit Alexa 555 (Invitrogen) was used at 1:1,000 and anti‐guinea pig Alexa 647 (Invitrogen) was used at 1:1,000. Images of fixed cultures were taken on a Zeiss LSM700 confocal using a 63× oil objective (NA 1.4) and a 20× water objective (NA 1.0).d‐glucose, 10 mM HEPES, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2, which was brought to a pH of 7.4 using NaOH. An inverted Zeiss Axiovert 200 microscope and a 63× oil objective (NA 1.4) coupled to a Photometrics Evolve camera were used to image frames with 30 ms exposure at either 1 frame per second or maximum frame rate (~18 frames per second) in the software Micro‐Manager Imaging experiments were performed at 37°C while perfusing the coverslips in external solution containing 125 mM NaCl, 10 mM F/F0 were plotted using Mathematica (Wolfram Research), and paired t‐test was used to calculate statistical significance, whereby terminals with and without mitochondria within the same axon were compared. Regions of interest were manually drawn, and after background subtraction, fluorescence was normalised to the first 10 frames. Mean stimulation fluorescence was calculated as an average across a plateau equating to stimulation duration. For co‐localisation analysis, a region measuring 40 × 40 μm was chosen at least 300 μm from the soma. Co‐localisation of SYN‐GFP and MtDsRed was quantified as the fraction of SYN‐GFP clusters which overlap with at least one MtDsRed‐positive pixel. Images were thresholded in ImageJ and, using the Image Calculator tool, a third image was generated showing those pixels which were positive in both input channels. Using the Particle Analysis tool, the size and number of the thresholded clusters were analysed. Microsoft Excel was used to calculate the fraction of MtDsRed‐positive SYN‐GFP clusters. In order to quantify the density of mitochondria and SYN‐GFP clusters within the axon, the whole axon was imaged using the 20× water‐immersion objective. Images were stitched together in ImageJ, to reconstruct the whole axon, and the longest process was traced and straightened. The MtDsRed and SYN‐GFP channels were thresholded and the number of thresholded clusters was analysed using the Particle Analysis tool within ImageJ. This was normalised to the straightened axon length. GraphPad Prism was used to perform ANOVAs and t‐tests and to visualise bar charts. Error bars represent SEM.Movies were aligned using the Cairn Image Splitter plugin in ImageJ. Graphs showing ΔVV, MJD, NFH and JTK designed experiments; VV, MJD and NFH collected and analysed the data; NFH performed molecular biology experiments; and VV, MJD, NFH and JTK wrote the manuscript.The authors declare that they have no conflict of interest.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.