Because NOS2 expression is determined by CaAbcc8−/− and Trpm4−/− mice, and immortalized N9 microglia, following activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS), using in situ hybridization, immunohistochemistry, co-immunoprecipitation, immunoblot, qPCR, patch clamp electrophysiology, calcium imaging, the Griess assay, and chromatin immunoprecipitation.We studied microglia in vivo and in primary culture from adult rats, and from wild type, Nos2 mRNA, NOS2 protein, and NO. Pharmacological inhibition of Sur1 (glibenclamide), Trpm4 (9-phenanthrol), or gene silencing of Abcc8 or Trpm4 reduced Nos2 upregulation. Inhibiting Sur1-Trpm4 increased the intracellular calcium concentration ([Ca2+]i), as expected, but also decreased NFAT nuclear translocation. The increase in [Ca2+]i induced by inhibiting or silencing Sur1-Trpm4 resulted in phosphorylation of Ca2+/calmodulin protein kinase II and of CN, consistent with reduced nuclear translocation of NFAT. The regulation of NFAT by Sur1-Trpm4 was confirmed using chromatin immunoprecipitation.In microglia in vivo and in vitro, LPS activation of TLR4 led to de novo upregulation of Sur1-Trpm4 channels and CN/NFAT-dependent upregulation of 2+-sensitive gene expression, including Nos2.Sur1-Trpm4 constitutes a novel mechanism by which TLR4-activated microglia regulate pro-inflammatory, Ca Toll-like receptor 4 (TLR4)-mediated neuroinflammation figures centrally in a growing list of inflammatory and degenerative conditions of the central nervous system (CNS), including traumatic brain injury, ischemic stroke, hemorrhagic stroke, Alzheimer’s disease, multiple sclerosis, Parkinson’s disease, and amyotrophic lateral sclerosis –4. These2+]i): (i) calcineurin, the Ca2+-sensitive phosphatase that, by dephosphorylating nuclear factor of activated T-cells (NFAT), promotes its nuclear translocation to induce Nos2 gene expression i may be crucial for nitrosative injury induced by activated microglia i linked directly to membrane depolarization, thereby providing negative feedback to Ca2+ entry through SOCE or ROCE channels  and FK506 were purchased from Enzo Life Sciences . Papain, dispase II, glibenclamide, 9-phenanthrol, diazoxide, SKF-96365, A23187, 1,2-bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), KN-93, and Percoll were purchased from Sigma-Aldrich . 11R-VIVIT was purchased from EMD Millipore . The TLR4 signaling inhibitor, TAK-242, was purchased from Invivogen . Artificial cerebrospinal fluid (aCSF) was purchased from Tocris Bioscience . All culture media, sera, antibiotics, DNase I, Fluo-4-AM, and pluronic were obtained from Thermo Fisher Scientific . All drugs and Fluo-4-AM were solubilized in dimethylsulfoxide (DMSO) vehicle. Papain, dispase II, and DNase I were solubilized in culture media. Sera and antibiotics were added directly to culture media.Lipopolysaccharide (LPS) , and the dura was opened sharply. A pre-loaded mini-osmotic pump was attached to a brain infusion kit, and the needle was advanced through the burr hole to a final depth of 5 mm under stereotaxic guidance. Cyanoacrylate glue was used to secure the applicator to the dorsal surface of the skull.Abcc8−/− and Trpm4−/− mice were obtained as described previously -1-propanesulfonate (CHAPS) lysis buffer  supplemented with freshly added protease and phosphatase inhibitor cocktail  and spermidine . Crude lysate was homogenized by centrifugation through a Qiashredder column , and the pellet was gently resuspended to minimize loss of hydrophobic membrane proteins. Prior to lysate collection for co-immunoprecipitation experiments, protein crosslinking was performed in cell culture dishes using 1 mM DSP (dithiobis(succinimidyl propionate); Thermo Fisher Scientific) according to the manufacturer’s instructions.We followed our previously validated approach to evaluate the expression of Sur1 and Trpm4 by immunoblot . To immuFor the analysis of the subcellular localization of proteins in N9 microglia, an optimized protocol for the fractionation of cytoplasmic versus nuclear protein was performed rapidly, as follows. First, adhered cells were incubated in ice-cold hypotonic buffer containing dilute detergent  for 3 min. With the aid of a cell scraper, cells were collected into a 1.5-mL tube and vortexed for 3 s. The contents were immediately centrifuged for 5 min, and the supernatant containing soluble cytoplasmic proteins was collected in a new tube. The remaining pellet containing intact nuclei was then resuspended in radio-immunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific) supplemented with PPI and left on ice for 10 min to allow for dissolution of nuclear membranes. Following this, a reduction in the viscosity of nuclear protein samples was carried out by homogenization through a Qiashredder column, as above. Successful fractionation was confirmed via immunoblot of the cytoplasmic protein lactate dehydrogenase (LDH) and the nuclear protein histone deacetylase 1 (HDAC1). For all other applications, unless otherwise stated, protein was harvested in RIPA lysis buffer supplemented with PPI and homogenized by centrifugation through a Qiashredder column.The following primary antibodies were used: mouse anti-NFATc1 ; rabbit anti-phosphorylated CaMKII ; rabbit anti-CaMKII ; mouse anti-NOS2 ; rat anti-HSC70 ; rabbit anti-phosphorylated calcineurin ; rabbit anti-calcineurin ; rabbit anti-LDH ; and mouse anti-HDAC1 . Protein was detected using species-appropriate horse radish protein-tagged secondary antibodies . Detection was performed using the ECL system  with routine imaging (Fuji LAS-3000) and quantification (ImageJ). For NFATc1, band densities of each isoform (1-3) were combined into a single value for quantification, which was done similarly for the two major isoforms of CaMKII (α/β). Acquired data were normalized to appropriate loading controls.2+ were assessed in N9 microglia using a Ca2+-sensitive indicator, Fluo-4-AM (Invitrogen), as previously reported i, and glibenclamide inhibition of Sur1 decreased [Ca2+]i  channels i and the transcription factor, NFAT, similar to TLR4-activated primary cultured microglia i using the Ca2+ ionophore, A23187, significantly increased nuclear NFATc1. TLR4 activation for 24 h, which increases [Ca2+]i   Fig. . Nuclearne) Fig. .2+]i induced by channel blockade caused activation of CaMKII. In vascular smooth muscle cells and cardiac myocytes, increasing [Ca2+]i activates not only CN but also CaMKII, with higher levels of Ca2+/calmodulin being required to activate CaMKII, compared to CN [2+]i, led to a significant increase in phosphorylated CaMKII (pCaMKII) . Inhibition of NFAT, either indirectly by FK506 inhibition of CN, or directly by 11R-VIVIT i and oscillatory Ca2+ signaling i, consistent with the absence of involvement of VOCE channels. The most parsimonious explanation for our combined observations is that Sur1-Trpm4 channels are the dominant contributors to the effects of Sur1-modulation in TLR4-activated microglia. Sur1-Kir6.2 channels also may present, but the net effects of Sur1-modulation in TLR4-activated microglia appear to be determined by Sur1-Trpm4.In TLR4-activated microglia, the dominant Sur1-regulated channel appears to be Sur1-Trpm4, not Sur1-Kir6.2. First, we showed that Sur1-activation by diazoxide has opposite effects in TLR4-activated microglia compared to quiescent microglia. In quiescent microglia, Sur1 activation 2+ homeostasis contribute to microglia-mediated progression of CNS disorders [2+]i is critical for the initiation and maintenance of distinct transcriptional programs underlying potentially harmful microglial phenotypes i linking directly to membrane depolarization, providing negative feedback that opposes additional Ca2+ entry. Co-assembly with Sur1 increases the apparent sensitivity of Trpm4 to intracellular Ca2+, thereby strengthening Trpm4’s role as a negative regulator of Ca2+ entry i than by continuous Ca2+ influx [2+]i accompanied by manifestations of the activated phenotype, including morphological changes, Sur1-Trpm4 upregulation, and Nos2/NOS2 induction.Calcineurin is a critical mechanism by which activated microglia shape their response to TLR4-ligation and control their phenotype , 87. Thefeedback , 89. NFA+ influx , 90. In 2+]i but that it significantly reduced activation of NFATc1 led us to consider alternative Ca2+-dependent mechanisms regulating CN. Importantly, sustained elevations of [Ca2+]i result in autonomous, persistent activation of CaMKII -1-propanesulfonate; ChIP, chromatin immunoprecipitation; CN, calcineurin; CNS, central nervous system; DAMP, danger-associated-molecular pattern; DAPI, 4′,6-diamidino-2-phenylindole; DIG, digoxigenin; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Gfap, glial fibrillary acidic protein; HBSS, Hank’s balanced salt solution; HDAC1, histone deacetylase 1; Iba1, ionized Ca2+-binding adapter molecule 1; IMDM, Iscove’s modified Dulbecco’s medium; IP, immunoprecipitation; ISH, in situ hybridization; KATP, ATP-sensitive potassium channel; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; NFAT, nuclear factor of activated T-cells; NO, nitric oxide; NOS2, inducible nitric oxide synthase; NS, normal saline; PPI, protease and phosphatase inhibitor cocktail; qPCR, quantitative real-time polymerase chain reaction; RIPA, radio-immunoprecipitation assay; ROCE, receptor-operated Ca2+ entry; ROI, region of interest; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SIP, standard isotonic Percoll; SOCE, store-operated Ca2+ entry; Sur1, sulfonylurea receptor 1; TLR4, Toll-like receptor 4; Trpm4, transient receptor potential melastatin 4; VOCE, voltage-operated Ca2+ entry; WT, wild type[Ca


We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca A representative example of a Ca2+/EGTA buffer curve is given in For determination of the concentration of Caescribed . K0.5, in a total volume of 1 ml at 25°C. The electrode was calibrated using O2-saturated water. Each assay contained 0.4 μM carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) and 1 mM NAD(P)H, and was started with the addition of 20 μg mitochondrial protein. For easier comparison, the activities were recalculated to NAD(P)H oxidation using a factor of 2 NAD(P)H/O2.O340-A400 in an Olis Aminco DW2a spectrophotometer (5 nm slit) at 22°C in a total volume of 1 ml, unless otherwise specified. NAD(P)H was used at 80 μM. For measurements with O2 as final electron acceptor, 0.4 μM FCCP was added, whereas assays with decylubiquinone (DcQ) as electron acceptor contained 1 mM KCN and 20 μM DcQ. Reactions were started with the addition of 0.01–0.04 mg mitochondrial protein or 0.02–0.03 mg E. coli membrane protein. Determinations of rate changes after shifts in Ca2+ concentrations were carried out in the same way, but in a 2 ml volume with constant stirring by a magnetic micro stir bar.NAD(P)H consumption was measured by dual wavelength spectrophotometry as A0.5(Ca2+) were extracted. R for the curve fits was on average 0.97 and in each case at least 0.90. The equation used for the curve fit wasv is the rate, a and b are minimum and maximum rates, respectively, c is –log (K0.5(Ca2+)) and d is a constant.For analysis of NAD(P)H oxidations activities at different free pCa, data from all biological replicates were pooled and fitted using sigmoidal curve fit in Kaleidagraph 4.1.3 . From the curve fits, values and standard errors for maximum rate and K2+ concentration, curve fitting was carried out using a least square exponential decay function in Kaleidagraph 4.1.3, and activity half-life values were calculated separately for each biological replicate before averaging. R was for each curve fit at least 0.97. The equation used for the curve fits wasv is the rate, a is the minimal rate, b is the maximum rate, c the rate constant and t the time. Half-life (t1/2) was then calculated based onFor determining the rate of activity changes after a rapid decrease in Ca-1 mg protein-1 (mean ± SE) and an Alm latency of 94 ± 1.5%. This indicates that the inner membranes of the mitochondria were practically impermeable to reduced pyridine nucleotides, and that a measured NADPH oxidation in the absence of Alm would only be due to the activity of the external NADPH DH.The inner membrane integrity of isolated mitochondria from potato tubers was tested as the Alm permeabilisation-dependence of EGTA-insensitive NADH oxidation, the activity of which depends on the access of NADH to membrane-bound DHs oriented only towards the matrix. We observed a high maximum activity of 480 ± 85 nmol min2+-dependent in the neutral pH range but not at low pH in spinach and H. tuberosus mitochondria  at pH 6.8 and 7.5 (2 at different concentrations of Ca2+ is shown in 0.5(Ca2+) determinations at pH 7.2 [0.5(Ca2+) for NADH oxidation is affected little by pH. In contrast, NADPH oxidation was halfway activated at pCa values of 5.19±0.12 and 3.95±0.05 at pH 6.8 and 7.5, respectively, corresponding to a 19-fold difference in K0.5(Ca2+) between the two pH values. Thus, NADH oxidation in potato mitochondria is activated by low Ca2+ concentrations, but NADPH oxidation demands higher Ca2+ concentrations, which at pH 7.5 is outside the physiological range. The large variation in K0.5(Ca2+) observed for NADPH oxidation indicates that the Ca2+ activation of NDB1 strongly depends on the pH.External NADH oxidation in t pH 7.2 ,29. To d and 7.5 . NAD(P)Ht pH 7.2 ,29 and f2+ and pH regulation in more detail we measured Ca2+ activation of the NADPH oxidation activity using different electron acceptors and media by dual wavelength spectrophotometry. The StNDB1 enzyme was assayed as present in isolated potato mitochondria and AtNDB1 in membranes isolated from the E. coli MWC008(DE3) double mutant lacking NADH DHs and expressing recombinant AtNDB1 [0.5(Ca2+) was lowest at pH 6.8 (0.95 μM) and increased at higher pHs, up to 24 μM at pH 7.5 (0.5(Ca2+) at pH 7.2 was similar independent of the buffering compound used, being 5.3–5.4 μM in both a zwitterionic buffer  and a BisTris/triethanol amine-based buffer system (Medium 2).To investigate the interaction of Cat AtNDB1 . For NADt pH 7.5 . The K0.0.5(Ca2+) for NADPH oxidation to DcQ by potato mitochondria was 1.5 and 6.2 μM at pH 6.8 and 7.2, respectively (0.5(Ca2+) for NADPH oxidation to O2 was, 4.4 and 27.5 μM at pH 6.8 and 7.2, respectively  values were observed, being 24.1 and 81.8 μM, with DcQ and O2 as final electron acceptors, respectively (0.5(Ca2+) values using DcQ as acceptor are highly similar in potato and A. thaliana, and substantially lower than what is observed for potato mitochondria using O2 as acceptor.The Kectively , i.e. siectively . Using tectively , which iectively . With th and 7.2 . At pH 7ectively . For allectively . The res2+ in the cytosol is strongly fluctuating, displaying upward transient spikes over a low “resting” concentration around 100 nM [H. tuberosus mitochondria has been shown to be slowly responsive to a decreased Ca2+ [2+ concentration. Under continuous measurement in a dual-wavelength spectrophotometer equipped with rapid stirring, an initial NAD(P)H oxidation rate was measured in the presence of 50 μM EGTA to certify minimal Ca2+-concentrations. Surplus amounts of CaCl2 and EGTA were then consecutively injected into the medium . Also, at pH 6.8 both NADH and NADPH oxidation responded rapidly to EGTA addition. However, at pH 7.2 NADH and NADPH oxidation deviated. It took substantially longer time for the NADH oxidation to reach steady state after the EGTA-induced decrease in free Ca2+ , whereas for NADPH oxidation half-maximum inhibition occurred in 5 s  in N. sylvestris was co-suppressed with the StNDB1 (Three families of type II NAD(P)H DHs are present in plant mitochondria, and among these only the NDB-proteins are external ,44,45. Te StNDB1 . Thus, pA. thaliana mitochondria, AtNDB1 and AtNDB2 contain canonical EF hand motifs [2+ [2+-independent AtNDB4 NADH dehydrogenase, but this domain contains protein segments that deviate from EF hand motifs in several positions [2+-dependent and -independent NADH DHs since NADH oxidation is variably dependent on Ca2+ in mitochondria from different tissues [H. tuberosus mitochondria, external NADH oxidation has a K0.5(Ca2+) of 0.2–1 μM at pH 7.2 [0.5(Ca2+) for NADPH oxidation has not been reported, but maximum activity has been denoted to be reached at 1 μM Ca2+ for H. tuberosus [2+ for potato cell and durum wheat mitochondria [Cytosolic NAD(P)H is known to be oxidised by plant mitochondria via separate NADH- and NADPH-specific DHs as evidenced by differential induction, tissue dependence and inhibitor sensitivities of the activities ,27,46. Id motifs  that medtifs [2+ . In eachtifs [2+ ,44. A siositions . Potato t pH 7.2 ,29. The chondria .0.5(Ca2+) for NADH oxidation to oxygen to be around 1 μM at both pH 6.8 and 7.5. However, the K0.5(Ca2+) for NADPH oxidation was substantially higher and also pH-dependent, ranging from 4–6 μM Ca2+ at pH 6.8 to 80–120 μM Ca2+ at pH 7.5, with pH 7.2 being intermediate  was 15–20 times higher at pH 7.0 than at pH 7.6 [0.5(Ca2+) for NADPH oxidation may be due to a direct pH effect on the NDB1 protein, changing its Ca2+ affinity. Using DcQ as electron acceptor in AtNDB1 and StNDB1, we observed similar pH dependences as for oxygen reduction via the native ubiquinone, further indicating that pH and Ca2+ affected the NDB1 enzyme directly. However, K0.5(Ca2+) with DcQ was approximately 4 times lower than with oxygen across the pH range. These observations suggest that the binding of Ca2+ to the EF-hand domain in StNDB1 affects binding of the natural ubiquinone and DcQ differentially. Enzyme kinetics of the type II NADH DH in Yarrowia lipolytica (NDH2e) have suggested a ping-pong mechanisms and NADH and DcQ interacting at overlapping sites [S. cerevisiae NDI1p type II NADH DH [2+ binding to the EF hand domain. The plant NDB1 EF-hand domain is attached to the NAD(P)H DH domain as an extension of a surface-exposed loop, which is juxtaposed to the dimerisation surface in the S. cerevisiae NDI1p [Using precisely measured Ca/EGTA buffers and potato tuber mitochondria we determined the K0.5Ca+ for NADate Figs . A pH-det pH 7.6 . Thus, tng sites . A close NADH DH . However NADH DH . If the ae NDI1p ,52, and ae NDI1p ,54.2+-binding proteins, including recoverin, calmodulin, and guanylate cyclase-activating proteins interact also with Mg2+, affecting the Ca2+ binding [2+ had no effect on NADPH oxidation nor K0.5(Ca2+) by potato mitochondria or AtNDB1  for external NADH oxidation was consistent with previous reports and also independent of pH, we observed that surprisingly high Ca2+ concentrations were needed to activate the NDB1 enzymes, and especially at a pH above neutral H fluorescence and Ca2+ in pollen tubes, though it is not known if the pool of fluctuating NAD(P)H was intra- or extramitochondrial [2+ levels does not exclude a role for NDB1 and changes in cytosolic NADPH/NADP+-ratio in growth. For example, addition of auxin to isolated protoplasts from leaves of wheat seedlings induced a rapid increase in the cytosolic Ca2+ concentration [Whereas the K0.5Ca+ for extral Figs . This inhondrial . The depntration .2+ concentrations by the NADPH DH and the rapid adjustment to Ca2+ variations indicate that the NADPH DH is highly responsive to changes in the cytosolic conditions . In contrast, NADH oxidation is more constitutively activated, being dependent on lower Ca2+ concentrations and slowly inactivated by a decrease in Ca2+.In conclusion, we observe that the regulation of the external NADPH DH in plant mitochondria is substantially different from that of the homologous NADH DHs. The pH-dependent demand for high CaS1 Fig2+ concentrations of the different Ca/EGTA-buffered media aliquots plotted against the potential determined for each aliquot using the Ca2+ electrode. For very low Ca2+ concentrations (pCa > 7), the response was not linear. One representative experiment using Medium 1 at pH 7.2 is shown. The apparent Ca/EGTA stability constant in this experiment was 5.742 106 M-1.The figure shows an example of the final calculated pCa values for free Ca(TIF)Click here for additional data file.S2 Fighttp://www.ebi.ac.uk) and used BoxShade (http://www.ch.embnet.org) for shading background according to conservation. Sequence numbering is according to the full-length protein sequences. GenBank accession numbers are as follows: StNDB1 (gi:5734587), AtNDB1 (gi:18417151), StNDB2 (gi:565360770), StNDB3 (gi:565382402), AtNDB2 (gi:18412775), AtNDB3 (gi:240256027), AtNDB4 (gi:15225428). The NAD(P)H binding motif and positions deviating between NADPH and NADH DHs are denoted by ** . EF-hand and EF-hand-like motifs in AtNDB1 and StNDB1 are denoted as previously described . The latter deviates in the NADH DH-types of homologues.Proteins were aligned using Clustal Omega ((TIF)Click here for additional data file.


To explain published data and to understand ROS effects during the onset of acute pancreatitis, a model using multi-valued logic is constructed. Formal concept analysis (FCA) is used to validate the model against data as well as to analyze and visualize rules that capture the dynamics.Recent findings suggest that in pancreatic acinar cells stimulated with bile acid, a pro-apoptotic effect of reactive oxygen species (ROS) dominates their effect on necrosis and spreading of inflammation. The first effect presumably occurs via cytochrome C release from the inner mitochondrial membrane. A pro-necrotic effect – similar to the one of CaSimulations for two different levels of bile stimulation and for inhibition or addition of antioxidants reproduce the qualitative behaviour shown in the experiments. Based on reported differences of ROS production and of ROS induced pore opening, the model predicts a more uniform apoptosis/necrosis ratio for higher and lower bile stimulation in liver cells than in pancreatic acinar cells. FCA confirms that essential dynamical features of the data are captured by the model. For instance, high necrosis always occurs together with at least a medium level of apoptosis. At the same time, FCA helps to reveal subtle differences between data and simulations. The FCA visualization underlines the protective role of ROS against necrosis.2+ increase, we implemented the commonly accepted hypothesis of ATP depletion after strong bile stimulation. Using an alternative model, we demonstrate that this process is not necessary to generate the dynamics of the measured variables. Opening of plasma membrane channels could also lead to a prolonged increase of Ca2+ and to necrosis. Finally, the analysis of the model suggests a direct experimental testing for the model-based hypothesis of a self-enhancing cycle of cytochrome C release and ROS production by interruption of the mitochondrial electron transport chain.The analysis of the model demonstrates how ROS and decreased antioxidant levels contribute to apoptosis. Studying the induction of necrosis via a sustained CaThe online version of this article (doi:10.1186/s12918-014-0101-7) contains supplementary material, which is available to authorized users. Increases of ROS occur early in the disease, and preclinical antioxidant treatments significantly reduced pancreatic injury and inflammation. However, clinical tests of antioxidants produced conflicting results and were even stopped because of excess adverse events  available at www.sbi.uni-rostock.de/resources/software/. Concept hierarchies were visualized with Concept Explorer [http://sourceforge.net/projects/conexp/] and – allowing a better manual layout – Siena from the ToscanaJ suite [http://sourceforge.net/projects/toscanaj/]. Concept Explorer was also used to convert formal contexts from *.csv to *.cxt, readable by ConImp [www.mathematik.tu-darmstadt.de/~burmeister/] that computed the stem bases.To generate the formal contexts from simulations and data, we programmed R scripts i is low , then at t + 1 the calcium concentration is enhanced to level 1. Without TLC-S or after medium [Ca2+]i, intracellular calcium remains at or decreases to level 0.The equation reads: If at a time point t = 1, at least a low bile stimulus is present , the ZG and through the plasma membrane . Within our model, however, this cycle can only be initiated by a prolongation of a Ca2+ pulse. The missing link in the causal chain is provided by influx of extracellular Ca2+ through store-operated Ca2+ plasma membrane channels: Without extracellular Ca2+, the strong calcium increase after high CCK stimulation is shorter and trypsinogen, the precursor of the digestive enzyme trypsin, is not activated :2+ → Pores —| Pot → ATP —| Ca2+:For the cycle CaThroughout the simulations, a basic ATP level is assumed, even for low potential. It is necessary for the initiation of apoptosis:The term !Caembrane H. An important hypothesis analyzed by our model is: "Increases in ROS … promote apoptosis but not necrosis" , Figures2+ has antagonistic effects on ROS. On the one hand, it activates the citric acid cycle, which leads to the production of NADH, that in turn fuels the ETC i oscillations occur during 200 μM bile stimulation for 55% of the cells H peak after the onset of 500 μM stimulation for human cells Figure F/G, then2+ oscillations with 200 μM TLC-S stimulation.For both stimuli, a medium fraction of all cells undergoes apoptosis . There is strong necrosis for 500 μM TLC-S. In accordance with , we concROS production but not release from the mitochondria increases for both stimuli.The following time profiles are reproduced by our simulations Figure :[Ca2+]i Another notable result is that apoptosis increases and necrosis decreases for 500 μM bile stimulation, if antioxidants are deactivated. This observation is reproduced by the respective simulation  leads to swelling of the mitochondrial matrix, rupture of the outer mitochondrial membrane (OMM) and a ROS burst, possibly caused by loss of antioxidants , p. 440.t + 3 ROSt = 2 CytCt+1.The ROS burst is supposed to cause the strong Cyt c release observed in liver cells under the same conditions  blocks ROS production, thus inhibiting cytochrome c release” i oscillations induced by medium bile stimulation promote ROS production, Cyt c release, activation of the caspase cascade and apoptosis. Increasing stimulation obviously does not influence these processes much. Instead, a large fraction of the cells undergoes necrosis via a positive Ca2+ feedback probably caused by enhanced mitochondrial pore opening, breakdown of the membrane potential and of ATP production, which hinders clearance of Ca2+.The model explains how global [Ca2+ between the cytosol, the ER, ZG and the extracellular space by a phenomenological rule generating oscillations H and antioxidants.We implicitly considered the exchange of Ca2+ increase. Even though the ATP depletion hypothesis is widely accepted and strong experimental evidence is given, the conflicting evidence from older experiments by [Also within our modelling framework, interesting biological questions still remain open. Theoretically, the model structure does not require ATP depletion. Instead, a sufficient long opening of plasma membrane channels could produce the same sustained Caments by  is still2+ during one  cell-wide Ca2+ oscillations, which have a frequency of approximately one per minute. There is evidence for single interactions of this hypothesis [Another testable hypothesis implied by the model is a self-enhancing cycle of Cyt c release and ROS production by interruption of the electron transport. This positive feedback should be activated by a primary ROS increase via Capothesis ,28, whicIn conclusion, this study contributes to a more complete picture of ROS regulation and effects, beyond its predominant negative role as a cell damaging agent within the classical free radical theory of ageing. Our analysis, combining logical modelling and FCA, describes how ROS shift the balance from necrosis to apoptosis at the onset of acute pancreatitis and thus counteract the spreading of inflammation. www.sbi.uni-rostock.de/resources/software/multi-valued-logic-and-fca/.R scripts for simulations and FCA analyses see In addition, the model was reimplemented with GINsim , exporte


Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA ethane-N,N,N′,N′-tetra-acetic acid) for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging.Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca In the case of Ca2+, the spatial restriction of subcellular Ca2+ gradients results from the combination of a localized and finely tuned Ca2+ entry across the plasma membrane (or from the release of Ca2+ from intracellular Ca2+ stores) and the strong cellular Ca2+ buffering capacity, limiting the subsequent diffusion by Ca2+-binding to mobile and fixed Ca2+ buffers 19] respeet al., 2014  was adjusted using the Invitrogen Ca Buffer kit . For HR-PiAC titration we used the universal pH buffer, see Supplementary 3, p. 830 in [Methods used for CaRubies2 have been published previously . Briefly. 830 in  which haConfocal Microscopy: Plated cells were incubated with the pH nanosensors (at 100 nM QDs in DMEM nutrient medium without serum) for 2 h, and then for 30 min with 500 nM Lysotracker Green. After they were washed twice with phosphate-buffered saline (PBS) alone , finally a HEPES buffered DMEM medium was reintroduced. Live cells were then immediately analyzed by confocal laser scanning microscopy using a Zeiss LSM operating system. pH probes (561 nm) and Lysotracker green (488 nm) were simultaneously excited and emission fluorescence was collected.2, 37 °C). After 2 h, the cells were harvested using TrypLETM Express Enzyme  and resuspended in a CO2-independent medium .The intracellular calibration of the pH sensors was performed using BHK-21 cell line. Plated 80% confluent BHK cells were incubated with 100 nM QDs for 1 h in the medium without antibiotics or fetal calf serum, and then the cells were allowed to rest in an incubator -1-piperazineethanesulfonic acid) (HEPES) for buffers in the pH range 7.0–8.0. DAPI  was added to exclude the dead cells. After incubation for 5 min at room temperature the samples were analyzed by a flow cytometer . For acquisition, a 532 nm or 405 nm wavelength lasers were used for H-Ruby direct or FRET excitation, respectively, and its emission fluorescence was collected in a 610 ± 20 nm spectral detection channel. Each time 10,000 events were acquired. A FlowJo vX.0.7 software was used for the analysis. First gating on the cells by forward/side scatter was done, then a population of live cells was selected and a mean fluorescence intensity of H-Ruby for each sample was determined. The K2+ and H+ Ruby sensors having absorption and emission spectra matching that of a green-emitting commercially available QD donor, CANdot®565 and leading to a calculated Förster radius of about 4.5 nm  thereby indicating an efficient FRET. The fluorescent acceptor combines an extended rhodamine fluorophore, an ion specific-chelating group and a linker arm allowing its conjugation to the QD surface  and 1 to 10 ion-sensitive acceptor (A) dye molecules A. For th surface B–D.F increases upon Ca2+ binding, its absorbance remains unchanged so that the FRET between donor and acceptor is independent of analyte binding and merely relays excitation, leading to an apparent large Stokes’ shift of 40 nm when excited in the blue (at 405–407 nm). However, ΦF changes from a quenched state at low analyte concentration, to a bright deep-red sensitized emission at higher analyte concentration. The FRET efficiency increases with increasing A:D ratio, as expected for multi-acceptor FRET that offers multiple non-radiant deexcitation pathways and acceptor orientations for the QD donor. As a result, at low acceptor concentrations, the nanobiosensor can be located upon blue excitation and detection of green/yellow QD emission, and the analyte concentration be monitored above 600 nm either by FRET or by direct excitation of the Rubies near 560 nm. For a multi-acceptor system (with a A/D >5), the nanoparticle is merely a carrier to up-concentrate the ion sensor, and the donor fluorescence is so much quenched that, even at resting low ion concentration, the ensemble can only be located by the red emission.In the Calcium Ruby probes, while the fluorescence quantum yield Φ2+-sensing moiety  or else a phenol-based moiety for H+ sensing (EP 13 199 575.5). In these compounds  rhodamine, incorporating either a BAPTA-based Caompounds B–D, ion  probes, ) or to t2+ binding affinity [All Calcium Rubies (CaRuby) bear  was constructed having the side arm attached to the ethylene glycol bridge between the two aromatic rings of the BAPTA ,16,17. TCaRuby2 . A fluorescence quantum yield of ФF = 0.64 at pH 4 as well as a large dynamic range of 86-fold makes it a promising tool for studying cytosolic pH gradients and signaling events. Of note, this new pH sensor has no sensitivity to divalent cations . After several attempts with different commercial and custom nanoparticles, we opted for these CdSe core/CdS-ZnS shell-type QDs that proved to be very stable, the hexane of a single initial stock solution being substituted for toluene when needed. While these non-functionalized peptides-coated QDs displayed no change in absorbance when changing from low to high ambient Ca2+ concentrations, their emission displayed a sensitivity to pH  crosslinking reaction was linked to the QDs as a maleimide-PEG-Ca/HRuby.To link Ca/HRuby, a NHSimilarly, the cell-penetrating peptides (CCPs) to be added were prepared with a maleimide function.®/CaRuby2-F-pair produced an efficient FRET with an increase in efficiency as the number of bound CaRuby molecules on the QD surface increased, as expected for multi-acceptor FRET  increased up to 80% while acceptor sensitization plateaued at 20% when the number of PEG5kDa-CaRuby was increased to 10.Measured mixed donor and acceptor emission spectra shown in A,C were 2+-sensitivity of the assembled FRET pairs with fluorimetric titrations in which we switched from a nominally Ca2+-free solution  to saturating  and we used the relative increase in CaRuby fluorescence (dynamic range DR = (F − F0)/F0) as an index of sensitivity. For H sensors, we proceeded analogously by switching from basic conditions (pH = 10) to an acidic solution at pH 4 and again we used DR as an index of sensitivity.As previously with CaRu1-Me, we systematically investigated whether the new second-generation CaRubies retained their ion sensitivity once they were coupled to the QD surface. We characterized the Ca2+vs. H+ sensors, respectively. Although CaRubies retained a high Ca2+-sensitivity after binding to PEG by click chemistry, QD-CaRuby pairs built from shorter PEGs were unexpectedly largely Ca2+-insensitive. This observation initially made with PEG0.3kDa -CaRu1-Me   also2+)  value for QD-HRu-PiAC was similar when measured upon direct or FRET excitation of the dyes (8.25 ± 0.06 and 8.4 ± 0.1 respectively), and only slightly shifted towards lower affinity as compared to the free dye (KD = 7.8 ± 1). The situation is different with QD-CaRu2 where FRET excitation yields a notably higher KD(Ca2+) than the one obtained by direct excitation, with a shift up to 1.3 ± 0.3 µM (as compared to 391 ± 2 nM). Such pKa difference may originate from the local environment of the probes and from their distance to the center of the nanocrystals: FRET emission excites preferably the closest probes prone to surface charge sensitivity—and the negative environment found there reduces the acidity of the probe—whereas direct excitation give a general and an average overview of the protonation status of the probes.Next, we examined the analyte binding affinity of Rubies once linked to QDs. As expected, the Kdyes see . KD(H+) + and Ca2+ nanobiosensors for the detection of local, ionic signals in live cells. We first showed the use of QD-HRuby to detect an induced cellular pH change and to reveal the acidic endosomal content macromolecules have the possibility to enter cells by different endocytotic pathways. Depending on the nature of their surface coating, QDs internalization can occur via lipid raft/caveolae endocytosis , via pha2+ transients, we favored a direct cytoplasmic delivery of our nanobiosensors by further linking the QDs to cell-penetrating peptides (CPPs) (see [1 and NR2 subunits of the Ca2+-permeant NMDA receptor (NMDAR), then trypsinized the incubated cells to replate them directly on a quartz slide and let them settle down and attach overnight [2+ transients were evoked by NMDAR activation  and responses were continuously monitored at 4 Hz during 3 min before and during agonist application. 2+ transients differed at different subcellular locations, but were comparable for each site upon successive applications of NMDAR agonists. In a second set of experiments aiming at detecting intracellular CaPs) (see  for a diPs) . Evanes2+ sensitive nanosensor, we now present a conceptually similar FRET-based pH-sensitive nanobiosensor. Both cation sensitive dyes used for this purpose are constructed on extended rhodamine scaffolds that are quenched by the Ca2+/H+-binding moiety, which itself is dequenched upon analyte binding, and in turn relieves the fluorescence quenching. This PET is relieved when a BAPTA motif binds Ca2+ in the former case, or when a phenol ring is exposed to an acidic medium in the latter case. The QD surface chemistry already developed [a of HR-PiAC was only slightly displaced to higher values, and, most interestingly, it kept its wide dynamic range (~30) with maximal fluorescence at pH 4. These observations provide guidelines for the design of a further improved dye for in cell studies. Of course, any modification reducing the high lipophilicity of the rhodamine will be a plus and a slightly more acidic Kd would allow monitoring pH changes in intracellular compartments. By these constructs, we extend the toolbox of smart sensors allowing in cellulo pH mapping [Based on the previous development of a FRET-based Caeveloped  to consteveloped . Therefo mapping ,24.2+ detection as opposed to the maintained good dynamic range of the pH sensors is the most intriguing finding in this study. This effect could probably be attributed to a change in local viscosity around the Qdot shell. In the case of Ca2+ detection, the efficacy of the PET quenching in the CaRuby relies on an efficient TICT (twisted-intramolecular charge transfer) effect, promoted by a fast rotation around the C-N bound leading to a geometrically favorable state  and viscosity (η): log ФF = C + x log η. This effect was well studied in a closely related 4-dimethylamino rhodamine by [For such molecular rotors, the Forster and Hoffman equation links quantum yields (Фamine by  using gl2+ but their sensitivity to pH incited us to introduce a corrective factor in our pH estimates. A shifted pK (8.25 instead of 7.7) of the QD-bound compared to the free indicator was maintained, even after this correction. A contaminating sensitivity of the QD luminescence itself to the analyte would bias measurements. The CdZn/Se QDs that we used here do not display a substantial sensitivity to Ca2+ and also Cl−) in a colloidal polymer [H-aggregates between two fluorescent acceptor molecules linked to the same QD. In view of the small number of dye molecules bound to a single QD, this process is, however, unlikely to occur in our constructs. An accurate reading of ion concentrations would also be made difficult by a surface charge effect, leading to unknown local analyte concentrations near the QD surface as previously suggested . This in polymer ,36,37,38


Huntington disease (HD) is caused by the CAG (Q) expansion in exon 1 of the IT15 gene encoding a polyglutamine (poly-Q) stretch of the Huntingtin protein (Htt). In the wild type protein, the repeats specify a stretch of up 34 Q in the N-terminal portion of Htt. In the pathological protein (mHtt) the poly-Q tract is longer. Proteolytic cleavage of the protein liberates an N-terminal fragment containing the expanded poly-Q tract becomes harmful to cells, in particular to striatal neurons. The fragments cause the transcriptional dysfunction of genes that are essential for neuronal survival. Htt, however, could also have non-transcriptional effects, e.g. it could directly alter Ca2+ homeostasis and/or mitochondrial morphology and function. Ca2+ dyshomeostasis and mitochondrial dysfunction are considered important in the molecular aetiology of the disease. Here we have analyzed the effect of the overexpression of Htt fragments  on Ca2+ homeostasis in striatal neuronal precursor cells (Q7/7). We have found that the transient overexpression of the Htt fragments increases Ca2+ transients in the mitochondria of cells stimulated with Ca2+-mobilizing agonists. The bulk Ca2+ transients in the cytosol were unaffected, but the Ca2+ content of the endoplasmic reticulum was significantly decreased in the case of mHtt expression. To rule out possible transcriptional effects due to the presence of mHtt, we have measured the mRNA level of a subunit of the respiratory chain complex II, whose expression is commonly altered in many HD models. No effects on the mRNA level was found suggesting that, in our experimental condition, transcriptional action of Htt is not occurring and that the effects on Ca2+ homeostasis were dependent to non-transcriptional mechanisms. Although the genetic cause of HD, Huntingtin (Htt), is expressed not only in neural cells but also in other tissues, the primary site affected in this pathology include several brain regions. Besides the cortex, cerebellum and thalamus, one of the hallmark of the disease is the loss of neuronal cells in the striatum (caudate and putamen), with selective damage to the GABAergic medium spiny neurons2+ homeostasis, have also been involved in the pathogenesis of HD. In particular, the involvement of ETC defects, more specifically of complex II, is supported by the finding that administration of the specific inhibitor of complex II 3-nitropropionic acid (3-NPA) induces a degeneration of rat striatal neurons that mimics that seen in the disease,,,,,,Defective mitochondria, which are key players in the maintenance of intracellular Ca2+ dyshomeostasis is also frequently considered as a factor in the aetiology of HD. Ca2+ signaling is crucial for a number of neuronal activities and also for the development and maintenance of neuronal circuits,2+ homeostasis associated to HD could also be due either to transcriptional or non-transcriptional defects. As an example, the increased binding of mHtt to the Inositol-1,4,5-trisphosphate receptor (InsP3R) has been related to a possible enhancement of Ca2+ release from the endoplasmic reticulum (ER)2+ released from the vicinal ER could exacerbate their dysfunction, even if a complex II defect would limit the functioning of the respiratory chain and consequently the ability of mitochondria to accumulate Ca2+2+ homeostasis appears to be more complicated, as increased mitochondrial Ca2+ uptake has been reported in other HD models,,Ca2+ homeostasis in a commonly used HD cell model, i.e. an immortalized striatal precursor cell line (Q7/7)2+ transients could be generated by the stimulation with the InsP3 linked agonists, e.g. the purinergic agonist ATP, or bradykinin2+] with aequorin targeted to the mitochondrial matrix or to the cytoplasm. Ca2+ transients were induced by the InsP3-linked agonist ATP. The data have shown that the overexpression of either the 18Q or the 150Q Htt fragments enhanced mitochondrial Ca2+ transient with respect to controls, i.e. the Q7/7 cells only overexpressing the red fluorescent protein Kate and the Ca2+ probe aequorin. Consistent with the results of Fernandes and coworkers2+ transients, whereas the ER Ca2+ content, evaluated by means of a probe specifically targeted to its lumen, showed a lower value in cells expressing the 150Q fragments.The aim of this work was to study the effects of the Htt fragments on Ca,,,2+ homeostasis by a non-transcriptional mechanism.A number of studies have reported transcriptional effects already occurring a few hours after the induction of mHtt expression Plasmids constructionWild-type (18Q) and mutant (150Q) cDNA of human IT15 exon 1 gene, encoding the N-terminal (1-90) region of the Htt protein, have been obtained by PCR amplification using as template the plasmid pIND-HD exon 1- EGFP 150Q  and the following primers, inserting the EcoRV and BamHI at the 5’- and 3’-ends, respectively: For (5’-CTCTAGATATCATGGCGACCCTGGAAAAGCTG-3’); Rev (5’-GTGGATCCGGTCGGTGCAGCGGCTCCTCAGC¬-3’). Since the intrinsic instability of CAG repeats, a series of PCR products of different size have been obtained, and the fragments carrying 18 and 150 Qs have been specifically selected and purified after agarose gel separation. Upon EcoRV/BamHI endonuclease digestion, DNA fragments have been cloned into the Kate-pcDNA3.1 plasmid digested with the same enzymes, giving recombinant vectors able to express in mammalian cells the Htt18Q- and Htt150Q-exon1 proteins fused at their C-terminus with the red fluorescent protein Kate. All the constructs have been finally verified by sequencing.Cell culture and transfection2. 24 h before transfection, cells were seeded onto 13 mm (for aequorin measurements and RT-PCR experiments) or 24 mm  glass cover slips and allowed them to grow to 70–80% confluence.Clonal striatal cell lines established from E14 striatal primordial of WT-HdhQ7 littermate mouse embryos (Q7/7) were used2+ measurements and RT-PCR experiments, Q7/7 cells were co-transfected with the cDNA encoding the red fluorescent protein Kate, representing our control, or with a cDNA encoding for exon 1 of the Htt gene containing 18Q or 150Q (both fused with Kate) together with the cDNA coding for the aequorin Ca2+ probe targeted to the mitochondrial matrix (mtAEQ) or to the cytoplasm (cytAEQ) in a 1:2 ratio. For FRET analysis, Q7/7 were co-transfected with the red fluorescent protein Kate or 18Q, 150Q expressing plasmids with ERD1 For CaAequorin Measurements2, 20 mM HEPES, 5,5 mM glucose, pH 7.4, 37°C)-containing 1 mM CaCl2 and then transferred to the perfusion chamber of a purpose-built luminometer. Ca2+ measurements were started in the KRB medium added with 1 mM CaCl2, and after 30 sec 100 µM ATP (Sigma) was added as indicated in the Figs. 2, 3. All the experiments were terminated by cell lysis with 100 µM digitonin (Sigma) in a hypotonic Ca2+-rich solution (10 mM CaCl2 in H2O) to discharge the remaining reconstituted active aequorin pool. The light signal was collected and calibrated off-line into Ca2+ concentration values, using a computer algorithm based on the Ca2+ response curve of wt and low affinity aequorin as previously describedMitochondrial aequorin (mtAEQ) and cytosolic aequorin (cytAEQ) were reconstituted by incubating cells with 5 µM wt coelenterazine (Invitrogen) for 1 h in Krebs Ringer modified buffer  with an immersion oil objective 60× UPLAN FLN . Excitation light at 425 nm was produced by a monochromator. The emitted light was collected through a beam splitter  and a 505 nm dichroic mirror. Images were collected by means of the Cell R imaging software (Olympus) and analyzed with ImageJ. FRET Ratio have been calculated according to the formula: YFP (background corrected)/CFP(background corrected).RNA isolation and reverse transcription PCR experimentsmRNA for SDHA and for the isoforms of Ryanodine receptor (RyR) were detected by the RT-PCR. Transfected and untransfected cells were washed three times with cold phosphate-buffered saline and collected using TRIzol reagent (Invitrogen). Total RNA was extracted, according to the manufacturer’s protocol, and 1 µg of total RNA was reverse-transcribed using a Superscript III reverse transcriptase (Invitrogen). An amount of cDNA corresponding to 1–10 ng of total RNA was amplified in 25 µl of a mixture containing 12.5 µl of Platinum SYBR Green qPCR SuperMix-UGD (Invitrogen), 2 µl of primers mixture (2.5 µM each). The PCR cycling parameters were: 94 °C for 7 min, 45 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 15 s. The used primers have the following sequences: SDHA forward, AGGCTTGCGAGCTGCATT; reverse, AATGCCATCTCCAGTTGTCC; RyR1 forward, CGAGAGGCAGAACAAGGCAG; reverse, GGTCCTGTGTGAACTCGTCA; RyR2 forward, GGAAGAAAATGAAGCGGAAA; reverse, AGGGGCACAGATGTTCAGTC; RyR3 forward, GGCCAAGAACATCAGAGTGACTAA; reverse, TCACTTCTGCCCTGTCAGTTTC; GAPDH forward, CAAGGTCATCCATGACAACTTTG; reverse, GGGCCATCCACAGTCTTCTG.Statistical analysisAll of the data are representative of at least ten different experiments unless otherwise indicated. Values are expressed as mean ± SEM. Statistical significance was determined using the Student’s t test. *p < 0.05; **p < 0.001; **** p<10-5, Student’s t-test. Localization of Htt fragments in Q7/7 cells,,To study the effects of Htt fragments on intracellular Ca 2+ homeostasis plasmids carrying the cDNAs of exon 1 of Htt, coding for either 18Q or 150Q fused to the red fluorescent protein Kate were used. As a control we transfected cells with only the plasmid encoding the Kate fluorescent protein. Fig. 1 shows that the cells overexpressing only the Kate protein  and those overexpressing the 18Q Htt fragment  had homogeneous staining in the cytoplasm. However, aggregate-like structures with particularly high fluorescence intensity could be detected in some cells expressing either the 18Q  or the 150Q fragment , suggesting that the strong overexpression of wtHtt somehow favored the formation of aggregates, mimicking what occurs in case of mHtt. These aggregates were similar to those observed in other HD cell models2+ transients in cells overexpressing Htt fragmentsMitochondrial Ca,Impairment of various aspects of the Ca 2+ uptake/extrusion system of mitochondria has been repeatedly described in HD model cells2+ level but not cytosolic Ca2+ transientsThe Htt fragments affect the ER CaTo understand whether the increased mitochondrial Ca2+ transients in 18Q and 150Q- overexpressing cells were linked to alterations of the global cytosolic Ca2+, the cells were transfected with cytAEQ2+ store, the ER: mitochondrial Ca2+ uptake senses Ca2+ released from the ER to generate microdomains of high Ca2+ concentration in close proximity of mitochondria,2+ content of the ER in the three Q7/7 cell populations  to correlate the observed mitochondrial Ca2+ transients to possible changes in the ER Ca2+ levels. The ER-directed FRET based Cameleon ERD12+ binding protein Calmodulin. Binding of Ca2+ to the probe results in a conformational change of D1ER that brings together the two fluorophores allowing FRET to occur. Ca2+ concentration is therefore proportional to the increased FRET Ratio. Our experiments revealed that cells expressing the 150Q fragment had lower levels of Ca2+ in the ER than those expressing 18Q or the controls . These results are in line with the findings by Tang et al3R to modulate its Ca2+ leak activity.We therefore turned our attention to the main intracellular Ca2+ is released by InsP3RER Ca2+ was related to action of Htt on InsP3R rather than on other (ER) Ca2+ release channels, i.e., the Ryanodine receptor (RyR), we explored the expression levels of the three isoforms of RyR  in Q7/7 cells. Interestingly, we found that these cells do not express any isoform of RyR  and Hela cells were used as positive and negative controls, respectively). Thus, Ca2+ was released from the ER of Q7/7 uniquely through the InsP3R.To support the idea that in our cell model, the alteration of mitochondrial and ER CaOverexpression of Htt fragments does not affect the transcription of SDHA,,2+ homeostasis in the mitochondria here described are likely due to a non transcriptional mechanism.Even if the protocol used was unlikely to produce transcriptional effects on mitochondrial membrane proteins; however others have been suggesting the possibility of early transcriptional defect in HD cell models,,,,,,,2+ handling have been described in most of the experimental HD models currently in use,,2+ dyshomeostasis in the development of the disease.The involvement of mitochondrial defects in the molecular aetiology of HD was suggested for the first time more than ten years ago, based on the evidence that 3-NPA, an inhibitor of complex II of the respiratory chain, was per se able to induce Htt-like symptoms2+ overload, i.e. to the induction of the permeability transition (PTP),,,,Opinions on the topic are divergent: some studies have claimed that mitochondria from HD model cells have increased propensity to depolarize and are more susceptible to Ca2+ overload, and were more susceptible to the mitochondrial permeability transition, as also found by other groups,3-controlling enzymes which were mostly downregulated2+ homeostasis in the Q cell experimental model could be explained in two alternative ways: either by transcriptional effects of the stably expressed mHtt, or by a compensatory process that counterbalanced the enhanced response to stress of the mitochondria by altering the levels of intracellular Ca2+ pools. Accordingly, we and others had found that the amount of Ca2+ released upon stimulation of the cells with a cell permeable derivative of InsP3In previous work on stable clones of Q7/7 cells2+ homeostasis by using the wt neuronal striatal precursors Q7/7 transiently expressing the N terminal portion of Htt, either in a wt form (18Q) or carrying a pathologically extended polyQ stretch (150Q)). It had been shown by others that the expression of the N terminal portion of Htt is sufficient to cause acute neuronal toxicity2+ handling defects observed in the presence of the pathogenic Htt forms.This work has explored the direct effects of mHtt on Ca2+ handling in primary striatal neurons from a murine model of HD2+ transients.Consistently with our previous published results comparing Q7/Q7 and Q111/Q111 immortalized striatal cell lines,,2+ peaks in these organells upon stimulation of cells expressing either the 150Q or 18Q exon 1 of Htt. Since cytosolic Ca2+ was not affected, the results were likely due to a direct action of the Htt fragments on the mitochondrial Ca2+ handling machinery or on the Ca2+ toolkit of the ER, that is responsible for the generation of Ca2+ hotspots in the proximity of mitochondria. The evidence that in our experiments both wtHtt and mHtt increased mitochondrial Ca2+ uptake, may suggest that the control of the mitochondrial functions involved in the sensing of the ER linked Ca2+ hotspots could be a physiological function of Htt per se, not specifically of mHtt. In this context, it is interesting that Htt has been found associated to the outer mitochondria membrane (OMM) by means of its N terminus,Further, in agreement with reports on isolated mitochondria2+ handling, we have found decreased ER Ca2+ levels specifically in the case of mtHtt. As previously claimed, Htt interacts with the Htt associated protein (HAP1A) to form a complex with the InsP3R, promoting its opening2+ release through InsP3R. Recent evidence underlines the importance of the specific transfer of Ca2+ to mitochondria by InsP3R for cell bioenergetics3R generates mitochondrial Ca2+ hotspots on the OMM surface, that drive Ca2+ uptake in the mitochondrial matrix,2+ between ER and mitochondria. Further studies on this aspect would be necessary to shed light on the mechanisms underlying Ca2+ dyshomeostasis in HD.As to the ER CaThe authors declare that there are no conflicts of interest.


In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.Calcium (Ca As a coe kidney . In the le (CNT) . This is(OH)2D3) . In the  (TRPV5) , 9. Subse (PMCA) , 11.2+ ATPase pumps. Four different genes encode for PMCA 1 to 4. Both PMCA1 and 4 are ubiquitously expressed and have been suggested to have a housekeeping function, extruding Ca2+ from the cell [2+ signaling in sperm motility, B-lymphocytes and cardiac nitric oxide signaling [PMCA is a member of the P-type ATPase family, and is related to the sarcoplasmatic/endoplasmatic reticulum Cathe cell . PMCA1 kthe cell , 14. Thiignaling –16.2+ transport in the late DCT and CNT [et al. investigated the exact localization of PMCA4 in the kidney, by co-staining the different tubular segments with representative markers [28k [2+ reabsorption.In the kidney, PMCA1 and 4 transcripts and protein are found in the proximal tubule, but higher expression has been shown at the distal part of the nephron , 18. PMC and CNT . Recentl markers . They veers [28k , 22. On 2+ handling. To this end, the PMCA4 KO mouse model was used and its Ca2+ balance was compared to wild type (WT) and heterozygous (HZ) littermates. There were no significant differences in serum Ca2+ levels or urinary Ca2+ excretion between groups or changes in expression of Ca2+-related genes.In this study we assessed the role of PMCA4 in renal CaThis study was carried out in strict compliance with the United Kingdom Animals (Scientific Procedures) Act 1986. All experimental procedures were approved by the University of Manchester Ethics Committee (permit-no: 40/3625) and all efforts were made to minimize suffering of animals. A completed ARRIVE guidelines checklist is included in ad libitum access to food and water. Experiments were performed with male WT (n = 10), HZ (n = 7) and KO (n = 10) mice when animals were aged 27–31 weeks old.PMCA4 germline null mutant mice, as previously described , were maImmunofluorescence staining was used to detect PMCA4 protein expression in kidney sections, and was performed as described previously . In shorad libitum access to food and drinking water. The amount of water and food provided to each mouse was recorded at the start and after the 24-hour period, to determine amounts ingested. Following the metabolic cage housing, blood was collected via the jugular vein under isoflurane anesthesia and stored overnight at 4°C. All animals were humanely sacrificed by cervical dislocation. Kidneys were isolated, decapsulated, subdivided and snap frozen in liquid nitrogen. Duodenum segments were extracted, cleaned with PBS and snap frozen in liquid nitrogen. All samples were stored at -80°C until required. Serum was obtained following centrifugation of whole blood samples at 500g for 10 min, and frozen at -80°C until required. One KO mouse presented with fluid filled kidney cysts, and was not included in data analysis.To collect urine, mice were housed individually in metabolic cages for 24 hours (from 10am to 10am) with 2+ concentrations as described previously [2+ measurements were verified using an internal control, which was a commercial serum standard . Serum and urinary phosphate (Pi) concentrations were determined by in-hospital services using automatic biochemical analyzers. Serum PTH levels were determined using the mouse PTH 1–84 ELISA kit  and serum Fibroblast Growth Factor (FGF)23 levels were determined with the mouse C-terminal FGF23 ELISA assay .Urinary osmolality was measured using an Advanced® Model 3320 Micro-Osmometer . A colorimetric assay was used to determine serum and urine Caeviously . Ca2+ me2+-related genes, as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control [Gapdh expression and calculated according to the ΔΔCT method [Total RNA was isolated from kidneys and duodenum using TRIzol® reagent (Invitrogen) according to the manufacturer’s protocol and dissolved in 150 μl diethylpyrocarbonate (DEPC)-treated deionized water, and stored at -80°C. Thereafter, RNA was treated with RQ1 DNase  and reverse transcription was performed using 1.5 μg RNA and Moloney murine leukemia virus reverse transcriptase  for 1.5 hours following the manufacturer’s protocol, except that RNasin® (Promega) was used as an RNase inhibitor . The obt control . All theT method . The PMC28k, respectively and blotted to a PVDF membrane . Immunoblots were incubated overnight at 4°C either with mouse anti-CaBP28k , mouse anti-NCX1  or mouse anti-beta-actin . Immunoblots were enhanced using chemiluminescence  and analyzed using a ChemiDoc XRS (Bio-Rad) system. Semi-quantification was performed as described previously [Total kidney protein lysates were prepared as described previously  and 30 μeviously .Values are expressed as mean ± S.E.M. Differences between the WT, HZ and PMCA4 KO mice were tested using a one-way ANOVA with Tukey post-hoc test. Differences were considered significantly different when P<0.05. Analysis of the dataset was performed using GraphPad Prism, version 6.0.2+ handling was investigated using PMCA4 WT, HZ and KO mice. By quantitative real-time PCR and immunofluorescence staining of the kidney cortex, PMCA4 mRNA and protein were not detected in kidneys from PMCA4 KO mice , and did not observe a disturbance in Ca2+ homeostasis, even though CaBP28k is involved in transcellular Ca2+ transport [2+ handling even though the PMCA4 KO mouse does not show a changed Ca2+ homeostasis.The Carocesses , 29. Disy stones , 31. Thextrusion ,11, 22. echanism , 32. How segment , 22. In of PMCA1 , 33. Thelobal KO , 34. On lobal KO , 35. Miche study , 36–38. ransport . This su2+-regulating genes, indicating that no compensation occurred. Recently Alexander et al., have shown that PMCA4 is located in the smooth muscle layer of the intestine, and not in the enterocytes, where PMCA1 was more highly detected [2+ transport in the duodenum [2+ to maintain a normal serum Ca2+ level; however, there was no indication of renal Ca2+ wasting in our mice studied here.At the intestinal level the PMCA4 KO mice did not show a difference in the Cadetected , suggestduodenum , 28, 39.duodenum . Bone co2+ homeostasis is mainly regulated by PTH and 1,25(OH)2D3 in response to low serum Ca2+ levels [2D3-regulated genes, such as TRPV5 and TRPV6, are not changed [2+ extruder, others have already shown the involvement of PMCA4 in signaling transducing pathways as well [Ca+ levels . The PMC changed . Moreove changed . In addi changed . These c as well , 46.2+ handling. However, the presence of PMCA4 in specific segments of the kidney points to the molecule having a different or additional role compared to PMCA1 and therefore, their exact roles should be investigated in more detail.In conclusion, PMCA4 does not seem to be vital for basal renal CaS1 Checklist(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 Figi (A) and 24-hour urinary Pi excretion (B) in wild type , heterozygous  and knockout  mice. Relative mRNA expression of NaPi-IIa (C), NaPi-IIc (D) and klotho (E) were determined in the kidney. Data represents mean ± S.E.M.Serum P(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


This process appears to be isoform-specific as expression of either RyR1 or RyR3 confers on myotubes different [Ca2+]rest. Using chimaeric RyR3–RyR1 receptors expressed in dyspedic myotubes, we show that isoform-dependent regulation of [Ca2+]rest is primarily defined by a small region of the receptor encompassing amino acids 3770–4007 of RyR1 (amino acids 3620–3859 of RyR3) named as the CLR (Ca2+ leak regulatory) region. [Ca2+]rest regulation by the CLR region was associated with alteration of RyRs’ Ca2+-activation profile and changes in SR Ca2+-leak rates. Biochemical analysis using Tb3+-binding assays and intrinsic tryptophan fluorescence spectroscopy of purified CLR domains revealed that this determinant of RyRs holds a novel Ca2+-binding domain with conformational properties that are distinctive to each isoform. Our data suggest that the CLR region provides channels with unique functional properties that modulate the rate of passive SR Ca2+ leak and confer on RyR1 and RyR3 distinctive [Ca2+]rest regulatory properties. The identification of a new Ca2+-binding domain of RyRs with a key modulatory role in [Ca2+]rest regulation provides new insights into Ca2+-mediated regulation of RyRs.Passive SR (sarcoplasmic reticulum) Ca 2+-binding domain of intracellular Ca2+ channels from muscle cells. This domain provides channels with distinctive properties that result in channel-specific modulation of the intracellular resting Ca2+ concentration.This paper reports the finding of a new class of Ca Furthermore, the elevation of [Ca2+]rest by expression of RyR1 appears to be the combined effect of both a passive Ca2+ leak from a ryanodine-insensitive pool of RyR1 channels (leak channels) and an enhanced basal sarcolemmal Ca2+ influx driven by the SR (sarcoplasmic reticulum) Ca2+ leak [[Ca+ stores –3. In skgulators –6. A rola2+]rest . More rea2+ leak .1S subunit suggest further that negative regulation of RyR1 function by the DHPR also contributes to modulating the passive Ca2+ leak responsible for [Ca2+]rest [2+]rest that are significantly higher than those restored by the expression of RyR1 [2+]rest similar to that of RyR1 [2+ leak responsible for [Ca2+]rest regulation.Studies in dysgenic myotubes that lack expression of the DHPR (dihydropyridine receptor) αa2+]rest . In appa of RyR1 . However of RyR1  despite 2+]rest modulation conferred by RyR1 and RyR3 [2+]rest regulatory properties of each isoform. Using a library of chimaeric RyR3–RyR1 and RyR1–RyR3 receptors expressed in dyspedic myotubes, we show that the ability of the RyR1 and RyR3 to confer isoform-specific [Ca2+]rest regulation can be traced to a small domain encompassing amino acids 3770–4007 of the C-terminal region of RyR1 (amino acids 3620–3859 of RyR3), which we referred to as the CLR (Ca2+ leak regulatory) region. Functional and structural analyses revealed the CLR domain contains a bona fide Ca2+-binding domain that modulates the Ca2+-sensing properties of RyRs. These data suggest that modulation of [Ca2+]rest by RyRs is under the direct control of a novel cation-binding region found within the RyRs with molecular properties unique to each isoform.To test this hypothesis, we took advantage of the remarkable differences in channel function and Ryanodine binding to crude membrane extracts (0.1 mg/ml) was performed at equilibrium (90 min at 37°C) in the presence of 250 mM KCl, 20 mM Hepes (pH 7.4) and 5 nM [3H]ryanodine (PerkinElmer Life Sciences) in the presence of a cocktail of protease inhibitors . Free Ca2+ concentrations were obtained by combination of 1 mM EGTA with specific amounts of CaCl2 according to calculation with the WEBMAXC Extended program (http://www.stanford.edu/~cpatton/maxc.html). Non-specific binding was determined by incubating the vesicles with 5 μM unlabelled ryanodine. Separation of bound and free ligand was performed by rapid filtration through Whatman GF/B glass fibre filters using a 24-well Brandel cell harvester as described previously [[eviously .2+]rest were obtained by direct measurements on myotubes differentiated in 35-mm-diameter culture dishes using double-barrelled Ca2+-selective microelectrodes as described previously  containing the same tag was also expressed and used as control protein. Clones were expressed in Escherichia coli BL21 strain in combination with pG-KJE8 vector (Takara™) encoding five molecular chaperones to optimize protein folding. Protein expression was induced by incubation with 0.2 mM IPTG for 6–8 h at 16°C in the presence of 1 mg/ml arabinose and 2 ng/ml tetracycline. Cells were then disrupted by sonication in solubilization buffer  and soluble proteins were then purified using Strep-trap affinity columns  after centrifugation at 100000 g for 90 min. Proteins were eluted with 3 mM desthiobiotin in 10 mM Tris/HCl (pH 7.4) and then washed/concentrated with 10 mM Tris/HCl (pH 7.4) using filtration units with a 10 kDa molecular-mass cut-off. The purity of the isolated protein domains was evaluated using SDS/PAGE as described previously rest conferred by RyR1 and RyR3 were associated to specific structural/functional domains of each isoform, we measured [Ca2+]rest of dyspedic myotubes expressing a series of chimaeric RyR3–RyR1 constructs spanning the entire primary sequence of RyR1 and RyR3 (2+]rest greater than that of dyspedic myotubes (>50 nM) were considered to be infected and therefore to express the receptor tested [2+-handling proteins SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1) and calsequestrin-1 .To assess whether the differences in myoplasmic [Caand RyR3 A. All my2+]rest of RyR1-expressing myotubes is approximately 110 nM, RyR3-expressing myotubes had significantly higher average resting free Ca2+ levels. Chimaeric constructs Ch4 and Ch3, containing the central and C-terminal region of RyR1 respectively, displayed a significant reduction in [Ca2+]rest when compared with wild-type RyR3. Average [Ca2+]rest conferred by Ch3-expressing cells was 158±18 nM (n=15), a concentration significantly lower than that conferred by chimaera Ch4 [188±20 nM ], but slightly higher than that observed in wild-type RyR1-expressing cells [114±7 nM ]. Consistent with these observations, we found that the reverse chimaeric constructs, in which the corresponding Ch4 and Ch3 regions of RyR3 were expressed in a RyR1 background (Ch4rev and Ch3rev), resulted in a significant increase in [Ca2+]rest when expressed in dyspedic myotubes (2+ homoeostasis and induced higher [Ca2+]rest than the Ch4rev region (168±8 nM compared with 138±5 nM for Ch3rev and Ch4rev respectively); however, the [Ca2+]rest reached by Ch3rev was lower than that observed in wild-type RyR3-expressing cells (P<0.05).myotubes A and 1C.2+]rest regulation was confined to a smaller domain of the C-terminal region, within amino acids 3770–4180 of RyR1  for Ch22rev compared with 114±7 nM (n=49) for RyR1 (P<0.001)]. The average [Ca2+]rest restored by Ch22rev, however, was still lower than those restored by wild-type RyR3 (P<0.01).The critical structural determinant of chimaera Ch4 was mapped further to the smaller overlapping region between chimaeras Ch17 and Ch21 A and 2B, of RyR1 A. Figure2+]rest regulatory domain of region Ch22 of RyR1 to its N-terminal half  that was significantly lower than that observed with the larger Ch22 region [162±14 nM ]. [Ca2+]rest displayed by Ch25-expressing cells was not statistically different from those observed in wild-type RyR1-expressing myotubes , for simplicity we refer to this domain of RyRs as the CLR region.Further analysis narrowed down the location of the [Ca Ch25 in A and 3C.2+ channel properties between RyR1 and RyR3 [2+]rest regulation of dyspedic myotubes was associated to alterations in channel properties, we further analysed the Ca2+-dependence of the chimaeric construct Ch25 using [3H]ryanodine-binding studies. 2+-dependence of [3H]ryanodine binding to membrane fractions from HEK (human embryonic kidney)-293 cells expressing either wild-type channels or chimaeric construct Ch25. As reported previously, Ca2+-dependence profiles of wild-type RyR1 and wild-type RyR3 indicate monophasic activation for wild-type RyR1 [EC50=0.41±0.18 μM (n=6)] and a biphasic activation profile for wild-type RyR3 [EC50(1)=0.39±0.04 μM and EC50(2)=35.1±18 μM (n=6)] with a plateau between 1 and 10 μM. Consistent with its predominant RyR3 background, the Ca2+-activation curve of chimaera Ch25 showed a biphasic activation profile, but with a more exacerbated biphasic Ca2+ response and a wider activation plateau =0.36±0.03 μM and EC50(2)=465.7±120 μM (n=4)]. [3H]Ryanodine-binding analysis of Ca2+ inhibition revealed that Ca2+-sensitivity for inactivation of RyR3 was 6–7-fold lower than that of RyR1 (IC50=0.44±0.22 mM for RyR1 compared with IC50=2.68±0.77 mM for RyR3). Analysis of Ca2+ inactivation for chimaera Ch25 showed a significantly reduced sensitivity to Ca2+ inhibition compared with wild-type RyR1, but similar to that of wild-type RyR3  shows important differences in channel function between wild-type RyR1 and wild-type RyR3, with RyR3 reaching noticeably higher activation levels than RyR1  and the corresponding fragment in RyR3 , were expressed in E. coli BL21 cells and analysed for their ability to bind Ca2+. In order to minimize misfolding of the protein domains, the CLR fragments were expressed in the presence of five chaperone proteins, to assist in protein folding, and then purified from the soluble fraction of the cell homogenate to avoid using inclusion bodies. All purified domains, including control region R1-(1–233), showed approximately 95% purity and molecular masses of approximately 30 kDa, consistent with their predicted 25–27 kDa molecular mass (, containing amino acids 1–233 of RyR1, showed only marginal fluorescence changes upon Ca2+ addition . The biphasic binding profile of domain CLR-1 was less pronounced with Kd1 of 11.0±1.8 μM and Kd2 of 21.6±2.7 μM. Whereas average Kd1 showed a slight, but significant, difference between CLR-1 and CLR-3, there was no difference seen in average Kd2, suggesting that the CLR region of RyR1 and RyR3 may present slightly different Ca2+-binding properties  and the other the CLR region encompassing amino acids 3770–4007 of RyR1 (amino acids 3620–3859 of RyR3). The impact of region 17/21 on [Ca2+]rest regulation was shown to be less dramatic than that of the CLR region in both the RyR3-based (Ch4) and the RyR1-based (Ch4rev) chimaeric constructs. Our previous studies of these chimaeric receptors have shown that the RyR1 region encompassing region 17/21 is directly involved in the cross-talk between DHPR and RyR1, and confers RyR3 with the ability to restore DHPR tetrad formation, DHPR retrograde signalling and excitation–contraction coupling [2+]rest observed in dyspedic myotubes expressing Ch4, Ch21 or Ch17 could be related to an enhanced orthograde regulation by DHPR that inhibited RyR1 function, thereby restricting overall SR Ca2+-leak, similar to the mechanism described by Eltit et al. rest should not be ruled out as all these chimaeric constructs have shown to affect both Ca2+-activation and Ca2+-inhibition profiles of the channel rest-regulatory properties distinctive of the specific CLR sequence being expressed, regardless of the overall amino acid background. In contrast with region 17/21, it is unlikely that the effect the CLR region has on [Ca2+]rest could be related to orthograde regulation by DHPR, as cumulative evidence indicates that the C-terminal region of RyR1 is not involved in cross-talk with the DHPR complex. Our previous studies involving several chimaeric RyR3–RyR1 channels, including Ch25 used in the present study, have shown that exchange of various RyR domains containing the CLR region had a negligible effect on either excitation–contraction coupling rest like the one caused by exchange of the CLR region could not be explained simply by changes in expression levels of the chimaeric channels.Importantly, and consistent with our previous studies in wild-type RyR3 and wild-type RyR1 , Western2+]rest is a common feature of skeletal muscle cells expressing RyR1 mutations linked to MHS . Extensive functional analysis of muscle cells expressing numerous MHS-linked mutations of RyR1 has revealed that elevated [Ca2+]rest is often associated with alteration of Ca2+ and Mg2+ regulation of RyR1 rest. These results are also in agreement with the cell boundary theorem that predicts permanent changes in cytosolic Ca2+ concentration as a result of steady changes of sarcolemmal Ca2+ fluxes ryanodine-binding studies have been reported previously in RyR3 rest regulation. Previous studies have associated this same region with interaction to S100A1 [2+-binding protein known to modulate Ca2+ cycling in skeletal muscle through its interaction with the calmodulin-binding site of RyR1 [2+]rest regulation observed in our study could be linked to binding of S100A1 because S100A1-null muscle fibres appear to present normal [Ca2+]rest regulation rest regulation. Because this modulatory role appears to be linked directly to the ability of RyRs to regulate passive SR Ca2+ leak [2+]rest could result from changes in RyR susceptibility to adopt the leak conformation. Our data, however, do not rule out the possibility that changes in gating properties of the functional pool of RyRs could also contribute to modulate the overall SR Ca2+ content that drives sarcolemmal Ca2+ fluxes and thereby [Ca2+]rest regulation. Lastly, the identification of this new Ca2+-binding domain in RyRs, which was found to play a key role in modulating myoplasmic [Ca2+]rest, provides new insights into Ca2+-mediated regulation of RyRs function.Overall, our data revealed that the region of amino acids 3770–4007 of RyR1 (amino acids 3620–3859 of RyR3) encompasses a novel Ca


The limited sensitivity of the historical treatment response criteria for acute myeloid leukemia (AML) has resulted in a different paradigm for treatment compared with most other cancers presenting with widely disseminated disease. Initial cytotoxic induction chemotherapy is often able to reduce tumor burden to a level sufficient to meet the current criteria for “complete” remission. Nevertheless, most AML patients ultimately die from their disease, most commonly as clinically evident relapsed AML. Despite a variety of available salvage therapy options, prognosis in patients with relapsed or refractory AML is generally poor. In this review, we outline the commonly utilized salvage cytotoxic therapy interventions and then highlight novel investigational efforts currently in clinical trials using both pathway-targeted agents and immunotherapy based approaches. We conclude that there is no current standard of care for adult relapsed or refractory AML other than offering referral to an appropriate clinical trial. Approximately 20,000 patients will be diagnosed with acute myeloid leukemia (AML) with greater than 10,000 AML patient deaths in the United States during 2015 . Patientde novo generation of AML due to toxicity from treatment. Experimental evidence to date suggests the first two mechanisms are the most common [It is now clear that AML is a diagnosis encompassing a wide range of myeloid malignancies with heterogeneous genetic etiology . It is at common ,10, althWhile a variety of different treatment regimens have been studied in an effort to improve outcomes of patients with RR-AML, there appears to be no single superior approach. We therefore believe that the only current standard of care for a patient with relapsed AML is to offer enrollment in a clinical trial . This arWhile most patients able to undergo initial cytotoxic induction therapy will initially achieve a CR, the most common cause of death is subsequent relapse of disease. Treatment options in this setting include aggressive therapy geared towards providing a bridge to an allogeneic hematopoietic stem cell transplantation , which is often considered the only potentially curative option for patients with RR-AML, or best supportive care with palliative intent in patients who are not candidates for an aggressive approach. Patients must have a suitable donor, good performance status, minimal comorbidities, and generally should be in at least a CR prior to undergoing alloHSCT.Most commonly used induction regimens involve a combination of infusional cytarabine, an anthracycline, and perhaps an additional agent. Cytarabine, a deoxycytidine analog that is actively metabolized to arabinofuranosylcytidine triphosphate (ara-CTP), serves as the backbone for many commonly utilized salvage regimens, demonstrating efficacy in relapsed AML both as monotherapy when given in high doses (HIDAC), and in combination with other therapeutic agents ,13. In gversus 63% ADE, p = 0.8), and in fact survival at 4 years was significantly inferior using this approach  [versus 61% no G-CSF, p = 0.7). Nevertheless, FLAG ± idarubicin remains an accepted alternative to HIDAC monotherapy in relapsed AML.FLA/FLAG—Fludarabine is a purine analog that acts by inhibiting ribonucleotide reductase (RNR), which augments the rate of synthesis of ara-CTP in circulating AML blasts when infused prior to cytarabine . It has  = 0.05) . LikewisCLAG/CLAG-M—Cladribine is another RNR-inhibiting purine analogue that was found to yield synergistic effects on inhibition of cell proliferation, induction of apoptosis, and disruption of mitochondrial membrane potential when combined with cytarabine . It has et al. evaluated 162 patients with RR-AML treated with CLAG versus MEC and found overall CR rates of 37.9% versus 23.8% (p = 0.05), with a median follow up of 20.3 months [MEC/EMA-86/MAV—Combinations of mitoxantrone, etoposide and cytarabine have been extensively evaluated in relapsed AML with multiple variations in the dose and schedule , resulting in CR rates between 18% and 66% ,27,28,933 months . An ongo3 months .2) have found CR rates 35%–51% [2), and G-CSF that has resulted in comparable CR rates (46% in a phase I/II study) [p < 0.0001) [GCLAC—Clofarabine is a second-generation deoxyadenosine analog that is characterized by high resistance to phosphorolytic cleavage by bacterial purine nucleoside phosphorylase, potent inhibition of DNA synthesis, prolonged retention of clofarabine triphosphate in leukemic blasts  and, sim 35%–51% ,34,35 (TI study)  Table 12 have fo 0.0001) . GCLAC a 0.0001) . Furtherversus 0% of refractory AML subjects) among 41 patients with RR-AML (including two patients with CML in myeloid blast crisis) [FLAD—Liposomal daunorubicin has been found to be at least as effective as free  daunorubicin in leukemic cells and could have decreased toxicity ,97. This crisis) . Fifty-eDysoxylum binectariferum), has properties as a cyclin-dependent kinase inhibitor and has been evaluated as timed sequential therapy in combination with cytarabine and mitoxantrone (FLAM) with a CR rate of 42.5% in a phase II study of 47 patients with RR-AML, including a CR rate of 75% in those with relapsed AML compared to only 9% in those with primary or multiply refractory disease [FLAM—Flavopiridol, a synthetic flavone derivative initially isolated from the stem bark of the Indian tree  and median OS  when compared to the placebo/cytarabine group. Overall survival (OS) benefit was greatest in patients aged 60 years or older  and those with early relapse  [Vosaroxin—Vosaroxin is a novel anticancer quinolone derivative that intercalates DNA and inhibits topoisomerase II . It was  = 0.04) . A separ = 0.04) . NotablyElacytarabine is an elaidic acid ester of cytarabine that was compared against seven commonly used salvage regimens (investigator’s choice) in a large phase III study but was not found to improve CR or OS rate . InteresIncreased understanding of AML biology has led to the identification of deregulated pathways that drive blast proliferation. These discoveries have, in turn, given way to the development of agents targeting these molecular pathways.Cancer Metabolism—Oncogenic mutations affecting two isoforms of the isocitrate dehydrogenase (IDH) enzyme, IDH1 and IDH2, have been reported in approximately 30% of de novo AML and appear to be an unfavorable prognostic factor . AG-221 CPI-613 is a lipoate derivative that disrupts mitochondrial metabolism by inhibiting pyruvate dehydrogenase and α-ketogluterate dehydrogenase . When coPlerixafor—In an effort to disrupt the interaction between leukemic blasts and the bone marrow microenvironment, which is postulated to be an important mediator of chemotherapy resistance , plerixaet al. examined the efficacy of six FLT3-ITD inhibitors for potency against mutant and wild-type FLT3 as well as for cytotoxic effect against a series of primary blast samples and found that the inhibitors could be ranked, from most to least selective against FLT3-ITD, as quizartinib, sorafenib, sunitinib, KW2449, and lestaurtinib [Kinase inhibition—Internal tandem duplication mutations resulting in constitutive activation of the FMS-like tyrosine kinase receptor 3 (FLT3-ITD) are present in approximately 20%–30% of cases of AML, represent a high-risk feature in normal karyotype AML, and are associated with a high risk of relapse following alloHSCT . Pratz eaurtinib .Quizartinib (AC220) has been shown in a phase I trial to be safe and efficacious in patients with both FLT3-ITD negative and positive RR-AML, with most of the responses being PR or CRi and occurring in FLT3-ITD positive patients . Multiplversus 22%, p = 0.013) [In the recently reported SAL-Soraml trial, sorafenib demonstrated safety and improved event free survival compared to placebo when used as an adjunct to standard induction chemotherapy in 267 younger (age <60) patients with newly diagnosed AML, only 17% of whom were FLT3-ITD mutated  . It also= 0.013) ,111. Add= 0.013) .et al. demonstrated that crenolanib has potent activity against FLT3-ITD as well as FLT3 D835 mutations in binding assays and in Ba/F3 cells from a mouse model. Several phase II trials evaluating crenolanib in patients with RR-AML and FLT3 mutations are ongoing (NCT01522469 and NCT01657682).Unfortunately, mutations of the FLT3 kinase domain can arise and limit the efficacy of FLT3 inhibitors . The mosin vivo in AML tumor models in preclinical studies [Another FLT3 inhibitor that has demonstrated inhibitory activity against FLT3-ITD/N676D, FLT3-ITD/F691L, and FLT3-D835Y mutants is G-749, which displayed potent antileukemic activity in bone marrow blasts from AML patients regardless of FLT3 mutation status . AMG 925 studies .JAK inhibition—The Janus kinase—signal transducer and activator of transcription (JAK-STAT) pathway has been found to be dysregulated in AML . Ruxolit2 d1 to d3, cytarabine 200 mg/m2 d1 to d7) in a phase Ib study of 28 patients (age <65) with relapsed AML and produced a CR rate of 68% [mTOR inhibition—Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth and proliferation by translational control of key proteins . Rapamyce of 68% .Vorinostat—Vorinostat is a histone deacetylase inhibitor that, despite promising data in phase I trials, was found in a phase II study to have a CR rate of only 4.5% in a group of 22 patients with RR-AML (16 patients) and untreated AML (6 patients) . Howeverde novo, untreated AML did not find a significant improvement in CR rates when compared to historical results [Statins—Interestingly, AML blasts frequently overexpress the genes for the low-density lipoprotein (LDL) receptor. Blockade of 3-hydroxy-3-methylglutaryl coenzyme reductase (HMG-CoAR) inhibits cholesterol uptake and synthesis and is believed to sensitize AML cells to cytotoxic therapy . A phase results .Proteasome Inhibition—Bortezomib, a proteasome inhibitor commonly utilized in multiple myeloma, was administered in combination with MEC chemotherapy in a phase I study, where 17 of 33 evaluable patients (52%) achieved either a CR or CRi and TRM was 9% . SeveralOther Pathways—Tosedostat is a novel oral agent that inhibits aminopeptidase activity, resulting in the depletion of cellular amino acid pools selectively in tumor cells . SeveralABT-199 is a selective, orally bioavailable small molecule BCL-2 inhibitor that is currently under evaluation in a phase II study with preliminary data revealing a CR + CRi rate of 15.5% among 32 patients .Hedgehog pathway—Aberrant activation of the hedgehog (Hh) pathway was found to be a feature of some CD34-positive myeloid leukemic cells in a preclinical study, where inhibition of Hh signaling was found to induce apoptosis in Hh-responsive CD34 cells . SeveralUnlike conventional chemotherapy, which primarily targets dividing cells, or small molecules that target specific pathways within blasts, immunotherapeutic interventions aim at directing an immune response against tumor cells. Several immunotherapeutic modalities are currently under evaluation and are detailed below and in versus 0.8%, p = 0.002) [Antibodies—Two targets in AML have served as the main focus for monoclonal antibody studies: CD123 and CD33. The latter is expressed on blasts in 80%–90% of patients with AML . Gemtuzu= 0.002) . Althoug= 0.002) . FurtherOther anti-CD33 antibodies include lintuzumab, an unconjugated humanized murine monoclonal antibody that failed to improve CR rates when combined with MEC in a phase III randomized multicenter study , and SGNCSL362 is a humanized second-generation anti-CD123 antibody with favorable preclinical data  that wasin vitro against human AML cells proportional to the level of cell-surface CD33 expression [Bispecific T-cell-engaging (BiTE) antibodies are single-chain antibodies designed to direct cytotoxic T lymphocytes at a predefined surface antigen on tumor cells. AMG 330 is a human BiTE antibody against CD33 and CD3 that has demonstrated potent cytolysis pression , which vpression . It has pression . Meanwhipression .in vivo and induced CR in 5 of 19 patients [Natural Killer (NK) cells are effector lymphocytes of the innate immune system capable of exerting anti-AML activity, as exemplified by its role in the graft-versus-leukemia (GVL) effect after alloHSCT . A humanpatients .et al., in which CART33 cells were administered to a patient with relapsed AML, found a temporal decrease in blast ratio from >50% to <6% two weeks after infusion. Unfortunately, the patient then developed cytokine release syndrome requiring a TNF-α inhibitor and subsequently died from disease progression 13 weeks after CART33 infusion [Adoptive Cell Therapy—Following favorable ongoing studies in lymphoid malignancies, chimeric antigen receptor-transduced T cells (CART), which are synthetic transmembrane constructs that combine the specificity of antibody target recognition with the potent effector mechanisms of T-cell immunity, have been designed to target CD33 (CART33) and CD123 (CART123) in AML. A study by Wang infusion . On the infusion ,121 and infusion . A phaseThe Wilms tumor 1 (WT1) gene, a zinc finger transcription factor implicated in leukemogenesis, is overexpressed in 70% of AML patients ,79 and hversus-host disease (GVHD) after DLI [Donor lymphocyte infusion (DLI) can be considered for patients who relapse after undergoing an alloHSCT in an attempt to induce a GVL effect. Generally patients who achieve a CR prior to DLI have a better chance of achieving a durable remission ,87. Intefter DLI . A recenfter DLI .Vaccines—A number of studies have looked at vaccination with different leukemia-associated antigens including WT1, PR1, proteinase 3, and RHAMM, with the goal of establishing an immunological response capable of eradicating malignant cells . These set al., who found an OS of 19% at 3 years among 1673 patients [versus MAC alloHSCT among 132 patients aged 35 years or older with AML  found a lower 4-year non-relapse mortality with RIC , a similar 4-year relapse rate  and similar overall survival  [Allogeneic Hematopoietic Stem Cell Transplant—AlloHSCT is an aggressive intervention that provides patients with RR-AML who have a good performance status and an adequate donor with the best chance of achieving a durable remission ,123. Relpatients . Patientpatients ,126,127.patients . A recen = 0.38) . Surviva = 0.38) . When po = 0.38) . Azaciti = 0.38) .Not all patients with RR-AML will be good candidates for the aggressive salvage therapy as described above due to reasons such as poor performance status, comorbidities, or cumulative toxicity from prior therapies. Treatment options in this setting are often palliative in nature and include best supportive care or less aggressive therapies such as hypomethylating agents or low-dose cytarabine (LDAC), which can induce CR rates of around 17% .2 days 1–7, while patients were able to proceed with an alloHSCT with reduced intensity conditioning [et al. [Hypomethylating agents—The hypomethylating agents 5-azacitidine and decitabine are cytidine analogs that act in part by inhibiting DNA methyltransferases  and are itioning . Decitab [et al. . Interes [et al. ,134. It Most patients who are diagnosed with AML will die of AML. Even within the favorable-risk subgroup of patients, as currently defined by cytogenetic and molecular criteria, many do not enjoy long-term survival. Although standard induction and consolidation chemotherapy regimens are undoubtedly intensive, a fundamental problem in the treatment of AML is not that available therapies are toxic but rather they are not effective enough . Moreoveversus CR2), may be most predictive of overall survival [In RR-AML patients with poor performance status, prohibitive comorbidities, or those who do not wish to undergo aggressive therapy, options are currently limited to best supportive care or low-intensity therapy with palliative intent. Although patients who are good candidates for aggressive treatment have a significant number of salvage therapy options available to them, none has categorically proven to offer superior outcomes and participation in clinical trials should be encouraged . In genesurvival . Unfortusurvival ,125. Patsurvival .associated with treatment resistance, they offer suboptimal predictive power for the individual patient [ex vivo drug sensitivity and resistance testing [AML has historically been approached as a homogeneous diagnostic entity with a resulting “one size fits all” treatment strategy, often resulting in disappointing outcomes. In reality, the acute myeloid leukemias are a heterogeneous group of diseases with distinct molecular and phenotypic characteristics. While factors such as patient age, secondary AML, WBC count at presentation, and cytogenetic and molecular markers all are  patient . Heterog testing ,140,141.In addition to AML clone biology characteristics that are postulated to contribute to therapy resistance and disease relapse, the bone marrow microenvironment has also been shown to affect therapeutic efficacy in patients with AML. A recent study found that AML cells can educate bone marrow-derived stromal cells to secrete Gas6, which fosters AML cell growth and chemoresistance via the receptor tyrosine kinase Axl . Overexp“Relapse” of AML is predicated on the concept of remission. The development of increasingly sensitive minimal or measurable residual disease (MRD) assays has demonstrated that current remission criteria, originally proposed in 1956, do not provide sensitive assessment of AML disease burden , as evid