Abnormal tau aggregation is a pathological hallmark of many neurodegenerative disorders and it is becoming apparent that soluble tau aggregates play a key role in neurodegeneration and memory impairment. Despite this pathological importance, there is currently no single method that allows monitoring soluble tau species in living cells. In this regard, we developed a cell-based sensor that visualizes tau self-assembly. By introducing bimolecular fluorescence complementation (BiFC) technique to tau, we were able to achieve spatial and temporal resolution of tau-tau interactions in a range of states, from soluble dimers to large aggregates. Under basal conditions, tau-BiFC cells exhibited little fluorescence intensity, implying that the majority of tau molecules exist as monomers. Upon chemically induced tau hyperphosphorylation, BiFC fluorescence greatly increased, indicating an increased level of tau-tau interactions. As an indicator of tau assembly, our BiFC sensor would be a useful tool for investigating tau pathology. Abnormal tau aggregation is a primary pathological hallmark in Alzheimer’s disease (AD) and multiple other neurodegenerative disorders, collectively called tauopathies . In a heFull-length human tau contains a microtubule-binding domain consisting of four conserved sequence repeats. Positively charged residues in the sequence repeats are important for binding with the highly negatively charged microtubules (20-30 electrons per αβ-tubulin dimer) . Tau’s bTo visualize tau-tau interactions, we have established a tau-BiFC cell model. The BiFC is a method to visualize protein-protein interactions that is based on the formation of a fluorescence protein complex from non-fluorescent constituents attached to proteins of interests . PreviouTo establish the tau-BiFC sensor, we used a Venus-based BiFC system. The Venus protein is a variant of yellow fluorescence protein (YFP), and is well suited for achieving spatial and temporal resolution of tau assembly because (i) it has fast and efficient maturation, (ii) its self-assembly rate is low compared to that of other BiFC pairs, and (iii) the fluorescence intensity of Venus-based BiFC is 13 times higher than that of EYFP-based BiFC ,16. To eNext, we investigated the cellular distribution pattern of tau-BiFC fluorescence. In the case of HEK293-tau-GFP cells, GFP-fluorescence was enriched all over the cytoplasm without showing a strong correlation with microtubules . In contTo validate the feasibility of tau-BiFC as an indicator of tau assembly, tau-BiFC cells were treated with forskolin and okadaic acid, which are known to induce tau hyperphosphorylation. Full-length tau has 79 putative serine and threonine residues, and 5 tyrosine residues. Phosphorylation of these residues is tightly regulated by protein kinases and phosphatases to maintain the microtubule dynamics required for neuronal plasticity. Among those regulating enzymes, the major tau phosphatase of the human brain is protein phosphatase 2A (PP2A), which regulates the activities of several protein kinases that phosphorylate tau. Okadaic acid is a potent inhibitor of PP2A, and is known to induce Alzheimer-like tau phosphorylation in rat brains ,19. ProtAn immunoblot assay showed that upon okadaic acid (30 nM) treatment, phosphorylation level increase at Ser199 and Ser202 of the full-length tau and a number of tau fragments , suggestIn contrast, while forskolin (20 μM) treatment also induced tau phosphorylation at Ser396 (1.7 fold), but neither tau fragmentation nor dimerization was not observed on the immunoblot . AlthougSince Alois Alzheimer discovered the presence of abnormal fibrous inclusions within neurons in a patient’s brain, neurofibrillary tangles are thought to be the pathological structure of tau. However, recent evidences indicate that (toxic tau aggregates are) smaller, soluble tau oligomers are the toxic aggregates, closely linked with neurodegeneration and memory impairment. The large inclusions might form as a survival strategy to protect neuronal cells by sequestrating the toxic aggregates. As such, preventing tau aggregation becomes a potential strategy to cure neurodegenerative disorders associated with tau. To identify the cause and molecular mechanism of tau aggregation and to reverse the processes, a reliable system capable of monitoring tau self-assembly processes is necessary. Our tau-BiFC system provides a chance to monitor and quantify tau aggregation processes by allowing direct visualization of tau-tau interactions in living cells. So, this tau-BiFC sensor would be a useful tool to investigate tau pathogenesis and to discover methods for the prevention and reversal of the aggregation process.5’-AATTCGGTCG ACCGAGATCT CTCGAGGTAC-3’, (VC155-R) 5’-CTAGTTGTGG TTTGTTTAAA CTCATCAATG TATC-3, (VN173-F) 5’-ATGACGACAA G CTCGAGGCC GCGAATTCAT CG-3’, (VN173-R) 5’-CTAGTTGTGG TTTGTTTAAA CTCATCAATG TATC-3’. pCMV6-hTau40-GFP and PCR amplified inserts were digested with XhoI/PmeI and ligated to generate pCMV6-hTau40-VN173 and pCMV6-hTau-VC155.A mammalian expression vector for pCMV6-hTau40-GFP was purchased from OriGene Technologies Inc. . To replace GFP with BiFC compartments, pBiFC-VN173 and pBiFC-VC155 were obtained from Addgene  and amplified by using PCR primers containing XhoI/PmeI restriction sequences : (VC155-F) 2. The day before transfection, HEK-293 cells were plated on 12-well plates with OPTI-MEM medium (Invitrogen). In order to generate HEK293-tau-GFP cell line, the cells were transfected with pCMV6-hTau40-GFP by using Lipofectamine®2000 reagent (Invitrogen) according to the manufacturer’s instructions. For the generation of HEK293-tau-BiFC cell line, the cells were co-transfected with pCMV6-hTau40-VN173 and pCMV6-hTau40-VC155. To establish the stable cell lines, the transfected cells were incubated with growth medium containing Geneticin for selection. Then, to enrich population, fluorescent cells were sorted by using FACSAria (BD Bioscience).HEK293 was purchased from ATCC  and was grown in Dulbecco’s modified eagle medium containing 10% fetal bovine serum and 10,000 units/mL penicillin and 10,000 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% COFor microscopic image analysis, cells were plated in a black transparent 96-well plate. The next day, tau-BiFC cells were treated with the okadaic acid or forskolin at various concentrations. After, 2, 9, 19, and 24 hrs of incubation, the entire 96-well plate was automatically imaged under same exposure by using Operetta® High Contents Screening System (equipped with a 10X and 20X dry lenses). The cellular intensities of tau-BiFC fluorescence were analysed using Harmony 3.1 software. Error bars indicate s.d. from two independent experiments. Each experiment was performed as triplicate.HEK293-tau-BiFC cells were incubated with okadaic acid or forskolin for 24 hrs at 37 °C. Then, cell lysates were prepared by using CelLytic M (Sigma) containing protease and phosphatase inhibitor cocktail. 10 μg of the protein lysates were separated on an SDS-PAGE gel (10%) and transferred to PVDF membrane for immuno-blot analysis. All tau antibodies were purchased from abcam : pSer199 and pSer202 (ab4864), pSer396 (ab64193) and Ser262 (ab32057).File S1Supporting figures. Figure S1, HEK293-tau-BiFC cell sorting by FACS. Figure S2, The association and reconstitution of tau-BiFC upon okadaic acid treatment. Figure S3, Colocalization of tau-BiFC fluorescence with anti-phosphorylated tau stain.(DOCX)Click here for additional data file.


Culb. & C.F. Culb. As conceivable source for novel anti-inflammatory compounds. Phytochemical investigation of the ethanolic crude extract resulted in the isolation and identification of 11 constituents, belonging to depsides and derivatives of orsellinic acid, olivetolic acid and olivetol. The two depsides imbricaric acid (4) and perlatolic acid (5) approved dual inhibitory activities on microsomal prostaglandin E2 synthase-1  and on 5-lipoxygenase tested in a cell-based assay  and on purified enzyme . Additionally, these two main constituents quantified in the extract with 15.22% (4) and 9.10% (5) showed significant inhibition of tumor necrosis factor alpha-induced nuclear factor kappa B activation in luciferase reporter cells with IC50 values of 2.0 and 7.0 µM, respectively. In a murine in vivo model of inflammation, 5 impaired the inflammatory, thioglycollate-induced recruitment of leukocytes to the peritoneum. In vitro screening of 17 Alpine lichen species for their inhibitory activity against 5-lipoxygenase, microsomal prostaglandin E4 and 5 a pronounced multi-target anti-inflammatory profile which warrants further investigation on their pharmacokinetics and in vivo efficacy. The potent inhibitory effects on the three identified targets attest  Lichens, wide-spread symbiotic associations between algae/cyanobacteria and fungi, occur in diverse environmental conditions, even in very inhospitable climates attended with extreme temperatures and aridity. Due to the various algal-fungal-combinations and the variable ecological and climatic growth conditions, lichens differ widely in appearance and produce a vast diversity of small chemical entities to protect the symbiosis partners against environmental influences and natural enemies. Accordingly, lichens are a rich source of bioactive secondary metabolites, such as mono- and diaromatics, terpenoids, steroids, anthraquinones, naphthoquinones, xanthones, and furans , with nu2 synthase-1 (mPGES-1) using pharmacophore-based virtual screening tools [2 synthases, mPGES-1, an inflammation induced-, membrane associated-enzyme, has been described as promising druggable target catalyzing the conversion of cyclooxygenase-derived prostaglandin (PG)H2 to PGE2, primarily affecting pathogenic processes [Recently, we identified lichen constituents from the chemical group of depsides and depsidones as potent inhibitors of microsomal prostaglandin Eng tools . Among trocesses .in vitro screening on mPGES-1. Additionally, we aimed to investigate the multi-targeting anti-inflammatory potential of lichen constituents in vitro, which is by far not fully investigated. Hence, the in vitro screening was extended to a further target within eicosanoid biosynthesis, namely 5-lipoxygenase (5-LO), since a dual inhibition of mPGES-1 and 5-LO is reported to provide safer and more effective anti-inflammatory properties [in vitro screening against the focused pharmacological targets followed by an in depth phytochemical analysis of the most promising lichen extract (C. monachorum), we identified two depsides (4 and 5) contributing to the multi-target in vitro effect. First results from an in vivo pilot study underpin the anti-inflammatory efficacy of this compound class. These previous findings prompted us to further identify bioactive lichen constituents and to explore their anti-inflammatory potential. For this purpose diverse Alpine lichen species were collected and investigated to complement the previously performed structure-based approach applying an operties ,11. MoreCetrelia monachorum (Zahlbr.) W.L. Culb. & C.F. Culb. was collected from maple bark in Grünau/Almtal, Upper Austria (47° 44.3’ N and 13° 56.82’ E) in July 2011. The lichen material was morphologically and microchemically identified according to Obermayer and Mayrhofer [Lichen material of ayrhofer .para-phenylendiamine and enabled the unambiguous assignment of species. Voucher specimens are deposited at the Institute of Pharmacy/Pharmacognosy, University of Innsbruck, Austria. Collected lichen species, date, locality of collection, voucher numbers, amount of dried thalli and extract yield, are listed in Table S1 in Further 16 Alpine lichens were collected between August 2010 and August 2011 in Halltal and Ötztal, Tyrol, Austria, and in Vinschgau, South Tyrol, Italy, and identified according to the key in Wirth . This inAll reagents were of purissimum or analytical quality and purchased from Merck  unless otherwise specified. C. monachorum (CM) was assessed using the HPLC method: 1100 Agilent system  equipped with photodiode array detector and auto sampler; stationary phase: Phenomenex Synergi Polar-RP 80A column ; mobile phase: aqueous 0.05 % formic acid (A) and MeOH (B); flow rate: 1.0 mL/min; oven temperature: 35 °C; detection wavelength: 235 nm; composition: start 10% B; 4 min 20% B; 9 min 60% B; 15 min 63% B; 30 min 63% B; 33 min 74% B; 48 min 75% B; 53 min 98% B; post time 10 min; fitted to MS-parameters: Bruker Esquire 3000plus iontrap ; split: 1:5; ESI, alternating mode; spray voltage: 4.5 kV, 350 °C; dry gas: N2, 10 L/min; nebulizer: He, 40 psi; scanning range: m/z 100-1500.Analysis of the metabolite profile of the ethanol crude extract of d4  or CDCl3  as NMR-solvents .NMR: 1D- and 2D-experiments were measured at a Bruker UltraShield 600  using methanol-in vitro screening of selected Alpine lichen species the air-dried thalli were ground with a ball mill  and extracted three times using EtOH 96% at room temperature and an ultrasonic bath . For C. monachorum 13 g dried and ground thalli were extracted at room temperature with EtOH 96% using an ultrasonic bath . Upon evaporation to dryness the crude extract (CM) yielded 1.68 g. Pure compounds 1 - 11 were isolated according to the schematic flow chart  UFLC-XR instrument, equipped with on-line degasser, auto sampler, column thermostat, and photodiode array detector using the HPLC protocol described above. The amounts of 4 and 5 were calculated as the percentage of dry weight of CM. Limits of quantification were determined as the signal-to-noise ratio of 10. To determine the contents of 2. Cells were harvested, sonicated and the homogenate was subjected to differential centrifugation at 10,000×g for 10 min and 174,000×g for 1 h at 4 °C. The pellet  was resuspended in 1 ml homogenization buffer , and the total protein concentration was determined. Microsomal membranes were diluted in potassium phosphate buffer  containing 2.5 mM glutathione. Test compounds or vehicle (0.1% DMSO) were added, and after 15 min at 4 °C reaction  was initiated by addition of PGH2 at the indicated concentrations. After 1 min at 4 °C, the reaction was terminated using stop solution . PGE2 was separated by solid-phase extraction and analyzed by RP-HPLC as described previously [Preparations of human A549 cells and determination of mPGES-1 activity was performed as described previously . In brieE. coli (BL21) was transformed with pT3-5-LO plasmid, and recombinant 5-LO protein was expressed at 30°C as described [2 plus the indicated concentrations of arachidonic acid (AA) were added to start 5-LO product formation. The reaction was stopped after 10 min at 37°C by addition of 1 mL ice-cold methanol, and the formed metabolites were analysed by RP-HPLC as described [4 (LTB4) and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-H(P)ETE).escribed . Cells wescribed . Semi-puescribed . 5-LO prg for 20 min at 20°C for preparation of leukocyte concentrates. Leukocyte concentrates were subjected to dextran sedimentation and centrifugation on LSM 1077 cushions . Contaminating erythrocytes of pelleted PMNL were lysed by hypotonic lysis. PMNL were washed twice in ice-cold PBS and finally resuspended in PBS pH 7.4 containing 1 mg/mL glucose and 1 mM CaCl2 (PGC buffer) (purity > 96-97%). For isolation of polymorphonuclear leukocytes (PMNL), human peripheral blood  was withdrawn from fasted (12 h) healthy donors that had not taken any anti-inflammatory drugs during the last 10 days, by venipuncture in heparinized tubes (16 IE heparin/mL blood). The blood was centrifuged at 4,000×6), cells were resuspended in 1 mL PGC buffer, preincubated for 15 min at 37°C with test compounds or vehicle (0.1% DMSO), and incubated for 10 min at 37°C with the indicated stimuli. Thus, the Ca2+-ionophore A23187 (2.5 µM) was added together with 20 µM AA and 10 min later the reaction was stopped on ice by addition of 1 mL of ice-cold methanol. Then, 30 µL 1 N HCL and 500 µL PBS, and 200 ng prostaglandin B1 were added and the samples were subjected to solid phase extraction on C18-columns . 5-LO products (LTB4 and its trans-isomers and 5-H(P)ETE) were analyzed by RP-HPLC and quantities calculated on the basis of the internal standard PGB1. Cysteinyl-LTsC4, D4 and E4 were not detected (amounts were below detection limit), and oxidation products of LTB4 were not determined. For determination of LO products in intact PMNL . The luciferase signal derived from the NF-κB reporter was normalized by the EGFP-derived fluorescence to account for differences in cell number. The transactivation of a NF-κB-driven luciferase reporter gene was quantified in HEK-293/NF-κB-luc cells  as previously described . Cells sTo estimate of cell viability one part of the experiments were performed in cells that were not transfected with pEGFP-C1, but instead stained for 1 h in serum-free medium supplemented with 2 μM Cell Tracker Green CMFDA  as described . Since tad libitum in the mouse facility of the Institute of Vascular Biology and Thrombosis Research . Animal care and all experimental procedures were approved by the Animal Experimental Committee of the Medical University of Vienna and by the Austrian Ministry of Science (license no. BMWF-66.009/0117-II/3b/2012).C57BL/6J male, 8-9 weeks old mice were housed in individually ventilated cages and fed with mouse chow and acidified water The mice of the treatment groups – sterile thioglycollate and perlatolic acid or thioglycollate alone -  were pre-treated i.p. with 1 mL of 6 µM perlatolic acid or 1 µL of DMSO dissolved in 1 mL of saline. Thirty minutes later the animals were injected i.p. with another 3 mL of saline with or without sterile thioglycollate (4%), and containing again 2 μL DMSO or perlatolic acid. Five hours after the second injection mice were sacrificed by carbon dioxide exposure and peritoneal cavities were washed three times with 2 mL of cold PBS containing 3 mM EDTA and collected as intra-peritoneal lavage. The leukocyte count in the peritoneum of untreated control mice was assessed by lavage. Mice without thioglycollate treatment were used as controls. The volume of the collected lavage was measured and the cell count determined by haemocytometer.50 values were calculated from averaged measurements at 3-5 different concentrations of the compounds by nonlinear regression using GraphPad Prism software  one site binding competition. To calculate the IC50 values regarding NF-κB inhibition at least three different concentrations measured in quadruplicate in three independent transfection experiments were used utilizing nonlinear regression with Data Analysis Toolbox software . Statistical evaluation of the data was performed by one-way ANOVA followed by a Bonferroni or Tukey-Kramer post-hoc test for multiple comparisons respectively. A p value < 0.05 was considered statistically significant. Data are expressed as mean ± S.E.. ICCetraria nivalis (L.) Ach., Cetraria pinastri (Scop.) Gray, Cetrelia monachorum (Zahlbr.) W.L. Culb. & C.F. Culb., Cladonia carneola (Fr.) Fr., Lepraria incana (L.) Ach., Lobaria linita (Ach.) Rabenh., Lobaria pulmonaria (L.) Hoffm., Nephroma resupinatum (L.) Ach., Parmelia caperata (L.) Ach., Parmeliopsis hyperopta (Ach.) Arnold, Peltigera leucophlebia (Nyl.) Gyeln., Peltigera rufescens (Weiss) Humb., Platismatia glauca (L.) W.L. Culb. & C.F. Culb., Stereocaulon alpinum Laurer, Thamnolia vermicularis (Sw.) Ach. ex Schaer., Umbilicaria cyindrica (L.) Delise ex Duby, and Xanthoria elegans (Link) Th. Fr. The ethanolic crude extracts of these species  were tested for their potency to inhibit two targets within eicosanoid biosynthesis, namely microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO), as well as NF-κB transactivation activity  and Lepraria incana (LI) showed outstanding pharmacological in vitro profiles in the applied assays , IC50 values of 1.4 µg/mL and 1.3 µg/mL, respectively, were determined for CM. In TNF-α-stimulated HEK-293 cells CM inhibited the NF-κB transactivation activity with an IC50 of 2.6 µg/mL thereby only moderately affecting the viability of this cell line . In fact, CM concentration-dependently inhibited PGE3 - 5) and eight monoaromatic derivatives. By interpretation of mass spectrometric and 1D and 2D NMR data the structures were identified as ethyl haematommate (1), methylbetaorcinolcarboxylate (2), atranorin (3), imbricaric acid (4), perlatolic acid (5), 4-O-metylolivetolcarboxylic acid (6), ethyl-4-O-methylolivetolcarboxylate (7), ethyl olivetolate (8), olivetolmonomethylether (9), olivetol (10) and divarinol (11). Their physical and spectroscopic properties are in accordance with those reported previously  as a major mPGES-1 inhibitor by using a pharmacophore based virtual screening . Along these lines, we could identify this compound as a major mPGES-1-inhibiting ingredient (IC50 = 0.4 µM) in CM. Additionally, two other depsides were analyzed, namely atranorin (3) and imbricaric acid (4). As reported before , 3 did not affect the mPGES-1 activity, whereas 4 exhibited a pronounced and concentration-dependent inhibition in our experiments , however, did not suppress enzyme activity up to a concentration of 10 µM. The isolates of 4 and 5 in the cell-free assay with remarkable IC50 values of 3.5 µM and 0.4 µM, respectively. For comparison, the reference drug zileuton (the only approved 5-LO inhibitor on the market) revealed an IC50 = 0.7 µM . In parallel to the results from the mPGES-1 evaluation, depside 3 and the monoaromatic constituents  did not affect 5-LO activity up to 10 µM.Analysis of 5-LO inhibition by the 11 lichen compounds revealed a significant inhibitory activity for the two depsides = 0.7 µM , which i3 - 5) were additionally tested for their potential to suppress NF-κB activation in TNF-α-stimulated HEK-293/NF-κB-luc cells. In contrast to atranorin (3), imbricaric acid (4) and perlatolic acid (5) showed pronounced and concentration-dependent inhibition with IC50 values of 2.0 µM and 7.0 µM, respectively  as previously described [4 and 5. Luciferase reporter gene expression reflects a downstream event of the NF-κB signaling cascade and cannot provide insights into the molecular targets contributing to the observed NF-κB inhibition. Thus, we tested the active compounds escribed , since i5 appeared the most potent anti-inflammatory candidate based on the in vitro analysis, we aimed to assess the inhibitory effect of 5 on inflammation in vivo. For this purpose, we employed the thioglycollate (TG)-induced murine peritonitis model [5) co-administered animals this inflammatory response was significantly reduced by 30-40%  a moderate selective cytotoxicity on UACC-62 and B16-F10 melanoma cells [in vivo model of acute inflammation performed in this study no hints of toxic effects such as signs of serious discomfort or distress, change in animal behaviour or mobility, increased breathing, elevated temperature or nasal discharge were observed.Because is model . Five hop< 0.05, . Brandaoma cells . In the 4 and 5 were disclosed as those constituents prominently contributing to the observed multi-potent effect of CM, their contents were quantified by HPLC-DAD analysis in the extract and determined to be 15.22% and 9.10%, respectively. Considering the contents of 4 and 5, and correlating their inhibitory effects with the measured bioactivities of the parent extract, the mPGES-1 inhibitory effectiveness of 4 and 5 are in full agreement with the original extract, assuming these depsides to be the major ingredients contributing to the suppression of PGE2 formation. Recalculations for the inhibitory activities on 5-LO and NF-κB attested CM more pronounced activities than can be achieved simply by considering the bioactivities of 4 and 5, which leads to the assumption of further minor constituents enhancing the inhibitory effects or a synergistic action between the bioactive depsides.Since C. monachorum as an Alpine lichen species with pronounced inhibitory activity against three major pro-inflammatory targets, namely mPGES-1, 5-LO and NF-κB. To our best knowledge, no other plant- or natural product-derived extract has been reported with such strong mPGES-1-inhibitory activity, and also among 5-LO-suppressing plant extracts [4) and perlatolic acid (5), which both potently interfere with all three targets.In this study, we identified extracts , CM beloC. monachorum represents a valuable source for novel bioactive ingredients with high potential for pharmacological intervention with inflammatory disorders.Based on these properties, 3 - 5) is in line with the SARs, which we previously established by means of structural insights from pharmacophore-based ligand-target interactions [n-pentyl side chains of depsides as important feature for interference. Underpinning this hypothesis, we determined in the present study a slightly weaker effect for the newly identified imbricaric acid (4) which is characterized by a three carbon chain substitution at C6’ in contrast to 5 that carries the n-pentyl residue. Atranorin (3) missing a mandatory free carboxylic acid group completely loses any mPGES-1inhibitory activity but is recognized to be the main active compound in a traditional lichen preparation, inhibiting COX-1 and COX-2 in a 45% range at 45 µM [Efficient inhibition of mPGES-1 by the isolated depsides  were revealed as the main bioactive chemical class of this extract. The order of their inhibitory activities in CM on both targets within eicosanoid biosynthesis is 5 > 4 >>3, indicating that the potencies against mPGES-1 and 5-LO run in parallel as potent dual inhibitors of these enzymes.Here, we show for the first time that ingredients of 5), with the low IC50 value of 0.4 µM for both 5-LO and mPGES-1, is even more potent as compared to the reference inhibitors MK886 (of mPGES-1) and zileuton (of 5-LO) in the applied assays. Such high potency, together with the dual functionality against 5-LO and mPGES-1, qualifies 5 as valuable pharmacological agent. Note that under the assay conditions chosen, effects of the compounds on transcription and translation of 5-LO can be excluded due to the short incubation period (15 min). Interestingly, perlatolic acid (3) was found to be inactive against 5-LO before [50 = 8.3 µM [50 ≥ 28.5 µM; [Whereas atranorin . 4 and 5 exhibited a pronounced suppression of NF-κB activation in the low micromolar range, with imbricaric acid (4) being the most potent inhibitor (IC50 2.0 µM) followed by perlatolic acid ; 3 only showed a moderate activity with an IC50 of 20 µM. Taking into account that IKK2 inhibition can be ruled out as molecular mechanism for the observed NF-κB inhibition by 4 and 5, the underlying mode of NF-κB suppression still needs to be elucidated. Inhibition of NF-κB transactivation activity by depsides described here is unprecedented. NF-κB inhibition has been reported only for some lichen constituents from other chemical classes, e.g. triterpenes , dibenzoin vitro assays, perlatolic acid (5), inhibits the inflammatory recruitment of leukocytes in a peritonitis model in vivo. This observed inhibition of leukocyte recruitment compares well with the reported inhibition of recruitment by other inhibitors of the NF-κB- [in vivo anti-inflammatory potential.By conducting a pilot study, we here show  that onee NF-κB-  and 5-LOe NF-κB-  and withe NF-κB- . More de4 and 9.10% of 5 in CM underpins the potent activities of the extract, which in this study could to be streamlined to these two multi-targeting depsides. Intriguingly, they bundle the inhibitory in vitro potential of three prominent targets involved in inflammatory processes and, thus, warrant further preclinical analysis as multi-target anti-inflammatory leads.Correlation of the contents of these most abundant constituents with 15.22% of Information S1Click here for additional data file.


By scaffolding, Pak1 delivers signals through an auto-phosphorylation-induced conformational change without transfer of a phosphate group to its immediate downstream effector(s). Here we review evidence for this regulatory mechanism based on structural and functional studies of Pak1 in different cell types and research models as well as in vitro biochemical assays. We also discuss the implications of Pak1 scaffolding in disease-related signaling processes and the potential in cardiovascular drug development.P21 activated kinase-1 (Pak1) has diverse functions in mammalian cells. Although a large number of phosphoproteins have been designated as Pak1 substrates from  For instance, Pak1 was found to phosphorylate both myosin regulatory light chain and myosin light chain kinase (MLCK) predictably resulting in both an increase and a decrease of myosin II activity . Likewise it would seem important with regard to understanding Pak1 control mechanisms, to know its physiological substrate(s). Yet studies with Pak1 mutants cast doubt on the role of phosphorylation of downstream targets as a prominent Pak1 signaling mechanism. These studies demonstrated that kinase dead mutants of Pak1 retained the ability to enhance formation of lamellipodia  to its downstream target(s). This has been seen in numerous protein kinases as with MLCK, whose name and function is primarily defined by its obligatory protein substrate – myosin regulatory light chain .Pak family serine/threonine protein kinases have six members divided intro group I and group II Paks. Interestingly, although Pak1 and Pak4 belong to two different groups and are less homologous to each other, compared with members in the same groups , they prFigure 1B). Two auto-phosphorylations in the AID also contribute to kinase activation  may interfere with auto-phosphorylation, dimerization and substrate specificity of Pak1. These issues emphasize the importance of employing different approaches with the objective to understand Pak1 catalytic activity in mammalian cells and animal models.Since its discovery in 1994, many phosphoproteins have been assigned as Pak substrates . The ide20 enhances the activity of myosin II and likely assembly of actomyosin in mammalian cells  of 10 and above . These observations appear to shed light on molecular mechanisms responsible for Pak1 initiated cytoskeletal reorganization inasmuch as phosphorylation of MLC20 may regulate assembly of myosin II as well as myosin II activities  with its partners in brain extract shifts the Pak1 signal to a higher molecular weight position in SDS PAGE . AssociaWhile phosphorylation of ADF/cofilin promotes polymerization of F-actin, MAP4 phosphorylation stimulates disruption of the microtubule network in cardiomyocytes. A Pak1 induced rise of phosphatase activity leads to increased formation of the microtubule network accompanied by dephosphorylation of MAP4, a tau-like protein . An incrin vitro. These results indicate that activation of PP2A may be through a scaffolding mechanism . PP2A dephosphorylates multiple targets and is involved in regulation of cardiac excitation and contraction. Moreover in Pak1 knock-out mice, phosphorylation of cofilin at serine-3 was elevated with altered regulation of glucose homeostasis  induced increases of pacemaker activity through regulation of activities of L-type Ca2+ and delayed rectifying potassium channels  for the same binding region in Pak1. This competition could dislocate the “native peptides” and attenuate the Pak1–Pak1 interaction. Thus, interruption of Pak1 homodimer and attenuation of auto-inhibition may occur. PRR-1 and PID are bioactive peptides interacting with Pak1 . Activation of Pak1 by low molecular weight molecules exists in nature, and it is likely that some of those may not have been discovered yet. Pak1 is directly activated by sphingosine in vitro and in vivo characterized by an increase of auto-phosphorylation, bypassing upstream protein signals (in vitro (Isaria sinclairii, a Chinese traditional medicine with a variety of therapeutic applications (ex vivo rat heart, FTY720 normalizes arrhythmias triggered by enhanced β-adrenergic stimulation through Pak1 activation (As a key regulatory molecule crucial for multiple physiological processes, it is not surprising that Pak1 is regulated at multiple levels by diverse mechanisms ( signals . Sphingo signals . C2 and in vitro . A sphinications . In humaications ,b. In extivation . A recentivation .in vivo. However, in endothelial cells, inhibition of sphingosine-1 phosphate receptors did not abolish FTY720 induced enhancement of barrier integrity (ex vivo hearts, FTY720 treatment induces an increase in Pak1 auto-phosphorylation (ex vivo heart responds to FTY720 instantly, which may suggest that Pak1 is directly activated by FTY720 (in vivo will not only have application to further define Pak1 function in vivo, but also may serve as a novel therapeutic approach for cardiovascular diseases. FTY720 activates PP2A and inhibits tumor cell growth and tumorigenesis (FTY720 is converted to FTY720-phosphate by sphingosine kinase and then binds to sphingosine-1 phosphate receptors , which hntegrity , suggestntegrity . In ex vrylation . The ex y FTY720 . Identifigenesis . In miceigenesis . Althougigenesis , activatin vivo and serve as drug candidates for novel therapeutics of major cardiovascular diseases.Employment of scaffolding in signal transduction by Pak1 challenges the conventional understanding of a protein kinase. Some protein kinases other than Pak family members may also use the same signaling mechanism, but are not yet uncovered. Better understanding of scaffolding of Pak1 not only provides novel insights and enriches our knowledge on diverse modes of signal transduction, but also serves as guide for drug design and therapeutics targeting at Pak1. Substantial efforts have been made to develop inhibitors for Paks. Highly selective agonists for Pak1 may be equally important. The unique structure and catalytic activity of Pak1 has provided great potential for design and development of Pak1 activators through disruption of Pak1 auto-inhibition. These molecular tools should be valuable in further exploring Pak1 function The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sunlight-induced non-melanoma skin cancer is the most prevalent cancer in the United States with more than two million cases per year. Several studies have shown an inhibitory effect of caffeine administration on UVB-induced skin cancer in mice, and these studies are paralleled by epidemiology studies that indicate an inhibitory effect of coffee drinking on non-melanoma skin cancer in humans. Strikingly, decaffeinated coffee consumption had no such inhibitory effect. Mechanism studies indicate that caffeine has a sunscreen effect that inhibits UVB-induced formation of thymine dimers and sunburn lesions in the epidermis of mice. In addition, caffeine administration has a biological effect that enhances UVB-induced apoptosis thereby enhancing the elimination of damaged precancerous cells, and caffeine administration also enhances apoptosis in tumors. Caffeine administration enhances UVB-induced apoptosis by p53-dependent and p53-independent mechanisms. Exploration of the p53-independent effect indicated that caffeine administration enhanced UVB-induced apoptosis by inhibiting the UVB-induced increase in ATR-mediated formation of phospho-Chk1 (Ser345) and abolishing the UVB-induced decrease in cyclin B1 which resulted in caffeine-induced premature and lethal mitosis in mouse skin. In studies with cultured primary human keratinocytes, inhibition of ATR with siRNA against ATR inhibited Chk1 phosphorylation and enhanced UVB-induced apoptosis. Transgenic mice with decreased epidermal ATR function that were irradiated chronically with UVB had 69% fewer tumors at the end of the study compared with irradiated littermate controls with normal ATR function. These results, which indicate that genetic inhibition of ATR (like pharmacologic inhibition of ATR via caffeine) inhibits UVB-induced carcinogenesis support the concept that ATR-mediated phosphorylation of Chk1 is an important target for caffeine’s inhibitory effect on UVB-induced carcinogenesis. More than two million cases of non-melanoma skin cancer occur each year in the United States  that lacked excision repair, post-UV administration of caffeine inhibited UV-induced mutations, and it was suggested that “caffeine could diminish mutagenesis in Hcr− strains by modifying the process of recombinational repair in a way that reduces its inaccuracy”  demonstrated a strong inhibitory effect of ellagic acid, quercetin, myricetin, tannic acid, and many other plant phenols on the mutagenic action of the bay-region diol epoxide that is an ultimate carcinogenic metabolite of benzo[a]pyrene  have appreciable UV absorption between 260 and 300 nm (with a peak at ∼273 nm), we studied the effect of topical application of these compounds prior to UVB irradiation on UVB-induced thymine dimers and sunburn lesions in the epidermis of SKH-1 mice. Topical application of caffeine or caffeine sodium benzoate 0.5 h prior to UVB irradiation inhibited UVB-induced formation of thymine dimers and inhibited UVB-induced sunburn lesions, and caffeine sodium benzoate was more effective than caffeine Figure .2) caused a dose-dependent increase in UVB-induced apoptosis . Pretreatment with oral green tea or caffeine enhanced UVB-induced increases in p53 positive cells, p21 positive cells, and apoptotic sunburn cells  or caffeine (0.44 mg/ml in the drinking fluid) for 2 weeks prior to irradiation with UVB 30 mJ/cm0 mJ/cm2.s Figure . Oral ads Figure . Oral adin vivo significance of these studies by doing studies in mice with UVB-induced p53 mutations in their epidermal cells. Studies of p21 expression and apoptosis in p53 knockout mice will also help define the relationship of UVB and caffeine to p21 in this particular situation.In normal human and mouse keratinocytes with functional p53, UVB irradiation increased the levels of p53 and p21 , and apoptosis was increased Figure  Heffern. Geneticin vivo importance of decreasing ATR function for inhibition of UVB carcinogenesis was demonstrated by showing that decreasing ATR function in transgenic mice inhibited UVB-induced carcinogenesis  for 1 week inhibited the UVB-induced increase in p-Chk1 (Ser 345) by 82 and 99%, respectively at 6 h post-UVB, and cyclin B1 was increased 153 and 201%, respectively at 6 h post-UVB Figure  Lu et a. The tim2) twice a week for 20 weeks resulted in mice without tumors but with epidermal hyperplasia and a high risk of developing tumors over the next several months in the absence of further treatment with UVB (“high-risk mice”) (Lou et al., Treatment of SKH-1 mice with UVB 30 mJ/cm twice a Topical applications of caffeine to UVB-pretreated high-risk mice inhibited carcinogenesis as described in Table Topical caffeine increased the percentage of mitotic tumor cells with cyclin B1 by 70% (Lu et al., /−− background. Unlike caffeine, this genetic approach was selective for ATR function and had no effect on ATM activation by DNA damage. Primary keratinocytes from these mice had diminished UV-induced Chk1 phosphorylation and a twofold increase in apoptosis after UV exposure. With chronic UV treatment, transgenic mice remained tumor-free for significantly longer and had 69% fewer tumors at the end of observation of the full cohort than in littermate controls (Figure To determine the effect of genetic inhibition of ATR on UVB-induced carcinogenesis, we generated transgenic mice with diminished ATR function in skin and crossed them into a UV-sensitive Xpcs Figure  (Kawasums Figure .Since oral administration of caffeine increased locomotor activity and decreased tissue fat in SKH-1 mice (Lu et al., Animal data indicate that caffeine administration inhibits UVB-induced carcinogenesis by functioning as a sunscreen as well as by enhancing UVB-induced apoptosis and apoptosis in UVB-induced tumors. The stimulatory effect of caffeine on UVB-induced apoptosis occurs by p53-dependent and p53-independent mechanisms. Inhibition of the ATR/Chk1 pathway by caffeine is a major contributor to caffeine inhibition of UVB-induced carcinogenesis. The inhibitory effects of caffeine on UVB-induced carcinogenesis in animal studies described here are paralleled by an inhibitory effect of regular but not decaffeinated coffee on non-melanoma skin cancer in humans (Jacobson et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The morphogenic Hedgehog (Hh) signaling regulates postnatal cerebellar development and its aberrant activation leads to medulloblastoma. The transcription factors Gli1 and Gli2 are the activators of Hh pathway and their function is finely controlled by different covalent modifications, such as phosphorylation and ubiquitination. We show here that Gli2 is endogenously acetylated and that this modification represents a key regulatory step for Hedgehog signaling. The histone acetyltransferase (HAT) coactivator p300, but not other HATs, acetylates Gli2 at the conserved lysine K757 thus inhibiting Hh target gene expression. By generating a specific anti acetyl-Gli2(Lys757) antisera we demonstrated that Gli2 acetylation is readily detectable at endogenous levels and is attenuated by Hh agonists. Moreover, Gli2 K757R mutant activity is higher than wild type Gli2 and is no longer enhanced by Hh agonists, indicating that acetylation represents an additional level of control for signal dependent activation. Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling. Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin. Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes. The Hedgehog (Hh) pathway regulates development and stem/progenitor cell fate and its deregulation is a major cause of some malignancies, such as medulloblastoma, the most frequent pediatric solid tumor In mammals, Hedgehog signaling is activated by the interaction of the ligand with the inhibitory receptor Patched (Ptch). This alleviates the repression upon the transmembrane transducer Smoothened (Smo), which promptly migrates to the tip of the primary cilium, a microtubule-based organelle indispensable for Hedgehog function Genetic studies have revealed that Gli2 and Gli3 are the primary mediators of Hh signaling and are essential for embryogenesis. Conversely, Gli1 is dispensable for development but plays a key role in tumorigenesis A key mechanism regulating Gli transcriptional activity is represented by post-translational modifications Regulation of Gli2 and Gli3 processing is also regulated by interaction with Sufu that protects them from cullin/SPOP-mediated degradation Therefore, despite the three Glis share the regulation by phosphorylation and ubiquitination, these covalent modifications appear to be modulated by distinct kinases and ligases and to bring about different outcomes.KCTD11In our recent work, we have found that Gli1 is acetylated at a single conserved lysine, and that this modification inhibits its transcriptional activity The mechanisms underlying the acetylation-dependent inhibition and the physiological conditions where this modification occurs are still not understood.Given the relevance of Gli2 in mediating the transcriptional output of Hedgehog activation, here we have sought to understand the function and regulation of Gli2 acetylation.We show that Gli2 is endogenously acetylated at a single conserved lysine and that this modification inhibits the transcriptional activity by preventing its promoter recruitment. Importantly, we provide evidence that Gli2 acetylation is a key conserved step, which regulates signal-dependent transcriptional activation and can be monitored during Hedgehog-mediated tissue development. Thus, in contrast to phosphorylation and ubiquitination, the acetylation/deacetylation checkpoint seems to operate though a conserved mechanism, involving an interplay between early and late activatory events.HEK293T and NIH3T3 cells were cultured as previously described The following plasmids were provided by other laboratories: 8xGli-Luc reporter vector was provided by H. Sasaki, RIKEN Center for Developmental Biology, Japan; 12xGli-Luc reporter was from R. Tofgard, Karolinska Institutet, Sweden; Myc-Gli2 was from A. Dlugosz, University of Michigan, Ann Arbor, MI, U.S.A.; GST HAT-pCAF and PCI-p300 were provided by M. Fanciulli, Regina Elena Cancer Institute, Italy; TIP60 was provided by O. Segatto, Regina Elena Cancer Institute, Italy.Myc-Gli2 K757R and K757Q were obtained through site directed mutagenesis  of the wild type Myc-Gli2 vector using the following primers:ACGCGGAACACCCAGCTGCCTChGLI2K757R forward: GAGGCAGCTGGGTGTTCCGCGThGLI2K757R reverse: ACGCGGAACACCAGGCTGCCTCCCCTChGli2K757Q forward: GAGGGGAGGCAGCCTGGTGTTCCGCGThGli2K757Q reverse: Mutant sequences were verified by sequencing reactions.HEK293T and NIH3T3 cells were transfected by using Lipofectamine 2000 Reagent or Lipofectamine Reagent and Plus reagent (Invitrogen) respectively, according to the manufacturer's protocol. Luciferase assays were performed as described previously Cells were lysed with RIPA buffer  supplemented with protease inhibitors and total protein extracts were quantified. For nucleus and cytoplasm extraction, cells were lysed in 100 µL Buffer A  and incubated on ice. 0.6% NP40 was added to the samples. After centrifugation, the supernatant (cytoplasmic fraction) was removed and the pellet resuspended into 500 µL Buffer B  and incubated on ice. 1% NP40 was added and samples centrifuged. The pellet was resuspended into 50 µL Buffer C , incubated on ice and centrifuged to isolate the nuclear fraction.Protein extracts were analyzed by SDS-PAGE and blotted onto a nitrocellulose membrane (Perkin Elmer). Membranes were blocked by 5% milk in Tris buffered saline with 0.1% Tween20, and incubated with primary antibodies.The following antibodies were used: rabbit polyclonal antibody against acetylated lysine , goat anti Gli2 , anti-Gli2 (C-10) , mouse anti Myc-HRP , mouse anti-Myc , anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin , anti-CREB .Plus-ECL (Perkin Elmer).Signals were detected using Western Lightning Acetyl-Gli2(Lys757) and Acetyl-Gli1(Lys518) antisera were generated by Eurogentec by rabbit immunization with specific peptide:Acetilated-Gli1(Lys518) H2N-IGS RGL K(Ac)LPSLT C- CONH2Acetilated-Gli2(Lys757) H2N-PHTRNTK(Ac)LPPLPC-CONH2The specificity of the antibodies was validated by competition assays with the immunogenic peptides, with or without lysine acetylation.Acetylation assays were performed as previously described HEK293T, transfected with the indicated plasmids, were lysed and overexpressed Gli2 was immunoprecipitaded with anti Myc (9E10) AC beads ; NIH3T3 cells were lysed and endogenous Gli2 was immunoprecipitated with goat anti-Gli2 . After immunoblott acetylation was detected with rabbit polyclonal anti acetyl lysine , or anti Acetyl-Gli2(Lys757) (1∶1000).Ptch1 (Mm00436026_m1), GAPDH (Mm99999915_G1), HPRT (Mm00446968_m1), GLI1 (Mm00494645_m1).Total RNA was extracted from NIH3T3 cells, reverse transcribed and analized by quantitative Real Time PCR as previously described HEK293T cells, transfected with the indicated coding vectors, were crosslinked and chromatin immunoprecipitation was carried out as previously described 5-GTATTGCATGCGAGAAGGTTGG-3;hPtch1 promoter, forward: 5-TTTCTGCGACGCGATTGGCTCG-3;hPtch1 promoter, reverse: 5-AGAACATCATCCCTGCCTCT -3;hGAPDH promoter, forward: 5-CACCTGGTGCTCAGTGTAG -3.hGAPDH promoter, reverse: Data are shown as fold difference vs Empty vector.Immunohistochemistry was performed as described previously Sections from paraffin-embedded tissues were deparaffinized with xylol, fixed with decreasing ethanol concentrations, blocked and incubated with anti Gli1 , or anti acetyl-Gli1(Lys518) or anti acetyl-Gli2(Lys757) antibodies (1∶50). Sections were counterstained with hematoxylin.U test for non-parametric values and a p<0.05 was considered significant.Statistical analysis was performed using StatView 4.1 software . Results are expressed as mean ± s.d. of at least three separate experiments, each performed in triplicate. Statistical differences were analyzed with the Mann-Whitney We first tested the ability of different histone acetyltransferases (HATs) to acetylate Gli2. We selected three different HATs, each representing a member of the main HAT families To study the functional consequence of Gli2 acetylation, we performed luciferase assays with a Gli responsive reporter (12× Gli-Luc) and the different HATs: exogenous p300 caused a strong inhibition of Gli2 mediated reporter activity, while TIP60 and pCAF did not display any effect . Thus, pGli2 contains a potentially acetylatable lysine (K757), which is conserved in Gli1 (K518) but not in Gli3 To determine whether lysine 757 is acetylated, we mutated it into arginine and performed in vivo acetylation assay with the mutant Gli2K757R. As shown in It has been shown that substitution of a lysine with a glutamine mimics a status of constitutive acetylation Having observed that Gli2 acetylation at K757 inhibits its transcriptional activity, we next wondered whether this modification occurs at endogenous level and how it is connected with the activation status of the Hedgehog pathway.Toward this end, we generated a rabbit polyclonal antibody against the acetylated K757 of Gli2, which was able to readily detect this modification and did not react against K757R mutant Gli2 .To determine whether acetylated Gli2 is detectable at endogenous levels and to study if this modification is perturbed in response to activated Hedgehog signaling, we performed acetylation assays in NIH3T3 cells treated with the Smo agonist SAG. As shown in To study the contribution of K757 deacetylation to the Gli2-dependent transcriptional activation, we analyzed the activity of K757R Gli2 mutant, mimicking a status of constitutive deacetylation in luciferase assays and by quantitative Real Time PCR . ComparePtch1, in NIH3T3 cells transfected with Gli2 WT or K757R mutant, and treated with SAG or vehicle control.We next performed a quantitative Real Time PCR to assess the levels of the Hh target mRNA Ptch1 transcript that was further increased by four fold upon Smo activation with SAG , where Hh pathway regulates development of the cerebellar granule cell progenitors and promotes their mitotic expansion in the External Granular Layer (EGL).Confirming the increased Hh activation status, incubation of sections with anti Gli1 antisera, strongly stained the EGL . In contActivation of Gli2 function involves at least three main steps: i) a modification of processing/stability with alterations of its half–life; ii) changes in cellular compartmentalization; iii) interaction with target promoters and transcriptional cofactors to induce transcription. Therefore, we next addressed whether acetylation of Gli2 affects one of these events leading to transcriptional activation.Ptch1 promoter We ruled out the possibility that acetylation could interfere with Gli2 stability since the steady state levels of WT and mutant proteins did not appear to be different  bottom. Remarkably, the promoter occupancy of Gli2 K757Q was abrogated, compared to WT Gli2, whereas the K757R mutant displayed a significant increase in chromatin binding . TherefoProtein acetylation is a critical regulatory step for histones and many non-histone proteins, including transcription factors, enzymes or structural proteins In the present work we have characterized the acetylation of Gli2, the early transcriptional effector of Hedgehog signaling. We have demonstrated that Gli2 is acetylated at lysine 757 by the co-activator p300 and that this modification inhibits Hh-target gene expression.While it is still unclear the type of signals that may induce this p300-mediated acetylation of Gli2, we have shown that removal of the K757 acetyl group occurs upon activation of Hh signaling. It is likely that the mechanism linking Hh activation to Gli2 deacetylation involves the activation of class I HDACs Interestingly, the acetylation/deacetylation equilibrium seems to contribute to the typical dynamic response of Hh-dependent transcription. Indeed, a Gli2 mutant mimicking a constitutive deacetylation status is hyperactive but no longer induced by Smo agonists.Thus, in addition to the balance between the full length GliA and the truncated GliR isoforms, the acetylation/deacetylation balance seems to be an additional regulated mechanism to switch from one status to another. This observation adds further complexity to the classical model of Gli activation and implies that acetylation represents a supplementary mechanism utilized by cells to modulate Hedgehog output. In support to this model, we show that, when monitored with a specific antibody, acetylated Gli2 cannot be found in territories of active Hedgehog signaling, such as the outer EGL, where HDAC1 levels have also been found to be upregulated It will be important to understand whether and to what extent acetylation also contributes to the graded response to ligand concentrations, thus representing a further level of control during morphogen dependent development.What are the mechanisms of transcriptional inhibition following Gli2 acetylation? In this work we have demonstrated that acetylation of Gli2 does not affect its intracellular localization and stability. Instead, acetylation appears to prevent Gli2 recruitment to target chromatin. Since the residue K757 maps outside the zinc finger domain, it is unlikely that acetylation affects Gli2/DNA affinity. A plausible explanation is that K757 acetylation prevents the association with chromatin-bound cofactors, which are essential for the promoter entry/transactivation of Gli2. In this regard, a potential candidate for this effect could be the ATP-dependent chromatin remodeling factor Brg, which has been reported to interact with both HDACs and Glis and to participate to their Hh-induced chromatin recruitment and target gene activation In conclusion, these data illustrate a novel mechanism of regulation of the Hh signaling, where acetylation of Gli2 at lysine 757 functions as a critical regulated step, controlling the activation status of Hh pathway. We suggest that this mechanism contributes to the early regulatory events immediately downstream of Smo , and is functionally coordinated with Gli1 acetylation, which represents a late regulated step of Hedgehog pathway activation .These results further emphasize the relevance of Gli acetylation as a key regulatory epigenetic modification, with promising therapeutic potential for diseases linked to aberrant Hh pathway activation.


Drosophila to identify transcriptional regulators of immunity and pathology and their associated target genes and physiologies. We show that Mef2 is required in the fat body for anabolic function and the immune response. Using genetic and biochemical approaches, we find that MEF2 is phosphorylated at a conserved site in healthy flies and promotes expression of lipogenic and glycogenic enzymes. Upon infection, this phosphorylation is lost, and the activity of MEF2 changes—MEF2 now associates with the TATA binding protein to bind a distinct TATA box sequence and promote antimicrobial peptide expression. The loss of phosphorylated MEF2 contributes to loss of anabolic enzyme expression in Gram-negative bacterial infection. MEF2 is thus a critical transcriptional switch in the adult fat body between metabolism and immunity.Infections disturb metabolic homeostasis in many contexts, but the underlying connections are not completely understood. To address this, we use paired genetic and computational screens in   •Mef2 is required in Drosophila for immune function and storage of fat and glycogen•MEF2 is phosphorylated in vivo at a conserved site (T20) to promote anabolism•Infection reduces phospho-T20, allowing MEF2 to bind TBP and an immune TATA box•MEF2 dephosphorylation leads to metabolic dysfunction in Gram-negative infection Drosophila, shifting upon infection from activation of anabolic enzymes to immune response genes.The transcription factor MEF2 acts as a central switch between metabolic and immune functions in  Metabolic regulation is tightly and ubiquitously linked with immune responses and inflammatory signaling . ProlongDrosophila as well as in vertebrates. We have previously shown that infection with Mycobacterium marinum causes metabolic disruptions in Drosophila resulting, in part, from a systemic loss of AKT activity  and with metabolic activities (cluster X). We further analyzed Mef2 as a potential nodal point between immune activation and metabolic disruption.To test in vivo function of predicted regulators, we performed a secondary functional screen, testing time to death after Mef2 are embryonic lethal , diacylglycerol acyltransferase (mdy), and fat body hexose kinase (Hex-C). In addition to being repressed by M. marinum infection, cluster X was rapidly and transiently downregulated 3–6 hr after septic injury (infection with a mixture of nonpathogenic Gram-positive and Gram-negative bacteria), with most genes returning to baseline within 24 hr , ATP citrate lyase (ATPCL), glycerol-3-phosphate dehydrogenase (Gpdh), glycerol kinase (Gk), and the mitochondrial citrate transporter (sea) , UDP-glucose pyrophosphorylase (UGP), glycogen synthase (GlyS/CG6904), and the glycogen branching enzyme (AGBE) (Mef2 (FASN and Hex-C) could also be repressed by inducible expression of a Mef2-engrailed repressor fusion (Mef2 for expression (er (sea) A. A simifat body ), phosphr fusion , confirmpression A. Howevepression A and S2.Hex-C, Pgm, UGP, GlyS, and AGBE) and lipogenesis , unchallenged Mef2 knockdown flies were almost entirely devoid of triglyceride and glycogen . As would be expected, Mef2 knockdown animals died more rapidly than wild-type flies when starved  from Drosocin and Metchnikowin upstream of enhanced GFP (eGFP). This region included NF-κB, DEAF-1, and GATA sites previously shown to regulate Mtk expression (Dro site 3 and Mtk site 2)   A and S5. pathway )  was a near-perfect match to the ideal MEF2A site as defined by SELEX (KCTAWWWWTAGM) (Mtk MEF2-TATA site (GCTATAAAAGC). This site eliminates one of the central A/T nucleotides in the MEF2 consensus, changing the spacing between the two putative half-sites. When assayed by EMSA, the Mtk core promoter was bound by nuclear extracts from S2R+ cells transfected with Mef2-HA, causing two distinct complexes to form: complex 1, at lower apparent molecular weight, was strong and consistently present, whereas the amount of complex 2 varied from experiment to experiment, though it was always less abundant than complex 1 (Mtk TATA sequence.We next asked whether the MEF2-TBP complex could bind the putative MEF2-TATA site. The WWWTAGM) , so we fomplex 1 C. MutatiMtk MEF2-TATA box  and other immune-inducible small peptides . Of thesTATA box . 17/27 ADrosophila TSSs   C.∗.HAMef2 expression. Because all transgenes were inserted into the same site, ∗Mef2 constructs were expressed at similar levels , or phosphomimetic MEF2 (T20E) specifically in the fat body. One week after eclosion, these flies were shifted from 18° to 29° to drive r levels A.∗.HA either before or 3 hr after mixed infection. As was seen for the endogenous protein, wild-type MEF2.HA associated with endogenous TBP only after infection (We tested whether T20 directly regulated the association of MEF2 with TBP by immunoprecipitating fat-body-expressed MEF2nfection D. NonphoDrosophila fat body. Others have observed MEF2C T20 phosphorylation by protein kinase A (PKA) and AKT  after either M. luteus or E. cloacae infection but had little effect on most metabolic genes. Conversely, expression of phosphomimetic MEF2 had little general effect on antimicrobial peptides and did not counteract the loss of most anabolic transcripts after M. luteus infection but rescued levels of anabolic transcripts after E. cloacae infection.Together, these data suggested that loss of MEF2 T20 phosphorylation might cause the loss of anabolic transcripts seen after infection and that loss of T20 phosphorylation might be permissive for antimicrobial peptide expression. To test this model, we measured expression of immune and metabolic MEF2 targets in uninfected and infected animals inducibly expressing wild-type controls A and S7.imd pathway activation  stems, at least in part, from the loss of T20-phosphorylated MEF2.Together, these data indicate that T20 phosphorylation can regulate the association of MEF2 with TBP, switching MEF2 between metabolic and immune functions. The loss of metabolic gene expression driven by Mef2 as a factor critical for energy storage and the inducible immune response in the Drosophila fat body. Many infection-induced antimicrobial peptides depend on Mef2 for normal expression. In consequence, flies lacking Mef2 activity in the fat body are severely immunocompromised against a variety of infections. Mef2 sites are also associated with genes encoding key enzymes of anabolism, and Mef2 is required for normal expression of these genes; consequently, flies lacking Mef2 function in the fat body exhibit striking reductions in the total levels of triglyceride and glycogen. These two groups of target genes are counterregulated during infection; the anabolic targets of Mef2 are reduced in expression when antimicrobial peptides are induced. We show that fat body MEF2 can exist in two states with distinct physiological activities. In uninfected animals, MEF2 is phosphorylated at T20 and can promote the expression of its metabolic targets. In infected animals, T20 is dephosphorylated, and MEF2 associates with the TATA-binding protein to bind a compound MEF2-TATA sequence found in the core promoters of antimicrobial peptides. The loss of T20-phosphorylated MEF2 promotes the loss of anabolic transcripts in flies with Gram-negative bacterial infection. These data, taken together, suggest that the central role of MEF2 in promoting fat body anabolism and immune activity reflects a switch between distinct transcriptional states regulated, at least in part, by differential affinity for TBP determined by T20 phosphorylation  in end-stage  samples . Probes  samples .Drosophila chr 3R as background sequence.For each cluster, extended gene regions  were analyzed with CLOVER , using DDrosophila Genome 1 array and treated the same as true clusters). Sites predicted to regulate more than two false clusters were eliminated from consideration.Binding sites were tested against 20 false clusters of genes (genes selected randomly from those represented on the Affymetrix http://go.princeton.edu/cgi-bin/GOTermFinder).Functional predictions were from Generic GO Term Finder | > 0.58, p < 0.01) in end-stage M marinum samples  as background sequence. We excluded sites with p > 0.02.For each cluster, extended gene regions  were compiled into a single textfile. Where adjacent genes appeared in a single cluster, no sequences were duplicated. This was analyzed using CLOVER  with a pDrosophila Genome 1 array and treated the same as the clusters above). Matrices that appeared frequently as regulators of these false clusters were eliminated from consideration.Each transcription-factor binding site matrix was analyzed using the same parameters against 20 false clusters of genes , E cloacae (NCTC 10005), and M luteus  were cultured overnight shaking at 37° in LB without antibiotics. L monocytogenes (NCTC 7973) was cultured overnight standing at 37° in BHI without antibiotics. C albicans was cultured overnight shaking at 30° in YT without antibiotics. Microbes were resuspended in sterile PBS for injection.escribed . E. coliWestern blots on whole-fly lysates were performed by smashing 3 adult male flies in 75 μl 2x Laemmli SDS-PAGE buffer with DTT; 5 μl of this lysate were used per gel lane.Drosophila in ice-cold 25 mM HEPES pH 7.6, 100 mM KCl, 1% Nonidet P-40, 0.1 mM EDTA, 12.5 mM MgCl2, 10% glycerol, 0.1 mM DTT, supplemented with phosphatase and protease inhibitors (Roche). These extracts were then incubated overnight at 4° with protein-A or G sepharose or dynabeads. Anti-MEF2  or anti-HA  were added in the morning and permitted to bind for at least one hour at 4°. Beads were then washed extensively in IP lysis buffer and finally resuspended in 2x Laemmli SDS-PAGE buffer with DTT for detection by Western blot.For immunoprecipitation, whole-fly lysates were prepared by smashing 20-50 adult male Drosophila S6 Kinase 9209, phospho-AKT substrate 10001 (Drosophila MEF2)  .Mtk and Dro enhancer/promoters were amplified from Drosophila genomic DNA and cloned upstream of eGFP in the vector pCS2P-eGFP-X/P, simultaneously eliminating the sCMV promoter. Mutants were generated using DpnI mutagenesis, sequenced, and a fragment containing the mutation was cloned into the parental plasmid. Site locations, relative to initiator ATG: Dro 1, −1240 to −1229; Dro 2, −947 to −936; Dro 3, −66 to −54; Mtk 1, −804 to −793; Mtk 2 and T, −62 to −51. Primers are in ∗ cells  acid sodium salt (Sigma). 5 pm unlabeled double-stranded oligo was used as a competitor. For supershift, nuclear extracts were preincubated with antibodies in binding buffer for 1 hr at 4° before the addition of biotinylated oligos; incubation with a control anti-GFP antibody did not disrupt observed binding (data not shown). EMSA reactions were run on 5% native acrylamide gel, containing 1% glycerol.The LightShift Chemiluminescent EMSA Kit (Thermo Scientific) was used according to the manufacturer’s instructions, with the following modifications. 2 femtomoles of double stranded oligo were mixed with 1-2 μg of nuclear extract and incubated 10 min at room temperature in 20 μl of 10 mM Tris pH 7.6, 75 mM KCl, 1 mM DTT, 2.5 mM MgClE. coli on Glutathione Sepharose-4B  and washed 3 times in kinase assay buffer . 2 μg GST fusion protein was incubated with 0.1U recombinant AKT  or 0.02U recombinant activated p70S6 Kinase (T412E)  and 2 μCi γ-32P-ATP in 1X buffer for 30 min at 30°C. Boiled reactions were resolved on 12.5% SDS-PAGE gels and stained with Coomassie Blue. Dried gels were exposed to autoradiography film.GST-MEF2tide-WT and GST-MEF2tide-T20A fusion protein constructs encoding GST, a glycine-rich linker and amino acids 14-24 of MEF2 were constructed using the Gibson Assembly Kit (NEB) in pGEX-KG and confirmed by sequencing. GST (from pGEX4T1) and GST-MEF2tide were purified from


Initially alanyl-tmRNA in complex with EF-Tu and SmpB enters the vacant A-site of the stalled ribosome like aminoacyl-tRNA but without a codon–anticodon interaction, and subsequently truncated mRNA is replaced with the tag-encoding region of tmRNA. During these processes, not only tmRNA but also SmpB structurally and functionally mimics both tRNA and mRNA. Thus trans-translation rescues the stalled ribosome, thereby allowing recycling of the ribosome. Since the tag-peptide serves as a target of AAA+ proteases, the trans-translation products are preferentially degraded so that they do not accumulate in the cell. Although alternative rescue systems have recently been revealed, trans-translation is the only system that universally exists in bacteria. Furthermore, it is unique in that it employs a small RNA and that it prevents accumulation of non-functional proteins from truncated mRNA in the cell. It might play the major role in rescuing the stalled translation in the bacterial cell.Transfer messenger RNA  is a small RNA molecule that is conserved among bacteria. It has structural and functional similarities to tRNA: it has an upper half of the tRNA-like structure, its 5’ end is processed by RNase P, it has typical tRNA-specific base modifications, it is aminoacylated with alanine, it binds to EF-Tu after aminoacylation and it enters the ribosome with EF-Tu and GTP. However, tmRNA lacks an anticodon, and instead it has a coding sequence for a short peptide called tag-peptide. An elaborate interplay of actions of tmRNA as both tRNA and mRNA with the help of a tmRNA-binding protein, SmpB, facilitates  Figure 1A; Figure 1A; trans-translation , serves as the target for cellular ATP-dependent proteases  does not terminate efficiently because peptide release factor does not function. Thus, the cell should have a system to cope with such emergencies. However, little attention was given to this issue until the mid-1990s, and therefore the discovery of tmRNA was a big surprise. Initially the tRNA-like structure and function of tmRNA were elucidated. Both terminal regions of tmRNA can form a secondary structure resembling the upper half of the cloverleaf-like structure of tRNA , which iroteases .Trans-translation requires a tmRNA binding protein called SmpB in addition to canonical elongation factors : initially, tmRNA in complex with SmpB is aminoacylated with alanine by alanyl-tRNA synthetase. Ala-tmRNA enters the A-site of the stalled ribosome on a truncated mRNA to receive the nascent polypeptide from peptidyl-tRNA in the P-site. Then peptidyl-Ala-tmRNA translocates to the P-site, which exchanges the template from truncated mRNA to the tag-encoding region on tmRNA. It can reasonably explain the missing origin of the alanine residue connecting the truncated polypeptide encoded by mRNA with the tmRNA-encoded tag-peptide: it is derived from the alanine moiety aminoacylated to tmRNA. This model was proposed on the basis of the results of an in vivo study showing that truncated polypeptides fusing the tmRNA-encoding tag-peptide in its C-termini accumulate in the cell when a truncated mRNA is expressed (in vitro study showing that the tag-peptide is synthesized using Escherichia coli cell extract depending on the addition of poly(U) and on the aminoacylation capacity of tmRNA . Ala-tmRNA·SmpB·EF-Tu·GTP enters the vacant A-site of the stalled ribosome. GTP hydrolysis induces a conformational change of the stalled complex to release EF-Tu.GDP, allowing accommodation of Ala-TLD·SmpB in the A-site. During this process, the C-terminal tail of SmpB interacts with the mRNA path extending towards the mRNA entry channel. Subsequently, Ala-TLD in the A-site receives the nascent polypeptide chain from peptidyl-tRNA in the P-site, and the resulting peptidyl-Ala-TLD·SmpB translocates from the A-site to the P-site. During this process, the C-terminal tail of SmpB dissociates from the mRNA entry channel and binds around the site of codon–anticodon interaction in the P-site with change in its conformation from the extended structure to the folded structure, which in turn promotes release of mRNA from the ribosome. The conformational change of the C-terminal tail concomitant with translocation makes the A-site free, thereby allowing introduction of the resume codon of tmRNA into the decoding region. This model has been supported by results of structural studies of several trans-translation intermediates. Cryo-EM studies have revealed three kinds of intermediates in the pre-accommodated, accommodated, and translocated states  around the start of the C-terminal tail of SmpB (E. coli SmpB (H136) has a crucial role in GTP hydrolysis, leading to the proposal that stacking of this residue with G530 triggers GTP hydrolysis.This model can explain why in vitro : the C-tin vitro . Howevernslation . In a cr of SmpB . Recentltrans-translation process  in the stalled ribosome since publication of the results of an earlier in vivo study using an artificial mRNA , including ClpXP, ClpAP, Lon and FtsH, and the periplasmic protease Tsp . ClpX or ClpA recognizes the C-terminal ALAA sequence of the tag-peptide to unfold the trans-translation products in an ATP-dependent fashion for degradation by its partner ClpP peptidase  sequence, a binding site of the repressor protein catabolite control protein A (CcpA), within the coding region of several mRNAs including TreP mRNA for trehalose phosphorylase , has also been suggested . Alanine might not be an absolute prerequisite for in vitro , althougin vitro . TLD andFigure 1A; C. crescentus belonging to α-proteobacteria has been shown to actually function in trans-translation  with the tag-encoding region in between and a 3′-amino acid acceptor piece, and the two pieces join together by base-pairing to form into a tRNA-like structure similar to that in one-piece tmRNA  has an activity to hydrolyze the linkage between tRNA and the nascent polypeptide of peptidyl-tRNA after it drops off from the ribosome. Drop-off is enhanced by RRF alone, RRF together with RF3  or RRF, E. coli ArfA or tmRNA is viable, whereas a double knockout is lethal, explaining why tmRNA is not essential in many bacteria  and YaeJ (ArfB), facilitate rescue of the stalled ribosome. A single knockout of either bacteria . The rib extract . However extract . RF-2 usypeptide . Thus, tin vitro  and in v operon is induced by tryptophan via the translational arrest of the leader peptide (TnaC) by inhibiting hydrolysis of peptidyl-tRNAPro by RF2 , ArfA or YaeJ would fail to access this stalled ribosome. Instead, the peptidyltransferase center is modulated by EF-P binding to the region between the P-site and E-site to resume peptidyl-transfer  is universally present in archaea and eukaryotes. EF4 is universally conserved among bacteria. Neither tmRNA nor SmpB is present in the cytoplasm of eukaryotes, where a complex of Dom34p (Pelota) and the GTP-binding protein Hbs1 promotes subunit dissociation of the stalled ribosome and drop-off of peptidyl-tRNA (trans-translation system is unique in that it employs a small RNA and that it prevents accumulation of non-functional proteins from truncated mRNA in the cell.Pth is essential for bacteria and is widely distributed among the other domains. While the dyl-tRNA  in concedyl-tRNA . The Domdyl-tRNA , althougdyl-tRNA , 2014. IThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of neurofibrillary pathology.In Alzheimer's disease (AD) and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A) activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol  Alzheimer's disease is the leading cause of dementia and as of 2006, the worldwide prevalence of the disease was 26.6 million in vitro and in vivoThe etiology of Alzheimer's disease is probably multifactorial, and there is growing interest in the potential relationship between anesthesia and the onset and progression of Alzheimer's disease et al.in vivo, as this would more closely mimic clinical anesthetic exposure. We observed that propofol leads to tau hyperphosphorylation under normothermic conditions both in mice and in human neuroblastoma cells.It has been recently demonstrated that anesthetics can also produce tau hyperphosphorylation in the absence of hypothermia P<0.001 vs. control). As expected, given that anesthesia-induced hypothermia inhibits the activity of PP2A We first wanted to confirm that propofol would induce hypothermia-mediated tau hyperphosphorylation, as it has not been reported previously. Thirty min following propofol administration, significant hypothermia occurred in the group of mice whose temperature was not controlled during anesthesia . The mouse rectal temperature decreased to 30.9±1.0°C in the hypothermia group versus 37.5±0.4 and 37.5±0.6°C in the control (n = 4) and temperature controlled  groups, respectively  following propofol, tau phosphorylation at AT8, PHF-1, and CP13 significantly increased compared to control ; however, by 6 h  following propofol, tau phosphorylation had returned to control levels at all 3 phosphoepitopes . There wHence, under normothermic conditions, propofol produces an immediate increase in tau phosphorylation at the AT8, PHF-1, and CP13 phosphoepitopes; however, at the AT8 phosphoepitope tau hyperphosphorylation persists 2 h following anesthesia but recovers 6 h following propofol treatment. Furthermore, the greater degree and longer duration of hyperphosphorylation at AT8, when compared to PHF-1 and CP13, suggests that this phosphoepitope is more susceptible to the hyperphosphorylation effects of propofol.To determine whether regional brain differences in tau phosphorylation exist, we also examined levels of tau phosphorylation at the AT8, PHF-1, pS262, MC6 and pS422 phosphoepitopes in cortical tissue harvested 30 min  or 2 h  following the administration of propofol or intralipid . In the cortex, tau phosphorylation at the AT8 phosphoepitope increased to 154±28% of control 30 min and to 144±29% of control 2 h following propofol administration , confirm9As our results demonstrate that tau hyperphosphorylation following propofol anesthesia occurs under normothermic conditions, we next examined the activation state of specific tau kinases as well as the protein expression levels of phosphatases commonly involved in the regulation of tau phoshorylation. Among the kinases capable of phosphorylating tau, glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5 (cdk5) and p35 (a specific activator of cdk5), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and c-Jun N-terminal kinase (JNK) are considered to be major physiological and pathological tau kinases We therefore examined next the levels of tau protein phosphatases (PP) catalytic subunits 30 min and 2 h after propofol administration. Tau can be dephosphorylated by PP1, PP2A, and PP2B, but PP2A shows a much stronger capacity to dephosphorylate tau We next determined the effect of propofol on tau phosphorylation in vitro using SH-SY5Y human neuroblastoma cells stably transfected to overexpress human tau (Tau-SH-SY5Y cells). These studies were performed to determine whether propofol-induced tau hyperphosphorylation is the result of a direct pharmacologic effect and not the consequence of an anesthetic-induced physiologic change. First, as a positive control, we examined the degree of tau phosphorylation in the cells at the AT8 epitope following 1 h incubation at 37°C  or 30°C (n = 3). Following a 1 h incubation of the Tau-SH-SY5Y cells at 30°C, tau phosphorylation increased to 245±18% of the control (37°C) group , confirmThe findings of the present study are novel and demonstrate that a common clinically used intravenous anesthetic, propofol, can increase tau phosphorylation in the mouse hippocampus and cortex, not only as a consequence of anesthesia-induced hypothermia, but also due to a direct effect of the drug itself. Furthermore, in the hippocampus, tau hyperphosphorylation at the AT8 phosphoepitope persists for at least 2 h following propofol administration before returning to control levels 6 h later, thus demonstrating that direct tau hyperphosphorylation by propofol can persist even following recovery from the sedative-hypnotic effects of the drug. The observation that normothermic hyperphosphorylation following anesthesia occurred within a clinically relevant time frame, 30 min and 2 h following exposure, is also novel.We previously observed that the hyperphosphorylation of tau following intravenous and inhalational anesthetics primarily involves the inhibition of PP2A by anesthetic-induced hypothermia et al., we observed that the degree of hyperphosphorylation observed following anesthesia varies as a function of the specific phosphoepitope. Specifically, we observed the greatest increase in tau phosphorylation following 30 min of propofol treatment at the AT8 phosphoepitope, and to a lesser extent at CP13, PHF-1, pS262, MC6, and pS422, in the hippocampus or the cortex. Furthermore, the persistence of tau hyperphosphorylation at AT8 (pSer202/pThr205) after 2 h in both brain regions, also suggests that propofol has a greater capacity to produce hyperphosphorylation at certain phosphoepitopes.More recent studies have observed that either ether or sodium pentobarbital anesthesia can directly lead to hyperphosphorylated tau both 30 sec and 5 min following treatment, as no evidence of hypothermia was observed by these investigators at either of these time points in vitro target epitopes on tau, see in vivo; for example, 10 days of diabetes induced by streptozotocin resulted in more than 1000% increase in p-CaMKII but no detectable phosphorylation at either pS262 or pS356, two sites demonstrated to be phosphorylated by CaMKII in vitroAs tau phosphorylation is regulated by the activity of several protein kinases Although it would be premature to extrapolate these preclinical findings to the clinical sphere at this juncture, the observation that propofol has direct effects on tau phosphorylation should not be underestimated. This is especially true given the fact that unlike anesthetics previously studied in the context of their capacity to produce tau hyperphosphorylation such as isoflurane, ether, choral hydrate, and sodium pentobarbital ad libitum, and they underwent an acclimatization period for a minimum of 24 h before being used in the experiments.The experimental protocol was approved by the Columbia University Animal Care and Use Committee and, in accordance with National Institutes of Health (NIH) guidelines, adequate measures were taken to minimize pain and discomfort. Seven to nine-week-old C57BL/6 male mice were purchased from a commercial vendor  and utilized in this study. The mice were housed in a temperature-controlled room at 22°C and were kept on a 12 h/12 h light/dark cycle. All animals had access to food and water On the day of the study, a 25 mg/ml solution of propofol  in intralipid (20% emulsion) was freshly prepared. In all of the experiments, the mice were exposed to either propofol  250 mg/kg or an equivalent volume of intralipid  via intraperitoneal (i.p.) injection. The control mice were returned to their home cage at room temperature after injection. All of the anesthetized mice were initially anesthetized in their home cage, and, once sedated, they were transferred to a Thermocare® ICS therapy warmer unit  either set to maintain a mouse body temperature of ∼37°C or with the heating element turned off, the latter setting used to achieve propofol-induced hypothermia. Rectal temperature was monitored using an electronic thermometer probe .Following propofol or intralipid exposure, the mice were killed by cervical dislocation either 30 min, 2 h, or 6 h following drug or vehicle administration, then the brain was immediately removed and dissected in ice-cold Tris-EDTA buffer . The hippocampal and cortical tissues were immediately frozen in liquid nitrogen and stored at −80°C. Protein extracts were prepared by mechanically homogenizing the hippocampal and cortical tissues in 5x vol/w RIPA buffer containing 50 mM Tris-HCl , pH 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 0.1% SDS (Sigma), 0.5% sodium deoxycholate, 1% NP-40 (Sigma), phosphatase inhibitors , and protease inhibitors . The samples were incubated on ice for 30 min, sonicated for 30 s in pulse mode, and then centrifuged at 11,000 g for 10 min at 4°C. Total protein content was determined in the supernatant using the bicinchoninate (BCA) method 2 flasks containing Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum, 3 mM L-glutamine, 1 mM nonessential amino acids as well as 50 units/ml penicillin/streptomycin , and were maintained in a 5% humidified incubator at 37°C. In preparation for anesthetic or vehicle exposure, the cells were transferred into 6-well plates and used at approximately 70% confluency.Propofol-induced tau phosphorylation was also examined using SH-SY5Y human neuroblastoma cells stably transfected to constitutively express human tau with 3 microtubule binding domains On the day of the experiment, the growth medium was removed by aspiration, and each well was washed 2 times with 1 ml of fresh DMEM containing no additives. Immediately following the wash, each well received either 1.5 ml of Propofol (3 µg/ml) in 10% intralipid in DMEM or 10% intralipid in DMEM (vehicle). The concentration of propofol utilized was prepared using the commercial formulation of propofol  and based on a concentration previously utilized in cell culture studies In preparation for SDS-PAGE and Western blot analysis, the cells were lysed on ice for 30 min and then sonicated on ice for 20 s in pulse mode at 30% power output. The lysate was then centrifuged at 1000 g for 10 min at 4°C, and the supernatant collected and analyzed for protein content using the bicinchoninate (BCA) method The expression of phosphorylated tau, total tau as well as the tau kinases and phosphatases was determined using SDS-polyacrylamide gel electrophoresis (PAGE) coupled with Western blot analysis. Brain homogenate or cell lysate aliquots containing 30 µg protein were separated on a SDS-10% polyacrylamide gel and then transferred onto nitrocellulose membranes . Non-specific binding sites were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature and then were incubated overnight at 4°C with antibodies directed against either total tau, phosphorylated tau, a specific tau kinase or phosphatase. The following day the membranes were washed 3 times and then incubated for 1 h at room temperature with a horseradish peroxidase-linked secondary anti-mouse antibody , and the immunoreactive band signal intensity was visualized by enhanced chemiluminescence . The immunoreactive bands were scanned using Fujifilm LAS 4000 imaging system  and densitometric analysis was performed on these scans with Image Gauge® analysis software .202/pThr205396/Ser404235202262, 1∶1000; Invitrogen, Camarillo, CA, USA), and pS422 . Total tau was detected using one of the following antibodies that detects all 6 six isoforms of tau: Tau46 , TG5 , SAPK/JNK , phospho-SAPK-JNK , p44/42 MAPK , phospho p44/42 MAPK . Changes in tau phosphatases were examined using PP2A-C subunit , pan-calcineurin A , PP1 catalytic subunit .Changes in tau kinases were examined using GSK-3β , and the following antibodies purchased from New England Biolabs, Pickering, Ontario, Canada: phospho-GSK-3β , according to the manufacturer instructions, and as described previously by us post hoc test applied when appropriate or by means of an unpaired t-test. The data were tested for normality using the Kolmogrov-Smirnov test. Statistical calculations were performed using Prism 4® software , and all data are reported as mean ± SD with a value of P<0.05 considered statistically significant.Group comparisons of immunoblot relative band intensities or PP2A assay results were performed using a one-way analysis of variance (ANOVA) with Newman-Keuls Multiple Comparison Tissue fixation was done according to the “cold Bouin's method” developed in our laboratory