The Gab2 docking protein acts as an important signal amplifier downstream of various growth factor receptors and Bcr-Abl, the driver of chronic myeloid leukaemia (CML). Despite the success of Bcr-Abl tyrosine kinase inhibitors (TKI) in the therapy of CML, TKI-resistance remains an unsolved problem in the clinic. We have recently shown that Gab2 signalling counteracts the efficacy of four distinct Bcr-Abl inhibitors. In the course of that project, we noticed that two clinically relevant drugs, imatinib and dasatinib, provoke distinct alterations in the electrophoretic mobility of Gab2, its signalling output and protein interactions. As the signalling potential of the docking protein is highly modulated by its phosphorylation status, we set out to obtain more insights into the impact of TKIs on Gab2 phosphorylation.Using stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS), we show now that imatinib and dasatinib provoke distinct effects on the phosphorylation status and interactome of Gab2. This study identifies several new phosphorylation sites on Gab2 and confirms many sites previously known from other experimental systems. At equimolar concentrations, dasatinib is more effective in preventing Gab2 tyrosine and serine/threonine phosphorylation than imatinib. It also affects the phosphorylation status of more residues than imatinib. In addition, we also identify novel components of the Gab2 signalling complex, such as casein kinases, stathmins and PIP1 as well as known interaction partners whose association with Gab2 is disrupted by imatinib and/or dasatinib.By using MS-based proteomics, we have identified new and confirmed known phosphorylation sites and interaction partners of Gab2, which may play an important role in the regulation of this docking protein. Given the growing importance of Gab2 in several tumour entities we expect that our results will help to understand the complex regulation of Gab2 and how this docking protein can contribute to malignancy. Chronic myeloid leukaemia (CML) is characterized by the increased and deregulated growth of myeloid cells in the bone marrow and their accumulation in the peripheral blood. CML represents about 20% of all cases of adult leukaemia and has an incidence rate of 1.6 per 100,000 people per year. This form of leukaemia is staged into three distinct phases. The first phase  is often asymptomatic and progresses over several years to the accelerated phase (AP), if undiscovered or left untreated. The AP ultimately progresses to the terminal blast crisis (BC) which is characterized clinically like an acute leukaemia. CML is driven by the oncogenic Bcr-Abl tyrosine kinase. This kinase originates from the spontaneous rearrangement of two human chromosomes (Philadelphia chromosome) causing the production of a chimeric protein with combined biological portfolios. The Bcr-moiety recruits novel interaction partners, which are modified on tyrosine residues with phosphate groups by the Abl moiety, a typical protein tyrosine kinase. This in turn activates numerous intracellular signalling pathways that are, under physiological circumstances, only activated by growth signals. As a result, cells, which harbour the Philadelphia chromosome, grow excessively, even in the absence of growth signals and thereby cause CML. This insight led to the development of drugs inhibiting the Bcr-Abl enzyme, which have revolutionised CML therapy. In some patients, however, these drugs do not cause remission or initially responsive leukaemia become drug resistant. Among several mechanisms, dysregulation of signal transducers downstream of Bcr-Abl, such as the Gab2 docking protein, contribute to drug resistance. In this manuscript, we provide a detailed analysis on the phosphorylation status and protein interaction repertoire of Gab2 in CML cells in the presence and absence of the clinically applied drugs imatinib (Gleevec) and dasatinib (Sprycel).The oncogenic protein tyrosine kinase Bcr-Abl, the product of the Philadelphia chromosome, drives chronic myeloid leukaemia (CML) . This diDespite the useful application of IM in cancer therapy, only 3 out of 4 CML cases respond adequately to IM in the first place and several of the initially responding tumours become TKI resistant due to the acquisition of Bcr-Abl mutations preventing TKI uptake and/or action ,8. ConseBCR and ABL reading frames extends the portfolio of the Abl kinase by interaction partners of the Bcr moiety such as the Grb2 adaptor [via its central SH2 domain to phospho-tyrosine 177 (Y177) in the Bcr moiety, while its C-terminal SH3 domain binds to a typical and an atypical Grb2 binding site in Gab2 [The fusion of the  adaptor ,10. As a adaptor ,12. One  adaptor ,14. Grb2 in Gab2 ,16. This in Gab2 ,17. Thes in Gab2 . The cri in Gab2 ,17-20. U in Gab2 ,13,14. I in Gab2 . However in Gab2 ,21-23.The important role of Gab2 downstream of Bcr-Abl is illustrated by the observations that its genetic depletion prevents the transformation of murine myeloid progenitors by the fusion kinase  and slowStable isotope labelling by amino acids in cell culture (SILAC) is a metabolic labelling strategy allowing, amongst other things the quantitative investigation of posttranslational modification (PTM) , proteinBcr-Abl ectopically [In order to obtain more insights into the plasticity of phosphorylation and protein interaction events in the Gab2 signalling complex in the context of Bcr-Abl signalling, we made use of our previously established model systems, the K562tet/Gab2-HA cell line and the murine pro-B cell line Ba/F3 expressing p210opically . K562tetopically . K562tetopically ,32, whicopically . Althoug50 values of the two drugs, we also included a second DST concentration, 0.01 μM, which lies in the range of observed plasma levels of DST, as well. The accelerated electrophoretic mobility of Gab2 was accompanied by a drastic reduction in its overall tyrosine phosphorylation, while the specific phosphorylation of Y452 was quite resilient in K562 cells , pY452 (PI3K recruitment) and the PI3K dependent feedback phosphorylation sites S159, S210 and T391 ,21. ThisS210A/T391A double mutant (Gab22xA), which cannot recruit 14-3-3 proteins [In agreement with our previous finding that both IM and DST hardly block AKT phosphorylation in K562 cells , we showproteins , displaybona fide phosphorylation sites  and Gab2  and the p85α/β subunits (PIK3R1/2) as it was also recently described for IM treated K562 cells . Commensain; see ,49 for fain; see , was preain; see .Our analysis also identifies several proteins as significantly decreased by TKI treatment that have not been linked to Gab2 signalling previously  and Casein kinase II α’  subunits Figure . In addiIn summary, our comparative analysis shows that the distinct efficacies of IM and DST are reflected by the differentially altered overall phosphorylation status of the Gab2 protein. Our analysis further underscores that large docking proteins with their extended disordered regions are very rich in phosphorylation sites, whose functions are just starting to emerge ,41,47,557 cells were electroporated with 30 μg FspI-linearized plasmid DNA. Selection of Ba/F3 and K562 cells was commenced 24 h post infection/electroporation .Plat-E cells were cultured as described previously ,61 and tBcr-Abl, a kind gift from Dr. Sebastian Herzog, Albert-Ludwigs-University, Freiburg (Germany). The Bcr-Abl cDNA was excised from pMIG/p210Bcr-Abl using EcoRI and subcloned into the EcoRI-linearized pBabe-Hygro expression vector. To generate the expression vectors pWZL/Gab2-HA-bsr, Gab2-HA was amplified by PCR from the retroviral pMIG-hGab2-HA [The retroviral pBabe/Bcr-Abl-Hygro vector was derived from pMIG/p210hGab2-HA  and thenhGab2-HA ,32. DetaK562 and Ba/F3 cells were processed to total cellular lysates by lysing in normal lysis buffer  and analyzed by western blotting as described previously .13C6-14N4  and L-lysine-2H4 , or “heavy” AA: L-arginine-13C6-15N4  and L-lysine-13C6-15N2  in SILAC-DMEM media , supplemented with L-proline, penicillin/streptomycin , glutamine, and 10% dialyzed fetal calf serum  which was used during labeling.K562 cells expressing Gab2-HA were cultured in DMEM  containing 4.5 g/l glucose, Na-Pyruvat, 3.7 g/l NaHCO3, supplemented with penicillin/streptomycin , glutamine, 10% fetal calf serum . Cell populations were labeled for 6 cell doublings with either “light” AA: L-arginine and L-lysine , or “medium” AA: L-arginine-SILAC labelled cells were lysed with normal lysis buffer , cell nuclei were pelleted and supernatants were used for affinity purification. Gab-HA complexes were enriched using anti-HA-sepharose (Roche Applied Science) in separate IPs, one IP per SILAC label. In the last washing step, sepharose beads were combined and protein complexes eluted by incubating beads for 10 min at 95°C in SDS loading buffer containing 1 mM DTT (Sigma-Aldrich). Reduced samples were alkylated using iodoacetamide (5.5 mM) (Sigma-Aldrich). Protein mixtures were separated by SDS-PAGE ), gel lanes were cut into 10 slices according to their protein content, samples in-gel digested using trypsin , and resulting peptide mixtures were STAGE tipped.6 and at resolution 60,000 at m/z 400. MS acquisition was done in data-dependent mode to sequentially perform MS/MS on the five most intense ions in the full scan (Top5) in the LTQ using the following parameters. AGC target value: 5,000. Ion selection thresholds: 1000 counts and a maximum fill time of 100 ms. Wide-band activation was enabled with an activation q = 0.25 applied for 30 ms at a normalized collision energy of 35%. Singly charged and ions with unassigned charge state were excluded from MS/MS. Dynamic exclusion was applied to reject ions from repeated MS/MS selection for 45 s.Samples for LC-MS/MS were fractionated by nanoscale–HPLC on either an Agilent 1200 or an Eksigent NanoLC-ultra connected online to a LTQ-Orbitrap XL (Thermo Scientific). Peptides were separated over a linear gradient from 10-30% ACN in 0.5% acetic acid with a flow rate of 250 nl/min. All full-scan acquisition was done in the FT-MS part of the mass spectrometers in the range from m/z 350–2000 with an automatic gain control target value of 10All recorded LC-MS/MS raw files (40) were processed together in MaxQuant  version Phosphosites with localization probabilities ≥0.75 (class I sites) were used for bioinformatics analyses.Imatinib mesylate and dasatinib were purchased from Santa Cruz Biotechnology. Doxycycline hyclate was obtained from Sigma Aldrich. Anti-HA Affinity Matrix was purchased from Roche.Antibodies used in this study were: anti-c-Abl (sc-131), anti-β-Actin (sc-47778) and anti α-Tubulin (sc-23948), all from Santa Cruz, anti-phospho-AKT S473 and T308, anti-p44/p42 MAP kinase, anti-phospho-p44/p42 MAP kinase T202/Y204, anti-phospho-Bcr Y177, anti-Gab2 (26B6), anti-phospho-Gab2 Y452 (C33G1) and S159, anti-CRKL, anti-phospho-CRKL and anti-phospho-STAT5 all from Cell Signaling Technology, anti-HA (3F10) from Roche Molecular Bioscience, anti-phospho-tyrosine (4G10) from Millipore and anti-phospho-Gab2 S210 and T391 antibodies were described previously  and obtaAP: Accelerated phase; BC: Blast crisis; CML: Chronic myeloid leukaemia; CP: Chronic phase; Dox: Doxycycline; DST: Dasatinib; HA: Haemagglutinin; IM: Imatinib; MS: Mass spectrometry; SILAC: Stable isotope labelling by amino acids in cell culture; TKI: Tyrosine kinase inhibitor.The authors declare that they have no competing interests.All authors contributed to the design, analysis and discussion of experiments. SH performed all cellular and biochemical experiments. SH, KTGR, BD, CG. and JD conducted the mass spectrometry analyses. TB wrote the manuscript together with SH, KTGR, FUW and JD. TB and JD are co-last authors. All authors reviewed and commented on the manuscript and accepted its final version.Kinase predictions from NetworKin. Amino acid sequences surrounding phosphorylation sites were employed to predict potential kinases responsible for phosphorylation using the NetworKIN algorithm.Click here for fileGab2 phosphorylation, imatinib and dastanib compared to DMSO treatment. Phosphosite identification and quantification information is shown. SILAC Gab2 phosphosite ratios of IM and DST treated cells compared to DMSO treated controls are depicted.Click here for fileGab2 phosphorylation, dastanib compared to imatinib treatment. Phosphosite identification and quantification information is shown. SILAC Gab2 phosphosite ratios of DST treated cells compared to IM treated cells are depicted.Click here for file(A-B) Correlation of protein quantifications of biological replicates for (A) imatinib treated cells versus DMSO treated controls and (B) dasatinib treated cells versus DMSO treated controls. Significant depleted proteins are annotated in red . (C-E) Correlation of phosphosite quantification. Plotted are Gab2 phosphosite ratios normalized to Gab2 protein ratios of (C) imatinib versus control, (D) dasatinib versus control, and (E) dasatinib versus imatinib treated cells of two biological replicates.Correlation of protein purification and quantification of biological replicates. Click here for fileGab2 protein-protein interactions, imatinib and dastanib compared to DMSO treatment. Protein identification and quantification information is shown. SILAC ratios of proteins identified in Gab2-HA immuno-precipitations of IM (1 μM) and DST (0.01 μM and 1 μM) treated versus DMSO treated cells are depicted. Proteins exhibiting inhibitor sensitive interactions are highlighted .Click here for file


In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1  and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs  respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP–PKD1 fusion protein expressed in NRVMs.  Protein kinase D has multiple roles in cardiac myocytes, where its regulatory mechanisms remain incompletely defined. In the present study we identify four-and-a-half LIM domains proteins 1 and 2 as novel binding partners and regulators of protein kinase D in this cell type. These PKD isoforms share the common structural features of a C-terminal catalytic domain and an N-terminal regulatory domain. Components of the regulatory domain autoinhibit the activity of the catalytic domain in unstimulated cells and promote PKD association with the plasma and intracellular membranes after stimulation with hormones, growth factors, neurotransmitters, chemokines and bioactive lipids  were clarified by centrifugation  in an Eppendorf 5417R centrifuge. Each supernatant was pre-cleared by incubation with glutathione–Sepharose 4B beads  for 1 h under rotation (4°C), then split in equal volumes and incubated for 2 h under rotation (4°C) with 5 μg of recombinant proteins  pre-bound for 1 h to glutathione–Sepharose 4B beads (4°C). The complexes were pelleted by centrifugation , washed three times in lysis buffer, resuspended in 2× SDS/PAGE sample buffer , boiled and stored at −20°C until use.For the GST pull-down assay, lysates from ARVMs (treated with vehicle or 100 nM PMA for 10 min) or NRVMs (treated with vehicle or 10 nM ET1 for 10 min) prepared in lysis buffer ATP in kinase assay buffer . Proteins were resolved on 12% acrylamide gels, which were subsequently dried and subjected to autoradiography.The eviously . Recombi2PO4, 0.8 mM MgSO4, 5.5 mM glucose, 32.1 mM NaHCO3 and 1.8 mM CaCl2, pH 7.2), after which time they were serum-starved for 3 h before treatment for 20 min with vehicle, ET1 (10 nM) or PE . Atenolol was used to inhibit any β-adrenergic receptor activation, which is known to counteract PKD activation ATP. Autoradiographic analysis revealed no detectable phosphate incorporation into GST, GST–FHL1 or GST–FHL-2, even though robust phosphorylation of cTnI was readily detectable . They also suggest that interaction with FHL1 or FHL2 does not inhibit the catalytic activity of caPKD towards an established substrate, cTnI.Our previous studies that built on the discovery of novel cardiac PKD1 interaction partners through a yeast two-hybrid screen  have ledtectable A. To exptectable B. Such fhttp://www.biochemj.org/bj/457/bj4570451add.htm). Similarly, transfection with FHL2 siRNA caused a comparable decrease in FHL2 protein expression relative to cells transfected with a scrambled siRNA sequence, with no compensatory change in FHL1 expression . We then examined the consequences of the selective knockdown of each FHL isoform on PKD activation in response to stimulation with ET1 or PE. As illustrated in 916 and transphosphorylation at Ser744/Ser748 in control cells transfected with scrambled siRNA. Knocking down FHL1 expression significantly attenuated the increase in PKD phosphorylation in response to ET1 (http://www.biochemj.org/bj/457/bj4570451add.htm).To explore the potential roles of FHL1 and FHL2 in regulating the activation and functions of endogenous PKD in cardiac myocytes, we applied a loss-of-function approach and knocked down FHL1 or FHL2 protein expression by RNAi through transfection of NRVMs with synthetic siRNA duplexes specific for each FHL mRNA sequence. Transfection with FHL1 siRNA caused an approximately 75% decrease in FHL1 protein expression relative to control cells transfected with a scrambled siRNA sequence, with no apparent effect on FHL2 expression . Thus the negative effects of reduced FHL isoform expression by RNAi on ET1- and PE-induced increases in PKD autophosphorylation at Ser916 and transphosphorylation at Ser744/Ser748 .PKD-mediated phosphorylation of HDAC5 at Seractivity ,14. We tOne possible explanation for this lack of effect of FHL isoform knockdown on MEF2 activation, despite a significant attenuation of PKD activation, is that under our experimental conditions PKD activity is not an important mediator of MEF2 activation in response to ET1 or PE. To help confirm the role of PKD activity in MEF2 activation in NRVMs, therefore, we also investigated the effects of the selective PKD inhibitor BPKDi ,23 underThe present study provides novel information concerning the regulation of PKD activity and functions in cardiac myocytes, through the following principal findings: (i) PKD interacts with two members of the FHL protein family, FHL1 and FHL2; (ii) FHL1 and FHL2 are not PKD substrates and do not inhibit PKD catalytic activity; (iii) FHL1 and FHL2 differentially regulate PKD activation after neurohormonal stimulation; and (iv) FHL1 and FHL2 do not regulate MEF2 activity after neurohormonal stimulation.fhl1 and fhl2, are associated with various skeletal and cardiac myopathies [FHL1 and FHL2 are non-enzymatic proteins that are expressed primarily in striated muscles, including the heart ,24,25. Topathies  and HIF-opathies . FHL proopathies , therebyopathies ,29, the opathies , the metopathies  and otheopathies ,32. Suchfhl1 and fhl2 in mice. Such studies have revealed that the global loss of FHL1 or FHL2 protein does not impair cardiovascular development, or cause any spontaneous cardiac phenotype [fhl1 or fhl2 does not induce a compensatory change in the cardiac expression of the other isoform [fhl1−/− mice have been reported to develop diminished cardiac hypertrophy following TAC (transverse aortic constriction) or cardiac-specific Gαq overexpression, suggesting a pro-hypertrophic role for FHL1 [fhl2−/− mice presented with a comparable magnitude of cardiac hypertrophy relative to wild-type controls after TAC [in vivo setting may be isoform-specific and dependent on the nature of the stress stimulus that triggers the hypertrophic response. Interestingly, at least in some settings, FHL1 and FHL2 appear to have differential effects on cardiac myocyte ERK activity [Previous studies have explored the roles that FHL1 and FHL2 may play in cardiac myocyte (patho)physiology through the targeted disruption of henotype –34. They isoform ,34, whicfor FHL1 . On the fter TAC , but moufter TAC , suggestactivity ,32, raisactivity . FHL2 reactivity . Loss ofactivity .in vitro studies in NRVMs, selective knockdown of either FHL1 or FHL2 inhibited PKD activation by pro-hypertrophic neurohormonal stimuli and attenuated the downstream phosphorylation of HDAC5, albeit with some differences in their impact on the responses to ET1 against PE (discussed later). Nevertheless, MEF2 activation by these stimuli was unaffected by FHL1 or FHL2 knockdown  through which FHL1 and FHL2 may regulate PKD activation also require consideration. Previous studies in rat cardiac myocytes have shown that neurohormonal PKD activation is achieved through the phosphorylation of its activation loop, principally by the novel PKC isoform PKCε ,21. Intent study , it is pd Ser916  suggest In conclusion, in the present study, we identify FHL1 and FHL2 as novel interaction partners for PKD in cardiac myocytes and we show that FHL1 and FHL2 regulate PKD activation in response to neurohormonal stimulation. Although such regulation affects HDAC5 phosphorylation, it does not control neurohormonal MEF2 activation, suggesting that PKD regulation by FHL proteins is likely to be of functional importance in PKD-mediated (patho)physiological processes other than MEF2-driven transcriptional reprogramming towards cardiac hypertrophy.


TGR5, an emerging G protein-coupled receptor, was identified as a membrane receptor for bile acids. The expression of TGR5 and its function are distinct from the previously identified nuclear bile acid receptor, farnesoid X receptor (FXR). These two bile acid receptors complement with each other for maintaining bile acid homeostasis and mediating bile acid signaling. Both receptors are also shown to play roles in regulating inflammation and glucose metabolism. An interesting finding for TGR5 is its role in energy metabolism. The discovery of TGR5 expression in brown adipocyte tissues (BATs) and the recent demonstration of BAT in adult human body suggest a potential approach to combat obesity by targeting TGR5 to increase thermogenesis. We summarize here the latest finding of TGR5 research, especially its role in energy metabolism and glucose homeostasis. Bile acids (BAs) are the main products of cholesterol catabolism, which constitutes a major route for the elimination of surplus cholesterol. There are four major types of BAs: cholic acid (CA) and chenodeoxycholic acid (CDCA) are primary BAs; deoxycholic acid (DCA) and lithocholic acid (LCA) are secondary BAs, which are converted from primary BAs by bacterial enzymes in the intestine . BA syntIt is now clear that, in addition to their important roles in nutritional absorption, BAs are signaling molecules that can activate BA receptors to initiate signaling pathways and regulate gene expression. Two major receptors for BAs have been identified: (1) the nuclear receptor, FXR, and (2) the G protein-coupled receptor TGR5). FXR is a member of the nuclear hormone receptor superfamily. Among the 48 currently identified nuclear hormone receptors, FXR belongs to a subcluster of nuclear receptors that play key roles in metabolic regulation . FXR is –10. FXR In vitro studies have demonstrated that activation of TGR5 transduces signal through Gs protein-mediated cAMP accumulation [TGR5 is a recently identified plasma membrane-bound, G protein-coupled receptor for BAs. TGR5 is encoded by a single exon that maps to chromosome 1C3 in mouse and 2q35 in humans. Homologs of TGR5 can also be identified in aquatic vertebrates, indicating a potentially conserved role of TGR5 during evolution. TGR5 is ubiquitously expressed, but its expression levels vary among different tissues, with high expression in liver, intestine, brown adipose tissue, and spleen , 21. In mulation . The ordThe role of TGR5 in bile acid metabolism was confirmed in TGR5 knockout mice. Total BA pool size in TGR5 knockout mice was significantly decreased by 21–25% compared with that of the wild-type mice, suggesting that TGR5 contributes to BA homeostasis . However3 umbrella [Another interesting study indicating a potential role of TGR5 in BA metabolism is its function in gallstone formation. TGR5 knock-out mice did not develop gallstones when they were fed a lithogenic diet . Furtherumbrella . In addiumbrella . TGR5 waumbrella , 30.In summary, BAs mainly signal through two receptors, FXR and TGR5. FXR is key to controlling the homeostasis of BAs and preventing the toxic effects of BAs on hepatocytes. In contrast, TGR5 may establish a secondary and backup pathway for maintaining BA levels and for preventing BA-induced toxicity. in vivo [κB-mediated hepatic inflammation [κB function during hepatic inflammation will be an interesting future study.TGR5 mRNA was detected in the resting CD14+ monocytes in fractionated human leukocytes, adherent alveolar macrophage cells , and Kupin vivo . Previouammation . WhetherTGR5 may play a potential role in type 2 diabetes, as suggested by a recent finding that OA treatment lowered serum glucose and insulin levels in mice fed with a high-fat diet. Moreover, OA treatment enhanced glucose tolerance . ActivatNew England Journal of Medicine demonstrate that adult humans have significant amount of metabolically active BAT [Overweight and obesity are now viewed as chronic diseases in the whole world that threaten public health if not treated . Obesitytive BAT –42. Is itive BAT –45. AnotTGR5 is a key factor in energy expenditure by regulating metabolism. Activation of TGR5 by BAs increases energy expenditure in brown adipose tissue, preventing obesity and resistance to insulin . This efOlea europaea, indeed abolished the gain of the body weight by the high-fat diet in an animal model [An interesting question is whether activation of TGR5 by natural or synthetic ligands would reduce body weight caused by the high-fat diet. Oleanolic acid (OA), a natural ligand of TGR5 isolated from al model . It imprBesides OA, several other types of secondary BAs are also natural ligands for TGR5, for example LCA, TLCA, DCA, CDCA, and CA . HoweverTGR5 is an emerging membrane receptor for mediating bile acid signaling. Recent studies highlight it as a novel target for regulating glucose and energy metabolism. The TGR5 signaling in BAT and muscle, two important sites of energy dissipation, suggests that it may be a novel target to combat obesity. We anticipate that, in the near future, more potent and specific TGR5 ligands will be identified and go into clinical trials for obesity and diabetes treatments.


Pedicle screws with PMMA cement augmentation have been shown to significantly improve the fixation strength in a severely osteoporotic spine. However, the efficacy of screw fixation for different cement augmentation techniques, namely solid screws with retrograde cement pre-filling versus cannulated screws with cement injection through perforation, remains unknown. This study aimed to determine the difference in pullout strength between conical and cylindrical screws based on the aforementioned cement augmentation techniques. The potential loss of fixation upon partial screw removal after screw insertion was also examined.8 array was employed to determine the significance of design factors. Conical and cylindrical pedicle screws with solid or cannulated designs were installed using two different screw augmentation techniques: solid screws with retrograde cement pre-filling and cannulated screws with cement injection through perforation. Uniform synthetic bones (test block) simulating severe osteoporosis were used to provide a platform for each screw design and cement augmentation technique. Pedicle screws at full insertion and after a 360-degree back-out from full insertion were then tested for axial pullout failure using a mechanical testing machine.The Taguchi method with an Lp = 0.0129 for conical screws; p = 0.005 for cylindrical screws). 2) For a given cement augmentation technique , no significant difference in pullout strength was found between conical and cylindrical screws (p >0.05). 3) Cement infiltration into the open cell of the test block led to the formation of a cement/bone composite structure. Observations of the failed specimens indicated that failure occurred at the composite/bone interface, whereas the composite remained well bonded to the screws. This result implies that the screw/composite interfacial strength was much higher than the composite/bone interfacial strength. 4) The back-out of the screw by 360 degrees from full insertion did not decrease the pullout strength in any of the studied cases. 5) Generally, larger standard deviations were found for the screw back-out cases, implying that the results of full insertion cases are more repeatable than those of the back-out cases.The results revealed the following 1) Regardless of the screw outer geometry , solid screws with retrograde cement pre-filling exhibited significantly higher pullout strength than did cannulated screws with cement injection through perforation (Solid screws with retrograde cement pre-filling offer improved initial fixation strength when compared to that of cannulated screws with cement injection through perforation for both the conically and cylindrically shaped screw. Our results also suggest that the fixation screws can be backed out by 360 degrees for intra-operative adjustment without the loss of fixation strength. Osteoporosis is a common disease in aging populations. Spinal surgeons unavoidably encounter patients with osteoporosis who need spinal decompression and instrumentation due to degenerative spinal diseases. However, pedicle screw instrumentation in a severely osteoporotic spine remains a challenge for orthopedic surgeons.Previous studies have demonstrated that the holding power of screw in non-augmented osteoporotic bone decreases with decreasing bone mineral density -3. ConsePMMA augmentation is regarded as an efficient method to enhance screw strength in osteoporotic bones -8. TradiOur review of the literature found that most of the current research on pedicle screw pullout has been performed with cylindrically shaped pedicle screws ,7,18-20.Studies have investigated the mechanical performance of pedicle screws in vertebrae, but only minimal data are available concerning the performance of conical screws in the presence of compromising events such as partial screw removal. In the present study, we address two features of the pullout strength of conical versus cylindrical screws: 1) the fixation strength between solid screws with retrograde cement pre-filling and cannulated screws with cement injection through perforation and 2) the effect of partial screw removal after full insertion.8 array was employed to determine the significance of design factors. Three factors were considered in evaluating the holding power of a fixation screw inserted into synthetic bone. The three factors were screw shape , cement augmentation technique (solid screws with cement pre-filling/cannulated screws with cement injection) and screw insertion type (full insertion/back-out). Each factor was further assigned into two levels. Therefore, a total number of 8 trials (23) were required to identify the relative significance of design factors using a full factorial approach. Table The Taguchi method with an L3, which simulates a cadaveric spinal bone with extreme osteoporosis [Synthetic bone  made from polyurethane foam was used as substitute for cadaveric spinal bone because of its consistent and homogeneous structural properties. The synthetic bone was supplied as rectangular shape (test block) with the dimensions of 13 cm × 18 cm × 4 cm; the material was open-cell rigid polyurethane foam with a density of 0.09 g/cmoporosis -29.Four screw designs were employed in the present study: conical-solid, conical-cannulated, cylindrical-solid and cylindrical-cannulated. The outer geometry of conical and cylindrical screws differed mainly in the taper of their major and minor diameters from the hub to the screw tip. The cylindrical screws maintained a constant diameter from hub to tip; in contrast, the conical screws tapered 20%, from 6.0 mm at the hub (major diameter) to 4.8 mm at the tip. For both screw types, the thread pitch was 2 mm and the thread depth was 0.8 mm. The thread contour, a proprietary characteristic, was identical for both screws. For the cannulated screws, two radial holes with a diameter of 2 mm were located at 5-mm increments along the length of screw starting at the screw tip. Two sets of outer threads were made, one with a length of 32 mm from the screw tip and another with a length of 12 mm from the screw head. The outer thread on the screw head was fixed in a cylindrical rod with a matched inner thread in the subsequent pullout test. The conical and cylindrical screws in the cannulated design are illustrated in Figure The allocation of specimens to experimental groups is shown in Table Group 1: Conical-solid screws, retrograde cement pre-filling and full screw insertion.Group 2: Conical-solid screws, retrograde cement pre-filling and a 360-degree screw back-out after full insertion.Group 3: Conical-solid screws with screw full insertion and no cement augmentation.Group 4: Conical-cannulated screws, cement injection through perforation and full screw insertion.Group 5: Conical-cannulated screws and cement injection through perforation with a 360-degree screw back-out after full insertion.Group 6: Cylindrical-solid screws, retrograde cement pre-filling and full screw insertion.Group 7: Cylindrical-solid screws, retrograde cement pre-filling and a 360-degree screw back-out after full insertion.Group 8: Cylindrical-solid screws with full screw insertion and no cement augmentation.Group 9: Cylindrical-cannulated screws, cement injection through perforation and a full screw insertion.Group 10: Cylindrical-cannulated screw, cement injection through perforation and a 360-degree screw back-out after full insertion.For cannulated screws , PMMA cement was injected into the test block after screw insertion. A pilot hole was drilled into the test block using a 3-mm drill bit, and a cannulated screw was then inserted into the test block through the prepared pilot hole. The insertion rate for all screws was 3 rev/min ; a countFor solid screws , the solid screw was inserted into the test block through the prepared pilot hole and then removed to create a hole with identical dimension as the screw contour . A total of 3 ml of cement was then retrogradely injected into the created hole using a 4-mm diameter bone biopsy needle . A mark was made with aseptic marking pen on the needle. The length from the marking point to the needle tip was 5 mm shorter than the length of selected screw. Next, the biopsy needle was inserted into the prepared pilot hole until the marking point approached the entry edge of the test block. Cement was then injected into the pilot hole accompanied by progressive needle retraction out of the test block, until a total volume of 3 ml of bone cement was injected. With this technique, a uniform distribution of cement can be achieved. Following the pre-filling of bone cement, the solid screw was then fully inserted into the test block. To evaluate the effect of partial screw removal, the screws were randomly rotated out by 360 degrees from full insertion four minutes after the introduction of PMMA cement.The method for screw pullout test was identical to that used in our previous study . The indt-tests were performed for the intergroup comparison. Differences were considered significant at p < 0.05.To evaluate the effects of screw designs  and different modes of screw implantation (cannulated screw with cement injection or solid screw with retrograde cement pre-filling) on the stability of spinal fixation, the magnitudes of the ultimate pullout force were statistically compared. Unpaired two-tailed Student's The radiological and physical examinations of the screws inserted into the test blocks and specimens after the pullout tests are shown in Figure p = 0.0129 for conical screws; p = 0.005 for cylindrical screws); whereas solid screws without cement augmentation exhibited the lowest pullout strength (p <0.001). For the conical screws, the solid type provided a 23% increase in the pullout strength when compared to the cannulated type (p = 0.0129). In contrast, for the cylindrical screws, the solid type provided a 41% increase in the pullout strength when compared to the cannulated type (p = 0.005). For a given screw augmentation technique , no significant difference in pullout strength was found between conical and cylindrical screws (p > 0.05).The average ultimate pullout strengths of fully inserted conical and cylindrical pedicle screws for the different cement augmentation techniques are shown in Table The average ultimate pullout strength for conical and cylindrical pedicle screws tested at full insertion and after a 360-degree screw backout is shown in Figure The Taguchi analysis indicated that the design factor "cement augmentation technique" was the main influential factor, whereas "screw shape" was the least influential factor affecting the pullout strength.Adequate pedicle screw fixation in the presence of compromised bone quality presents a challenging problem for spine surgeons. Cancellous bone is more profoundly affected by osteoporosis process than cortical bone, and it is not surprising that the anchoring strength is significantly decreased in patients with low bone mineral density.3 [3, corresponding to the porosity of osteoporotic and normal bones, respectively. Their results revealed a significant correlation between peak pullout resistance and insertion torque. Zehnder et al. [3 polyurethane foam (used to simulate severely osteoporotic cancellous bone). They concluded that in a severely osteoporotic model, failure in cantilever bending at low forces would take place regardless of fixation methods used and the added benefit of oblique screw placement observed in healthy bone is not observed in osteoporotic bone. In addition, Ramaswamy et al. [3) simulating osteoporotic, osteopenic and normal bones, respectively. Their results indicated that the holding power of screws is directly correlated to bone density, thread design and number of threads engaging the bone. Reinsertion through the same hole could reduce the ultimate pullout strength. Additionally, Patel et al. [3) of polyurethane foams to examine whether the commercially polyurethane foams are suitable for mimicking human osteoporotic cancellous bone. The fracture stresses of these foams enable them to be used as models for normal (0.32 g/cm3), osteoporotic (0.16 g/cm3) and very low density osteoporotic cancellous bone (0.09 g/cm3). They concluded that the 0.16 g/cm3 polyurethane foam is a good alternative for in vitro testing because it has compressive Young's modulus and yield strength values similar to osteoporotic bone that has also been tested in compression. In the current study, in considering of the ease of cement injection, the polyurethane foam with a density of 0.09 g/cm3 was chosen for emphasis of extremely osteoporotic cancellous bone. We believe that although the pullout test did not measure the actual screw/bone interfacial strength, the 0.09 g/cm3 polyurethane foam provides a uniform platform to compare the mechanical behavior of pedicle screws with various designs.Cancellous bone is generally reported to have a density in the range of 0.09 to 1.25 g/cm3 . The var3 . The tes3 . Consequ3 ,29,32. Il et al.  performeFew reports have addressed the effects of partial screw removal on the bone/screw interfacial strength ,33. HowePMMA cement infiltration into the open cell of the test block led to the formation of a composite cement/bone structure in the area of cement infiltration Figure . For all8 array analysis revealed the design factor "cement augmentation technique" was the main influential factor, whereas "screw shape" was the least influential factor affecting the pullout strength. In addition, the highest pullout strength was found in combination of cylindrical-solid-full insertion. The results of Taguchi analysis were consistent with our conclusion that the PMMA augmentation technique for solid screws with retrograde cement pre-filling offers improved initial fixation strength.The Taguchi method utilizing an orthogonal array has proven to be a useful tool to markedly reduce the total number of required experiments. The method contains a well-chosen subset of all possible test condition combinations and can achieve a balanced comparison of levels of any factor -36. In tOur study is an in vitro analysis of specimens prepared in a laboratory environment, which does not necessarily represent clinical circumstances. There are limitations to this study. First, a test block was used as a substitute for human osteoporotic vertebrae. Although the synthetic bone provides a platform for comparison of pullout strength, the material properties of the test block are somewhat different from those of actual osteoporotic cancellous bone; the extrapolation of our results to clinical utilization should be performed with caution. Second, the measurement of pullout strength at the screw-bone interface did not take into account the cortical shell of the spinal vertebrae, which may have an impact on interfacial bonding strength. However, our specimens were all prepared and tested in a uniform, reproducible manner, and we believe that our results provide a comparison of the mechanical performance of various screws in severely osteoporotic bone. Third, the volume of injected cement tested was constant (3 ml). The amount of injected cement might be an important influential factor in determining the screw holding power. The effects of the amount of injected cement on bone/screw interfacial strength deserved to be conducted in the future. Last, the present work is limited to static loading (pullout in test block) without considering of other physiological loadings. In actual physiological situations, the screw/bone interface is subjected to complex dynamic multi-directional loading. Our results cannot be used to predict the biomechanical performance of screw fixation under cyclic loading in the long term. Therefore, the possible future work could be to investigate the fatigue properties of pullout strength of the solid vs. cannulated pedicle screws in animal models.We conclude that solid screws with retrograde cement pre-filling offer improved initial fixation strength when compared to that of cannulated screws with cement injection through perforation for both conically and cylindrically shape screws. Our results also suggest that the fixation screws can be backed out by 360 degrees for intra-operative adjustment without the loss of fixation strength.The authors declare that they have no competing interests.LHC and CLT participated in the study design, in collecting the data and drafting of the manuscript. DML participated in the Taguchi analysis. PPL and YCL participated in the experimental work. CCN participated in revising critically the manuscript. WJC advised and assisted drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/33/prepub


AHP) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP, resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc.The slow afterhyperpolarizing current (sI AHP, that contributes to the late phase of spike frequency adaptation in hippocampal pyramidal neurons -protein kinase A (PKA) pathway has been extensively studied  that has three kinetically distinct phases: fast (fAHP), medium (mAHP), and slow AHP (sAHP)  in radioligand binding assays  belongs to a superfamily of structurally related peptide hormones that include glucagon, glucagon-like peptides, secretin and growth hormone-releasing hormone Harmar, . PACAP cin situ hybridization revealed the presence of PAC1 , and protocols were reviewed and approved by the University College London Animal Ethical Committee.2, 1.5 MgCl2, 1.25 KH2PO4, 25 NaHCO3, and 16 glucose) and the sIAHP was recorded at room temperature with patch pipettes made of borosilicate glass and containing a gluconate-based intracellular solution . The liquid junction potential for the intracellular solution is −11 mV. Voltage values reported here are not corrected for the liquid junction potential. Only cells with a resting membrane potential ≤−50 mV and a series resistance ≤30 MΩ were included in this study.Whole-cell patch clamp recordings were performed from CA1 pyramidal neurons using the blind patching technique  to block voltage-gated sodium channels, and 1 mM tetraethylammonium (TEA) to block a subset of voltage-gated potassium channels to increase the calcium influx and thereby the calcium dependent sIAHP. Traces were filtered at 0.25 kHz and sampled at 1.25 kHz and the stimulus was repeated with a 30 s interval. Bovine serum albumin (BSA) was added to the ACSF (25 µg ml−1) when peptides were used extracellularly to minimize unspecific binding. Peptides were applied by bath perfusion. It is worth noticing that the actual concentration of the peptide/s at the relevant receptors might differ from that nominally applied, due to the size and diffusion of the peptide/s to neurons located at different depths in the brain slice and, possibly, to the action of extracellular peptidases. Protein kinase inhibitors were applied intracellularly through the patch pipette. DMSO up to 0.13% did not affect either the sIAHP amplitude or charge transfer, or the effect of PACAP-27 on the sIAHP amplitude or charge transfer when applied intracellularly through the patch pipette as a vehicle to dissolve some protein kinase inhibitors.The sIAHP was determined as changes in amplitude and charge transfer of sIAHP. The sIAHP amplitude was measured by calculating the mean current between two cursors placed 50 ms apart, 1 s after the initiation of the voltage step to avoid contamination by other, faster potassium currents. Charge transfer was determined as the area under the curve starting from the sIAHP peak until full decay had occurred.Cells were excluded if the series resistance changed significantly (>25%) in the course of experiments. Modulation of sIIsoproterenol hydrochloride and tetraethylammonium chloride (TEA) were obtained from Sigma (UK); PACAP-27 amide, SB 203580, phorbol-12,13-dibutyrate (PDBu), bisindolylmaleimide I (BIM-1), and chelerythrine chloride from Calbiochem ; PACAP-38 from Peninsula Laboratories Europe ; PACAP-(6–38) from Bachem ; tetrodotoxin (TTX) from Latoxan ; and Rp-cAMPS and 8-(4-chloro-phenylthio)−2'-O-methyl-cAMP (8CPT-O-Me-cAMP) from BioLog Life Science Institute . Maxadilan and max.d.4 were a generous gift from Dr. Ethan Lerner at Harvard Medical School.Statistical analysis was performed with GraphPad InStat. Data are presented as mean with error bars indicating the standard error of the mean (S.E.M.), when not otherwise specified.AHP was recorded in the whole-cell patch-clamp configuration from 152 CA1 pyramidal neurons. CA1 neurons had an average resting membrane potential of −59.8 ± 3.5 mV  and an input resistance of 175.4 ± 51.1 MΩ . The sIAHP amplitude was on average 40.6 ± 24.2 pA and the charge transfer 101.4 ± 63.8 pC .The sIAHP of VIP and other neuropeptides that activate the cAMP-PKA pathway in hippocampal pyramidal neurons are well studied , coupled to the cAMP-PKA pathway  reduced the sIAHP amplitude (n = 7) and the charge transfer by 77.3% ± 5.2% (n = 7) in CA1 pyramidal neurons.The modulatory effects on the sImplitude A,C by 67mplitude E, n = 7 % ± 5.2% E, n = 7 AHP. PACAP-27 suppressed sIAHP within 15–20 min of bath application (AHP amplitude by 86.1% ± 4.7% and the charge transfer by 85.8% ± 5.1% (n = 4). No significant difference between PACAP-27 and PACAP-38 was found with regard to the reduction in sIAHP amplitude  and charge transfer . We therefore decided to use PACAP-27 in the following experiments.The region responsible for receptor activation of PACAP-38 resides in its N-terminal 27-amino acid sequence, which corresponds to the naturally occurring isoform PACAP-27 , the reduction with 250 nM was more substantial  and comparable to the effect of 500 nM PACAP-27 . PACAP-27 at 250 nM caused a suppression of the sIAHP amplitude by more than 90% in 50% of the neurons . An inward current of 14 ± 3.3 pA  was observed in response to 250 nM PACAP-27 in 16 out of 18 cells. The inward current might be due to the modulatory action of PACAP on background (leak) channels, HCN channels, and/or other nonspecific cationic channels, in analogy to what has been observed for other neurotransmitters inhibiting the sIAHP in hippocampal neurons.Beside inhibiting sIn = 18; P = 0.96, Wilcoxon matched-pairs signed-ranks test).PACAP-27 at 250 nM did not significantly affect the input resistance of CA1 pyramidal neurons, which was 164.5 ± 9.7 MΩ before and 163.9 ± 8.1 MΩ after PACAP-27 application , in agreement with its inhibitory effect on the sIAHP.The inhibition of the sI1, selective for PACAP and coupled to both adenylyl cyclases and phospholipase C, and VPAC1 and VPAC2 receptors, activated by both PACAP and VIP and coupled to adenylyl cyclases  and charge transfer by 24.4% ± 4.7%   D. This iAHP induced by maxadilan could be prevented by a PACAP receptor antagonist, we used PACAP-(6–38), a truncated PACAP-38 lacking six amino terminal amino acids. PACAP-(6–38) is a selective antagonist at PAC1 and VPAC2 receptors  or charge transfer  (AHP by a specific PAC1 receptor agonist (maxadilan) in the presence of a PAC1 receptor antagonist further supports a contribution by PAC1 receptors.To investigate whether the reduction of sI t test) C,E. The AHP as a consequence of PAC1 receptor activation is not supported by the results obtained upon 250 nM PACAP-27 application in the presence of PACAP-(6–38). Under this condition, PACAP-27 reduced the sIAHP amplitude  and the charge transfer  to a comparable extent  as observed in the absence of PACAP-(6–38) (AHP by maxadilan but the lack of antagonism of PACAP-27 by PACAP-(6–38) suggests the activation of more than one receptor subtype in the PACAP-mediated inhibition of this current.Surprisingly, the reduction of the sIP-(6–38) . The sup1 receptor antagonist that is more potent than PACAP-(6–38)  and charge transfer  to an extent that was comparable (amplitude and charge transfer: P > 0.5) to the effect of PACAP-27 alone  was observed upon application of PACAP-27 (250 nM) in the presence of Rp-cAMPS.To address this question, we used the PKA inhibitor Rp-cAMPS added to the intracellular solution  was significantly reduced in the presence of Rp-cAMPS . When isoproterenol was applied after PACAP-27 in the presence of Rp-cAMPS, the sIAHP was inhibited to 43.1% ± 7% (n = 5) of the amplitude left after PACAP-27 inhibition, showing that Rp-cAMPS inhibited PKA. These results show that PKA contributes to the PACAP-27-mediated inhibition of the sIAHP.To ensure that Rp-cAMPS inhibited PKA under our experimental conditions, we used isoproterenol (1 µM), a β-adrenergic receptor agonist that inhibits the sIAHP could be mediated by the activation of the ERK or p38 MAPK pathway.In a Drosophila mutant PACAP modulates a potassium current through the activation of a mitogen-activated protein (MAP) kinase pathway Zhong, . PACAP hAHP  to the same extent as in intercalated control experiments performed with 0.13% DMSO in the pipette solution . Similarly, inhibition of MEK by UO126 did not affect the suppression of sIAHP by 500 nM PACAP-27 .To first test for a potential contribution of the MEK-ERK1/2 pathway, we applied intracellularly the MEK inhibitor UO126 (40 µM) , a p38 MAPK inhibitor  when compared with the sIAHP inhibition induced in the absence of the p38 MAPK inhibitor . SB 203580 largely prevented the inward shift in the holding current  caused by PACAP-27 (250 nM).Next, we tested the hypothesis of an involvement of p38 MAPK in the PACAP-mediated inhibition of sIal., AHP A. FigureAHP in CA1 pyramidal neurons, while a contribution by the MEK/ERK1/2 pathway seems unlikely.Taken together, these results indicate that p38 MAPK is involved in mediating the inhibitory effect of PACAP on the sIAHP suppression. This prompts the question as to whether PACAP-27 activates p38 MAPK and PKA as part of the same or two parallel pathways leading to sIAHP suppression. If PKA and p38 MAPK operate sequentially as part of the same pathway, their concomitant inhibition should not have a stronger impact on the effect of PACAP-27 than inhibition of each single kinase. Conversely, if PKA and p38 MAPK act through parallel, independent pathways their combined inhibition should have a stronger impact on the effect of PACAP-27.Both the inhibition of PKA by Rp-cAMPS  and of pAHP . The observation that the combined inhibition of PKA and p38 MAPK does not prevent the PACAP-27 reduction of the sIAHP to a larger extent than the inhibition of each single kinase suggests that they act sequentially as part of the same signal transduction pathway.The PKA inhibitor Rp-cAMPS and the p38 MAPK inhibitor SB 203580 were included together in the intracellular solution, and 250 nM PACAP-27 was applied for ∼15 min, causing a partial inhibition of the sIAHP B. The PAB 203580 A. The re compare C and 3C  compare C and 4C AHP by PACAP-27, as suggested by the combined inhibition of PKA and p38 MAPK (AHP amplitude by 10.3% ± 5.2% (n = 7). This reduction was not significant (P > 0.05). To assess the integrity of the PKA meditated modulation of the sIAHP we applied to the same cells the membrane permeable PKA activator 8CPT-cAMP   linked signaling cascades  by PACAP-27 (250 nM) was not significantly different compared to recordings performed in the absence of chelerythrine and BIM-1 . An inward shift in the holding current of 29.7 ± 9.1 pA  was observed upon application of PACAP-27 (250 nM) in the presence of the PKC inhibitors. To validate PKC inhibition under our experimental conditions, we activated PKC by phorbol-12,13-dibutyrate (PDBu). Under control conditions, PDBu (500 nM) caused a strong inhibition of the sIAHP amplitude , which was significantly reduced  in the presence of chelerythrine (20 µM) and BIM-1 (500 nM). This indicates that PKC was indeed inhibited under these experimental conditions.To obtain maximal PKC inhibition, a combination of two specific inhibitors acting at different sites on PKC, 20 µM chelerythrine . The inhibition of the sIAHP amplitude by PACAP-27 was similar in the presence of Rp-cAMPS alone and the combination of Rp-cAMPS, chelerythrine and BIM-1 . By contrast, inhibition of PKC alone by chelerythrine and BIM-1 with no Rp-cAMPS did not reduce the PACAP effect to a similar extent (P = 0.003 Mann–Whitney test).The lack of PKC involvement in the suppression of the sIAHP is unlikely also as part of a crosstalk.These results suggest that the involvement of PKC in the PACAP-mediated inhibition of the sI2+-activated K+ current sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings reveal a novel modulation of the sIAHP by p38 MAPK in CA1 pyramidal neurons, adding to the role of this current as a convergency point for modulatory inputs mediated by multiple protein kinases in central neurons.This study shows that PACAP increases the excitability of CA1 pyramidal neurons by inhibiting the slow CaAHP have been reported for CRF, VIP and PACAP in layer II/III and V neocortical pyramidal neurons   did not prevent the inhibition of the sIAHP by PACAP. This is different from what has been observed in the neocortex, where PACAP has been reported to activate the cAMP/PKA pathway by acting mainly through PAC1 receptors  and small G-proteins . Our res1 and VPAC1 receptors are coupled to the phospholipase C (PLC) pathway, leading to the activation of PKC  of synaptic transmission at several synapses in the hippocampal formation (Otto et al., AHP via PAC1 and VPAC1 receptors might contribute to the complex effects of PACAP on synaptic plasticity in the hippocampal formation and ultimately on learning and memory consolidation.In conclusion, our study shows that PACAP effectively modulates the intrinsic excitability and firing behavior of CA1 pyramidal neurons through the suppression of sI


Cdc55 phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to PP2ACdc55 in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2ACdc55 activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2ACdc55, timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2ACdc55, which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms.Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2A In all eukaryotic cells chromosome partition during mitosis requires a number of processes, including the formation of the mitotic spindle, i.e. the machinery that drives chromosome segregation to the daughter cells. Mitotic entry requires a delicate balance between protein phosphorylation, driven by cyclin-dependent kinases (CDKs), and protein dephosphorylation, carried out by specific phosphatases that counteract CDK activity. A critical threshold in CDK activity is indeed required for mitotic entry. In the past few years the Greatwall kinase has also been implicated in mitotic entry through phosphorylation of proteins of the endosulfine family, which in turn inhibit the activity of the PP2A phosphatase that would otherwise dephosphorylate CDK targets. Whether Greatwall and endosulfines have a mitotic function in budding yeast, where PP2A promotes, rather than inhibits, mitotic entry has not been established. Here we show that the Greatwall-endosulfine-PP2A regulatory module is conserved also in budding yeast and that endosulfines from different species are interchangeable for their mitotic function. However, in budding yeast cells endosulfines contribute to full activation and proper localization of PP2A, suggesting that they act as both inhibitors and activators of PP2A. Our data emphasize how the same regulatory module is adapted to meet specific mitotic features in different organisms. Entry into mitosis in eukaryotic cells is driven by cyclin-dependent kinases (CDKs) bound to B-type cyclins. Many targets of cyclinB/CDKs have been identified in different organisms and include proteins involved in mitotic spindle formation/elongation, nuclear envelope breakdown, chromosome condensation and segregation.Several mechanisms contribute to the rapid raise in cyclinB/CDKs activity at the onset of mitosis: i) cyclin accumulation, through transcriptional activation and inhibition of their proteolysis; ii) phosphorylation of their catalytic subunit Cdk1 by Cdk1-activating kinases (CAKs); iii) removal of Cdk1 inhibitory phosphorylations on Thr14 and Tyr15 , that are phosphorylated by the Greatwall-related kinase Rim15 on the same serine (Ser64 of Igo1 and Ser63 of Igo2) that in Xenopus Arpp19 and ENSA is phosphorylated by Greatwall IGO1 and IGO2, as well as of RIM15, does not affect cell viability but compromises the establishment of the quiescence (G0) state. Indeed, Igo proteins phosphorylated by Rim15 bind to an mRNA decapping factor to shelter degradation of specific mRNAs during initiation of the quiescence program C. elegansBudding yeast possesses two redundant endosulfines, called Igo1 and Igo2 , structural (A) and regulatory (B55) subunit of PP2A from CSF-induced mitotic extracts . However, rim15Δ and igo1Δ igo2Δ mutant cells where sensitive to thermal stress  and were hypersensitive to cell wall stressors, such as caffeine, calcofluor white and SDS . Thus, although Igo proteins are PP2ACdc55 inhibitors, in vivo they sustain full PP2ACdc55 activity. However, they did not seem to affect the basic composition of the hetero-trimeric PP2ACdc55 complex, as shown by the similar levels of interaction between endogenous HA-tagged Cdc55 and the PPh21 and Tpd3 subunits in wild type and igo1Δ igo2Δ cells . The phosphorylation status of Cdk1 was monitored by western analysis using a phospho-specific antibody that recognizes phosphorylated Cdk1-Y19. In agreement with previous reports cdc55Δ mutant cells under these conditions  in wild type, rim15Δ and igo1Δ igo2Δ mutant cells, as well as cdc55Δ cells used as control, and analysed Mih1 phosphorylation by western blot. Consistent with a previous report RIM15 or IGO1 and IGO2 led to accumulation of hyperphosphorylated forms already at 25°C, albeit not to the same levels as deletion of CDC55 .If budding yeast endosulfines were required for full PP2As YPGal, . Loss ofs YPGal,  without Cdc55, at least for what concerns some of its mitotic targets, thereby tuning the levels of Cdk1 phosphorylation to promote mitotic entry.Altogether, these data indicate that Rim15 and Igo proteins contribute to activation of PP2Ain vitro and in vivo , raised the possibility that in yeast Igo1/2 modulate PP2A activity at a different/additional level. For instance, the Zds1 and Zds2 proteins, which bind PP2ACdc55 in a stoichiometric complex and inhibit its activity in vitroigo1Δ igo2Δ cells at various cell cycle stages, namely in G1 (unbudded cells), in S, G2 and early M phases (budded mononucleated cells) and in middle/late M phase (budded binucleated cells). As previously shown rim15Δand igo1Δ igo2Δ cells than in the wild type in all cell cycle stages , which regulate cell growth in response to nutrient availability and cell stress In several organisms endosulfine-like proteins bind to PP2A-B55 upon Greatwall-dependent phosphorylation of a conserved serine and inhibit it Cdc55 participates to the positive feedback loop for Cdk1 activation (see below). In agreement with recently published data RIM15 reduce significantly Igo1 interaction with PP2ACdc55, indicating that, like in higher organisms, endosulfine phosphorylation is required for its interaction with PP2A. In spite of its cell cycle-regulated interaction with PP2ACdc55, Igo1 phosphorylation on Ser64 appears to be constitutive during the cell cycle, raising the interesting possibility that cell cycle-controlled factors are involved to make the Igo1-Cdc55 binding periodic. Along the same line, it is also interesting to notice that phosphorylation of Ser64 of Igo1 and Ser63 of Igo2 is stimulated after inhibition of Cdk1 Cdc55 complexes.The peak of interaction between Igo1 and Cdc55 is cell cycle-regulated and peaks in late S or G2 phase (note that it is not possible to discriminate between late S and G2 phase in budding yeast due to the lack of specific markers), i.e. when PP2Ain vitro human ENSA and Arpp19 Xenopus Greatwall can phosphorylate yeast Igo1 (this manuscript), indicating that Rim15 and Igo proteins are the true counterparts of vertebrate Greatwall and endosulfines, respectively. Thus, the Greatwall-Endosulfine-PP2A regulatory module appears to be conserved in all organisms analysed to date, with the notable exception of the nematode C. elegans where an obvious Greatwall-like kinase seems to be missing The evolutionary conservation of the Greatwall-Endosulfine pathway is further strengthened by the finding that yeast Rim15 can phosphorylate Xenopus egg extracts Xenopus and human cells and for mitotic progression in Drosophilain vitro and induces interphase Xenopus egg extracts to enter mitosis, indicating that budding yeast endosulfines can inhibit PP2A, like their vertebrate counterparts. PP2ACdc55 inhibition in rapamycin-treated yeast cells is important to establish a quiescence-specific transcriptional program Cdc55 for mitotic entry in vivo. First, lack of Igo1 and Igo2 causes a slight reduction, rather than an increase, in PP2ACdc55 phosphatase activity. Taking into account that only about 1% of Cdc55 is bound to Igo1 (and presumably a similar fraction of Cdc55 is bound to Igo2), the 15–20% reduction in PP2ACdc55 activity in cells lacking Igo proteins would imply a prominent role for this proteins in the full activation of PP2ACdc55 complexes. Second, deletion of RIM15 or IGO1 and IGO2 delays accumulation of active cyclinB/Cdk1 and dephosphorylation of Cdk1 Tyr19, similarly to inactivation of PP2ACdc55Cdc55CDC55 overexpression RIM15 or IGO1 and IGO2 deletion. Yet, not only phosphorylated Igo1 can inhibit PP2A activity in vitro, but expression of human ENSA or Arpp19 rescues the temperature-sensitivity of igo1Δ igo2Δ cells, suggesting that vertebrate and yeast endosulfines are interchangeable for their mitotic function(s). Therefore, the apparently opposite modes of PP2A regulation by endosulfines in yeast versus vertebrates are unlikely linked to intrinsic differences in the structure of endosulfines and/or in their ability to bind and inhibit PP2A. Several other examples of proteins that behave as activators or inhibitors of their binding partner(s) depending on the organism and/or the conditions have been reported. Similarly to Igo1 and Igo2, budding yeast Zds proteins were shown to inhibit PP2ACdc55 phosphatase activity Cdc55 and assist its proper folding while keeping the complex inhibited. The presence of phosphorylated substrates could competitively bind to PP2ACdc55 and overcome its inhibition. The exact biochemical mechanism of PP2A-B55 inhibition by endosulfines is currently unknown and will be likely clarified by structural and biochemical data.ENSA and Arpp19 inhibit PP2A activity in Cdc55 regulation for mitosis, we find that the phosphatase activity of PP2ACdc55 complexes in igo1Δ igo2Δ cells is reduced compared to wild type cells. This results in delayed phosphorylation of Swe1 and Mih1, which are known targets of PP2ACdc55rim15Δ and igo1Δ igo2Δ cells are unable to timely dephosphorylate Tyr19 of Cdk1 and to efficiently raise cyclinB/Cdk1 activity in mitosis, resulting in a delayed spindle elongation, which requires high cyclinB/Cdk1 levels rim15Δ and igo1Δ igo2Δ mutants display lower activity of PP2ACdc55 complexes, nuclear retention of Cdc55 and accumulation of Tyr19-phosphorylated Cdk1 at physiological temperatures (e.g. 25°C), while progressing through mitosis with normal timing, is somewhat puzzling. Since mitotic defects of rim15Δ and igo1Δ igo2Δ mutants become apparent only under stress conditions, it is possible that the physiological state of cells can make them differentially responsive to increased levels of Tyr19-phosphorylated Cdk1. Deletion of SWE1 or expression of the non-phosphorylatable Cdc28-Y19F mutant protein rescues the temperature-sensitive growth and mitotic defects of rim15Δ and igo1Δ igo2Δ cells, whereas deletion of MIH1 aggravates them. The role of PP2ACdc55 in the nucleolar retention and inhibition of Cdc14 Consistent with a positive role of Igo proteins in PP2ACdc55 has been recently proposed to be spatially regulated by nuclear export of Cdc55, which is regulated by Zds1 and Zds2 and promotes mitotic entry Cdc55 might be instrumental to trigger efficient Swe1 degradation RIM15 or IGO1 and IGO2. The increased concentration of Cdc55 in the nuclei of rim15Δ and igo1Δ igo2Δ cells might be a consequence of decreased PP2ACdc55 activity or be caused by a more direct function of yeast endosulfines in the control of Cdc55 nuclear-cytoplasmic shuttling. Our finding that SWE1 deletion restores proper subcellular localization of Cdc55 in the absence of Rim15 and Igo proteins argues against the latter possibility. We thus favor the idea that yeast endosulfines only impact on PP2ACdc55 activity, participating in the positive feedback loop for Cdk1 activation like their Xenopus counterparts Cdc55 substrates, including Swe1 and Mih1. In fact, Cdc55 mislocalization could keep PP2ACdc55 spatially segregated from its substrates. Further experiments will be required to understand the links between PP2A activity and its subcellular localization.The activity of PP2ACdc55Cdc55 activity to Mih1 IGO1 and IGO2 causes synthetic growth defects when combined to deletion of ZDS1 and ZDS2.Similar to endosulfines, the Zds1 and Zds2 proteins bind tightly to PP2AIn conclusion, our data emphasize the plasticity of the Greatwall-Endosulfine-PP2A module. This module is conserved, but it has adapted to account for the differences in mitotic regulation depending on the cellular context. In cells where PP2A antagonizes Cdk1 activity the Greatwall-endosulfine module only inhibits PP2A, whereas in budding yeast where PP2A promotes timely mitotic entry the same module also stimulates PP2A activity, resulting in both cases in a sharp mitotic switch ade2-1, trp1-1, leu2-3,112, his3-11,15, ura3, ssd1), except for strains used for phosphatase assays that were derivatives of S288C. Cells were grown in either synthetic minimal medium (SD) supplemented with the appropriate nutrients or YEP  medium supplemented with 2% glucose (YEPD) or 2% galactose (YEPG). Unless differently stated, alpha factor was used at 4 µg/ml, hydroxyurea (HU) at 200 mM and nocodazole at 15 µg/ml. Standard techniques were used for genetic manipulations All yeast strains  were dert-test, assuming unequal variances.In situ immunofluorescence was performed on formaldehyde-fixed cells expressing HA-tagged Cdc55 using anti-HA monoclonal antibody , followed by indirect immunofluorescence using Cy3-conjugated goat anti-mouse antibody . To detect spindle formation and elongation, anti-tubulin immunostaining was performed with the YOL34 monoclonal antibody (Serotec) followed by indirect immunofluorescence using rhodamine-conjugated anti-rat antibody . Digital images were taken with an oil 63X 1,4-0,6 HCX Plan-Apochromat objective (Zeiss) with a Coolsnap HQ2-1 charge-coupled device camera (Photometrics) mounted on a Zeiss AxioimagerZ1/Apotome fluorescence microscope controlled by the MetaMorph imaging system software. Fluorescence intensity of HA3-Cdc55 in the nucleus and the cytoplasm was quantified with ImageJ on a single focal plane. Significance of the differences between fluorescence intensities was statistically tested by means of a two-tailed 7 cells/ml) were lysed at 4°C with acid-washed glass beads in lysis buffer , 1 mM Na-orthovanadate and 60 mM ß-glycero-phosphate). Total extracts were cleared by spinning at 12000 rpm for 10 minutes and quantified by NanoDrop. Same amounts of protein extracts were subjected to immunoprecipitation with an anti-Pk antibody (from AbD serotec) pre-adsorbed to protein A-sepharose. Inputs represent 1/50th of the IPs final volumes.TCA protein extracts were prepared as previously described For Clb2/Cdk1 kinase assays protein extracts were prepared as previously described Xenopus egg extracts E.coli for the in vitro phosphorylation assay (Hyperactive human Gwl (Gwl-K72M) purified from baculovirus-infected cells or endogenous Gwl immunoprecipitated from CSF-treated on assay  and the on assay , respectYeast protein extracts were prepared according to GGGSGGGGS) was expressed from the strong constitutive TPI1 promoter on an episomal centromeric plasmid 32P-labeled phosphorylase a (32P-labeled histone H1 (http://www.licor.com). For 2, 1 mM ATP, 30°C for 30′) was added as indicated and phosphatase activity assays toward 32P-labeled phosphorylase a were conducted. The activities of the different samples are presented as percent of activity with respect to the activity of the untreated control, which was set to 100%. Data are presented as mean values ± standard deviation (SD), and were analyzed using two-tailed Student's t-test, assuming unequal variances. Differences with p-values lower than 0.05 were considered statistically significant .For immunoprecipitation assays, yeast whole-cell extracts were prepared as described previously rylase a , 4A or tstone H1 , as prevNuclear division was scored with a fluorescence microscope on cells stained with propidium iodide (Sigma Aldrich). Flow cytometric DNA quantification was performed according to Figure S1in vitro by human Gwl using P32γATP. Phosphorylated Igo1 was visualized after SDS page and autoradiography.Igo1 interacts with the PP2A B subunit Cdc55 and not with its B′ subunit Rts1. A. Interaction between Igo1-Pk3 and either HA3-Cdc55 or Rts1-HA3 was assessed by immunoprecipitation with anti-Pk antibodies followed by western blot analysis with anti-HA and anti-Pk antibodies. B. Bacterially purified GST-Igo1 or GST-Igo1-S64A were phosphorylated (PDF)Click here for additional data file.Figure S2igo1Δ igo2Δ cells carrying the empty vector (YEp) or a high copy number plasmid carrying human ARPP19 (YEp-ARPP19) or human ENSA (YEp-ENSA) under the control of the IGO1 promoter Cells lacking Rim15 or Igo1 and Igo2 are sensitive to different stress conditions and their temperature-sensitivity is suppressed by expression of human endosulfines. A. Serial dilutions of strains with the indicated genotypes were spotted on YEPD plates and incubated at the indicated temperatures (top panel) or were spotted on YEPD containing 10 mM caffeine (Caff) or 0.01% calcofluor white (CFW) or 0.001% SDS and incubated at 25°C (bottom panel). B. Wild type and (PDF)Click here for additional data file.Figure S3RIM15 or IGO1 and IGO2 affects Swe1 hyperphosphorylation. Cycling (cyc) cultures of wild type, rim15Δ and igo1Δ igo2Δ cells expressing HA-tagged Swe1 (Swe1-HA3) were arrested in G1 by α-factor and released in fresh medium at 25°C in the presence of nocodazole (A) or at 38°C (B). At the indicated time points cells were collected for FACS analysis of DNA contents (not shown), kinetics of budding, spindle assembly/elongation and nuclear division (graphs), as well as to prepare TCA protein extracts for western blot analysis of Swe1-HA3 using anti-HA antibodies. Similar results were obtained in (A) for rim15Δ and igo1Δ igo2Δ cells (not shown).Deletion of (PDF)Click here for additional data file.Figure S4rim15Δ and igo1Δ igo2Δ cells. Serial dilutions of strains with the indicated genotypes were spotted on YEPD plates and incubated for 48 hours at the indicated temperatures.Expression of the non-phosphorylatable Cdc28-Y19F mutant protein suppresses the temperature-sensitive growth defects of (PDF)Click here for additional data file.Figure S5IGO1 and IGO2 causes synthetic sickness to cells lacking Zds proteins. A. A heterozygous IGO1/igo1Δ IGO2/igo2Δ ZDS1/zds1Δ ZDS2/zds2Δ diploid strain was induced to sporulate and its meiotic segregants were analysed in 103 independent tetrads for the presence of genetic markers identifying the different gene deletions. The table reports for each indicated genotype the total number of expected and found meiotic segregants, and among the latter the number of healthy, sick  and inviable segregants. As previously reported ZDS1 and ZDS2 decreases spore viability and increases sickness with variable penetrance, whereas deletion of IGO1 and IGO2 is well tolerated. B. Stationary phase cells of meiotic segregants with the indicated genotype that were classified as “healthy” in (A) were spotted on YEPD plates and incubated for 2 days at 25°C and 37°C. Deletion of IGO1 and IGO2 increases the temperature-sensitivity of healthy zds1Δ zds2Δ cells . C. Images of logarithmically growing cells with the indicated genotypes grown in YEPD at 25°C and shifted for 3 hours to 37°C.Deletion of (PDF)Click here for additional data file.Table S1List of strains used in this study.(PDF)Click here for additional data file.


Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.Ure2p is the protein determinant of the  Constitutive overexpression of Ssa1p but not the nearly identical Ssa2p cures cells of [URE3] [URE3] propagation [PSI+], excess Ssa1p protects [PSI+] from curing induced by overexpression of HSP104 [PSI+] stability is normal in strains that lack SSA1 or SSA2 [PSI+] formation [PSI+] by increasing the size of prion aggregates and hence hindering prion transmission to daughter cells  . Additiopagation . With ref HSP104 . [PSI+]  or SSA2  and overormation . Howeverer cells . In vitror Sis1p . The actor Sis1p . It has or Sis1p ,58. The or Sis1p ,59. The of Ure2p ,59,60.in vitro methods to dissect out the relative contribution of the different Ssa1p structural domains, by investigating the effect of a series of deletion mutants of Ssa1p on Ure2p fibril formation. We also investigated in further detail the mode and mechanism of interaction between Ssa1p and Ure2p.The mechanism of how Ssa1p inhibits Ure2p fibril formation is complex and is not yet fully understood. It remains unclear how specific domains of Ssa1p function independently or in cohort to produce the observed inhibition of fibril formation of Ure2p. To address these questions, we used the power of 2.(a)1,4-Dithiothreitol (DTT), β-mercaptoethanol, Tris, thioflavin T (ThT), ATP, ADP, creatine kinase (CK) and creatine phosphate (CP) were purchased from Sigma. TEV (tobacco etch virus) protease was from Invitrogen. Recombinant firefly luciferase and luciferase assay kit were from Promega. All other reagents were local products of analytical grade. Deionized distilled water was used throughout.(b)g, 30 min). The supernatant was then loaded onto a Ni affinity column  and washed with buffer A containing 100 mM imidazole. Protein was eluted using buffer A containing 350 mM imidazole and dialysed into 50 mM Tris–HCl buffer pH 8.4 containing 150 mM KCl, 5 mM MgCl2 and 1 mM DTT with or without 5 per cent glycerol (henceforth abbreviated to ‘Tris buffer pH 8.4’). Treatment with TEV protease to remove the His-tag was followed by a further round of purification by Ni affinity chromatography. It was found that the presence or absence of the His-tag had no effect on the ability of Ssa1p to inhibit Ure2p fibril formation. Therefore, the His-tagged protein was used in subsequent experiments. In vivo, when it is the sole HSP70-Ssap in the cell, His-tagged Ssa1p is well expressed and is able to provide essential cellular functions  in vivo; hence, it has been suggested that the curing effect of Ydj1p on [URE3] is indirect and occurs via its effect on Ssa1p [URE3] by Ydj1p  prion state in yeast cells  prion state, Ure2p. It has been reported that mutations in the NBD of Ssa1p affect [URE3] propagation in vivo, but not the process of [URE3] elimination  by overexpression in vivo [in vitro [URE3] led to the suggestion that the curing effect of Ydj1p occurs indirectly via its stimulation of HSP70 [URE3] [in vitro, which accounts for the reduced extent of curing in the presence of Ssa1p  bound states show less variability in conformation and the open state is preferred ,89–92. Hbundance . Much ofmologues ,92, meanmologues . NeverthΔA Ssa1p) interacted with Ure2p in a similar manner to the full-length protein. We then went on to examine the effect of progressive truncation of the Ssa1p C-terminal domain, in order to further refine the structural regions that are important for the inhibition effect on Ure2p.Having found that the presence or absence of nucleotide had no significant effect on the ability of Ssa1p to inhibit Ure2p fibril formation, we tested whether or not entirely deleting the ATPase domain reduced the inhibition ability of Ssa1p towards Ure2p and found it did not. We found that the ATPase domain-deletion mutant (in vitro. Further deletions around the border between the C-terminal random coil region and the α-helical bundle reduced the ability of Ssa1p to inhibit fibril formation, indicating that this region may contribute to the structural integrity of the α-helical bundle. Successive deletion of helices D and C of the α-helical bundle resulted in incremental weakening of the inhibition effect of Ssa1p on Ure2p fibril formation. On the other hand, the α-helical bundle of Ssa1p did not influence fibril formation of Ure2p in isolation from the SBD. Taken together, this indicates that the SBD of Ssa1p is primarily responsible for inhibition of Ure2p fibril formation, and the α-helical bundle of the CTD facilitates this effect. Interestingly, our results suggest that the α-helical bundle does not have to be fully formed to contribute to this effect. These findings are similar to the inhibitory effect of human HSP70 on aggregation of α-synuclein, which also relies primarily on the action of the SBD, but is facilitated by the presence of the CTD [The C-terminal lid is thought to contribute to the oligomerization of Ssa1p  and prov the CTD .ΔA Ssa1p interacts with the native Ure2p protein in solution which is consistent with previous work that proposed an interaction between Ssa1p and the native Ure2p protein [ΔA Ssa1p co-pelleted with Ure2p filament seeds, which provides an explanation for the delay in the lag time observed in the ThT experiments. Results from the pull-down assay also help to explain the incomplete recovery of the elongation rate after chaperone treatment observed by QCM, indicating that ΔA Ssa1p interacts with Ure2p fibril seeds, forming complexes that could not be completely dissociated by washing. The small amount of chaperone that remained bound to seeds on the QCM sensor reduced the growth rate of Ure2p fibrils. HSP70 has been proposed to adopt an ‘intermediate-open’ conformation upon binding to bulky substrates such as protofibrils or fibrils, where the substrate binding groove is not completely sealed by the lid [Ssa1p inhibition of fibril formation by Ure2p is efficient when Ssa1p is added during the lag phase or early exponential phase of fibril growth, suggesting that Ssa1p interacts with species present during these phases of growth. Our DLS data indicate that  protein ,60. We a protein ,96,97 to the lid . In thisΔA Ssa1p are able to interact directly with both native Ure2p and Ure2p fibril seeds, providing an explanation for the observation of inhibition of fibril formation even in the absence of nucleotide. We show that this inhibitory effect is primarily reflected in a reduction in the rate of fibril elongation. Further, our results concerning the involvement of the HSP70 CTD in binding are consistent with other recent results that indicate that the SBD is crucial for substrate interactions, but binding is enhanced by the presence of the CTD, either due to direct interaction between the substrate and the CTD, or due to the action of the CTD as a lid to facilitate capture of substrate molecules.Taken together, our results indicate that WT and


Accordingly, we found that GSK3β inactivation by a protein kinase C- (PKC-) dependent mechanism is involved in the NMDA-induced reduction of Tau phosphorylation at Ser199-202 epitopes. Taken together, these data indicate that NR2A receptor activation may be important in limiting Tau phosphorylation by a PKC/GSK3β pathway and strengthen the idea that these receptors might act as an important molecular device counteracting neuronal cell death mechanisms in various pathological conditions.The molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. In the present study, pharmacological inhibitors were deployed to investigate potential processes by which the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors modulates Tau phosphorylation in rat hippocampal slices. Our results demonstrated that Tau phosphorylation at Ser199-202 residues was decreased in NMDA-treated hippocampal slices, an effect that was not reproduced at Ser262 and Ser404 epitopes. NMDA-induced reduction of Tau phosphorylation at Ser199-202 was further promoted when NR2A-containing receptors were pharmacologically isolated and were completely abrogated by the NR2A receptor antagonist NVP-AAM077. Compared with nontreated slices, we observed that NMDA receptor activation was reflected in high Ser9 and low Tyr216 phosphorylation of glycogen synthase kinase-3 beta (GSK3 Inhibitors of protein kinase C , phosphoinositide 3-kinase , cyclin-dependent kinase 5  as well as protease and phosphatase inhibitor cocktails were acquired from Calbiochem .β (dilution 1 μg/mL), GSK3β Ser9 , GSK3β Tyr216 , and β-actin antibody were also purchased from AbCam. Goat anti-rabbit or goat anti-mouse peroxidase-conjugated antibodies  and SuperSignal chemiluminescent substrate kits were from Pierce Chemical Co. .Most antibodies reacting with Tau proteins were purchased from Abcam . The mouse polyclonal antibody Tau-5 (dilution 1 : 500) served to estimate total Tau protein levels in hippocampal extracts, along with rabbit polyclonal antibodies recognizing Tau phosphorylated at Ser199-202 , Ser262 , and Ser404 . Total GSK32PO4, 2.3 mM MgCl2, 1 mM CaCl2, 25 mM NaHCO3, and 11 mM glucose, saturated with 95% O2/5% CO2 (pH 7.4). Coronal brain sections of 350 μm, containing the hippocampus, were sliced in a Vibratome Series 1000 tissue sectioning system . Sections were then transferred to artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 3.5 mM KCl, 1.2 mM NaH2PO4, 1.3 mM MgCl2, 2 mM CaCl2, 25 mM NaHCO3, and 11 mM glucose, bubbled continuously with 95% O2/5% CO2 at 32°C. The brain sections were preincubated for 60 min before pharmacological treatment. After pharmacological treatment, hippocampal slices were dissected from the brain sections and homogenized in ice-cold RIPA lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, and 1 mM EDTA supplemented with protease and phosphatase inhibitor cocktails.Sprague-Dawley rats were anesthetized by isoflurane inhalation  and decapitated. Their brains were quickly removed and placed in cold cutting buffer containing 126 mM NaCl, 3.5 mM KCl, 1.2 mM NaHμg) were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Separated proteins were transferred onto nitrocellulose membranes, and nonspecific binding sites were blocked by incubation for 1 hour at room temperature in phosphate-buffered saline (PBS), pH 7.4, containing 5% bovine serum albumin (BSA fraction V) purchased from Fisher Scientific . Then, selected primary antibodies were incubated overnight at 4°C. After several washes with 0.1% Tween 20, the blots were incubated for 1 hour at room temperature in specific secondary horseradish peroxidase (HRP)-conjugated antibody solution. Both primary and secondary antibodies were diluted in Tris buffered saline ((TBS)/0.1% Tween 20/1% BSA). Immunoreactivity was visualized by chemiluminescence reactions and densitometric scanning with Vision Work LS software , and the intensity of the bands was quantified by ImageJ . The densitometry data were expressed as relative optical density.Protein levels extracted from rat hippocampus sections were measured by Bradford assay . Protein lysates . μM, and Tau phosphorylation was then processed according to Western blotting procedures. In initial experiments, we observed that the Tau isoform, estimated to be 62 kDa, became progressively less phosphorylated at Ser199-202 residues when exposed to increasing NMDA concentrations. The threshold concentration for reducing Tau phosphorylation at Ser199-202 was in the order of 5 μM with a maximal effect obtained at 10–50 μM , which normally did not alter phosphorylation levels of Tau at these epitopes. In contrast, we observed that blockade of NR2A receptors with NVP-AAM077 induces Tau phosphorylation at Ser199-202 epitopes. These data are indeed consistent with the notion that, when pharmacologically isolated, NR2A- and NR2B-containing receptors may exert opposite effects on Tau phosphorylation [From a pharmacological perspective, it has been proposed that NR1/NR2A receptor activation could favour the action of prosurvival mechanisms as well as biochemical processes limiting Tau phosphorylation. The possibility that stimulation of NR2A-containing NMDA receptors is responsible for downregulating Tau phosphorylation was then considered. rylation .β, known to influence the phosphorylation of these epitopes [β were examined. After NMDA treatment of hippocampal slices, total GSK3β protein was found to be unchanged, while its phosphorylation status was clearly affected. NMDA not only increased the phosphorylation of Ser9 residues of GSK3β but also noticeably reduced phosphorylation at the Tyr216 epitope of this kinase, suggesting functional blockage of GSK3β during NMDA treatment  and 10-DEBC hydrochloride (2.5 μM), respectively. In these experiments, the inhibitors were applied 30 minutes prior to NMDA exposure to ensure optimal enzymatic inhibition. μM). In contrast, we noticed that the capacity of NMDA to reduce Tau phosphorylation was totally prevented in hippocampal slices preincubated with the PKC inhibitor Chelerythrine chloride (1 μM), indicating that NMDA-induced regulation of Tau phosphorylation probably involves GSK3β  blockade by the PKC system , suggesting the contribution of the NR2A subunit (data not shown).Given that NMDA-induced reduction of Tau phosphorylation seems dependent on GSK3rocesses . To asseC system . In acconhibitor . Notablyβ inactivation.Several lines of evidence indicate that NMDA receptor activation is associated with dephosphorylation of cytoskeletal proteins , 11. Forβ, a site which tightly controls both enzymatic activities and other GSK3β phosphorylation sites, such as Tyr216 [β activity is downregulated after NMDA treatment of cerebellar granule neurons by mechanisms involving GSK3β hyperphosphorylation at the Ser9 residue [β activity, and from a mechanistic perspective, we demonstrated that this effect is possibly dependent on calcium mobilization. The exact mechanisms by which calcium could reduce GSK3β activity remain to be clarified, but the existence of pathways whereby calcium-dependent processes can downregulate GSK3β activity is plausible. Here, we observed that NR2A-induced Tau dephosphorylation and Ser9 phosphorylation were completely reversed by preincubation of slices in the presence of Chelerythrine chloride, suggesting that the ability of NMDA to reduce Tau phosphorylation likely depends on the activation of classical PKC and subsequent blockade of GSK3β activity. Several PKC isoforms are expressed in the hippocampus , and itβ during NMDA receptor activation may exist. Recently, gamma-aminobutyric acid type A (GABAA) receptors were implicated in the regulation of Tau phosphorylation at Ser199-202 epitopes. In this particular case, increased Tau phosphorylation at these residues was evoked after GABAA receptor activation by a mechanism requiring cdk5 and, consequently, reduced protein phosphatase 2A (PP2A) association with Tau [β activity, namely Akt/PKB and PI3K. Among other possibilities, it has recently been demonstrated that GSK3β phosphorylation at its Ser389 residue by the highly-enriched enzyme p38 mitogen-activated protein kinase (p38MAPK) attenuates GSK3β activity similarly to that implicating Ser9 phosphorylation [β inhibition via the p38MAPK mechanism.Of course, additional pathways that could inactivate GSK3with Tau . Howeverrylation . Therefoβ-targeted epitopes, NMDA treatments did not seem to influence sites located in the proline-rich (Ser 262) and C-terminal (Ser 404) domains of Tau proteins.Tau proteins contain three (R3) or four R4) tandem repeats of 31 or 32 amino acids at the carboxyl terminus and 29 or 58 amino acid inserts near the amino terminus  tandem r, 44–46. β are not yet understood. One possible explanation of these selective effects could be the differential localisation of Tau proteins. Interestingly, it was demonstrated previously that the antibody directed against Ser199-202 epitopes  is more inclined to interact with GSK3β-targeted Tau proteins located in the somatodendritic compartment of neurons, whereas other antibodies, such as those directed against Ser404 and 396 epitopes, appear to react more strongly with Tau isoforms distributed within axonal pools [β pathway could mainly interact with Tau proteins located in the somatodendritic compartment. On the other hand, the present studies examining the relationship between Tau phosphorylation and NMDA receptors indicate that NR2A subunits predominantly regulate Ser199-202 epitopes. Of course, the fact that NR2A-induced reduction in Tau phosphorylation was dominant as NMDA concentration increases suggests that NR2A receptors might be strategically located to initiate this downstream signalling. Mechanistically, it has also been reported that high NMDA concentrations can lead to activation of other signaling pathways, such as protein phosphate-1, which has the potential to limit NR2B receptor activation, calcium overload, and neurodegeneration during an excitotoxic event [The exact molecular mechanisms underlying epitope-and isoform-specific Tau regulation by NR2A-containing NMDA receptors and GSK3al pools . In thisal pools , 48. Accal pools , reducedic event . These pβ inactivation strongly suggests that the effect reported here may have interesting implications from a therapeutic perspective. Several experiments have demonstrated that hyperphosphorylation is linked with dissociation of Tau from microtubules, accentuating (mis)localisation of Tau proteins in dendrites [β activity by PKC isoforms. Consistent with this scenario is a recent study demonstrating that Tau dysfunction and neurofibrillary tangle formation are abolished by silencing GSK3β activity in Alzheimer's disease transgenic mouse models [The current study supports our previous observations that NR2A-containing NMDA receptors are potentially important for controlling Tau phosphorylation in the hippocampus. In fact, the present findings argue that stimulation of these receptors might function as a molecular device limiting Tau phosphorylation in neuropathological conditions, a notion which appears to be consistent with overwhelming evidence that NR2A receptors are coupled to neuroprotective mechanisms likely to reduce cell death . Thus, tendrites , 50–52. e models , and mane models , 53–56.β activity could also be important for limiting the progression of other cellular damages  which are associated with Alzheimer's disease [β pathway may provide novel perspectives for the treatment of excitotoxicity in pathologies such as ischemic stroke.Of course, more detailed studies are required to address whether this NR2A-mediated reduction in GSK3 disease , and fur