• Introduction • Pathological modifications of tau - Tau phosphorylation - Tau truncation - NFTs are not the primary toxic species • Tau turnover and possible deficits in Alzheimer disease • Tau toxicity in Alzheimer disease • Potential ‘tau-centric’ therapeutic strategies - Tau aggregation inhibitors - Microtubule stabilizing agents - Tau immunotherapy - Autophagy activators - Mitochondria targeted therapies • Summary and conclusions It has been almost 25 years since the initial discovery that tau was the primary component of the neurofibrillary tangles (NFTs) in Alzheimer disease (AD) brain. Although AD is defined by both β-amyloid (Aβ) pathology (Aβ plaques) and tau pathology (NFTs), whether or not tau played a critical role in disease pathogenesis was a subject of discussion for many years. However, given the increasing evidence that pathological forms of tau can compromise neuronal function and that tau is likely an important mediator of Aβ toxicity, there is a growing awareness that tau is a central player in AD pathogenesis. In this review we begin with a brief history of tau, then provide an overview of pathological forms of tau, followed by a discussion of the differential degradation of tau by either the proteasome or autophagy and possible mechanisms by which pathological forms of tau may exert their toxicity. We conclude by discussing possible avenues for therapeutic intervention based on these emerging themes of tau’s role in AD.
In Alzheimer disease (AD) mitochondrial abnormalities occur early in the pathogenic process and likely play a significant role in disease progression. Tau is a microtubule-associated protein that is abnormally processed in AD, and a connection between tau pathology and mitochondrial impairment has been proposed. However, few studies have examined the relationship between pathological forms of tau and mitochondrial dysfunction. We recently demonstrated that inducible expression of tau truncated at Asp-421 to mimic caspase cleavage (T4C3) was toxic to immortalized cortical neurons compared with a full-length tau isoform (T4). In this study we investigated the effects of T4C3 on mitochondrial function. Expression of T4C3 induced mitochondrial fragmentation and elevated oxidative stress levels in comparison with T4-expressing cells. Thapsigargin treatment of T4 or T4C3 cells, which causes an increase in intracellular calcium levels, resulted in a significant decrease in mitochondrial potential and loss of mitochondrial membrane integrity in T4C3 cells when compared with cells expressing T4. The mitochondrial fragmentation and mitochondrial membrane damage were ameliorated in T4C3 cells by pretreatment with cyclosporine A or FK506, implicating the calcium-dependent phosphatase calcineurin in these pathogenic events. Increased calcineurin activity has been reported in AD brain, and thus, inhibition of this phosphatase may provide a therapeutic target for the treatment of AD. In Alzheimer disease (AD) mitochondrial abnormalities occur early in the pathogenic process and likely play a significant role in disease progression. Tau is a microtubule-associated protein that is abnormally processed in AD, and a connection between tau pathology and mitochondrial impairment has been proposed. However, few studies have examined the relationship between pathological forms of tau and mitochondrial dysfunction. We recently demonstrated that inducible expression of tau truncated at Asp-421 to mimic caspase cleavage (T4C3) was toxic to immortalized cortical neurons compared with a full-length tau isoform (T4). In this study we investigated the effects of T4C3 on mitochondrial function. Expression of T4C3 induced mitochondrial fragmentation and elevated oxidative stress levels in comparison with T4-expressing cells. Thapsigargin treatment of T4 or T4C3 cells, which causes an increase in intracellular calcium levels, resulted in a significant decrease in mitochondrial potential and loss of mitochondrial membrane integrity in T4C3 cells when compared with cells expressing T4. The mitochondrial fragmentation and mitochondrial membrane damage were ameliorated in T4C3 cells by pretreatment with cyclosporine A or FK506, implicating the calcium-dependent phosphatase calcineurin in these pathogenic events. Increased calcineurin activity has been reported in AD brain, and thus, inhibition of this phosphatase may provide a therapeutic target for the treatment of AD. Tau is a microtubule-associated protein which in a hyperphosphorylated state forms paired helical filaments; the major component of neurofibrillary tangles (NFTs) 3The abbreviations used are: NFTneurofibrillary tangleADAlzheimer diseaseMTGMitotrackerGreenTMTMRMtetramethylrhodamine methyl esterROSreactive oxygen speciesHRHKrebs-Ringer HEPES bufferFCCPcarbonyl cyanide p-trifluoromethoxyphenylhydrazonePTPpermeability transition poremPTPmitochondrial PTPCsAcyclosporine A2,7-DCF2,7-dichlorofluorescin diacetate. (1.Kosik K.S. Joachim C.L. Selkoe D.J. Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 4044-4048Crossref PubMed Scopus (1148) Google Scholar, 2.Grundke-Iqbal I. Iqbal. K. Tung Y.C. Quinlan M. Wisniewski H.M. Binder L.I. Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 4913-4917Crossref PubMed Scopus (2896) Google Scholar). These NFTs are one of the primary pathophysiological hallmarks of Alzheimer disease (AD) and were originally suggested to play a major role in facilitating neuronal degeneration (1.Kosik K.S. Joachim C.L. Selkoe D.J. Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 4044-4048Crossref PubMed Scopus (1148) Google Scholar). However, recent studies now suggest that mature tangles may not be the toxic species (3.Ding H. Johnson G.V. J. Alzheimers Dis. 2008; 14: 441-447Crossref PubMed Scopus (21) Google Scholar, 4.García-Sierra F. Mondragón-Rodríguez S. Basurto-Islas G. J. Alzheimers Dis. 2008; 14: 401-409Crossref PubMed Scopus (74) Google Scholar). For example, in a repressible tau overexpression transgenic mouse model, turning off tau expression attenuated memory impairment and neuronal loss, whereas NFTs continued to accumulate (5.Santacruz K. Lewis J. Spires T. Paulson J. Kotilinek L. Ingelsson M. Guimaraes A. DeTure M. Ramsden M. McGowan E. Forster C. Yue M. Orne J. Janus C. Mariash A. Kuskowski M. Hyman B. Hutton M. Ashe K.H. Science. 2005; 309: 476-481Crossref PubMed Scopus (1587) Google Scholar). Furthermore, reduction of endogenous wild type tau attenuated behavioral abnormalities in an APP transgenic AD mouse model, in which substantial NFT pathology is absent (6.Roberson E.D. Scearce-Levie K. Palop J.J. Yan F. Cheng I.H. Wu T. Gerstein H. Yu G.Q. Mucke L. Science. 2007; 316: 750-754Crossref PubMed Scopus (1508) Google Scholar). These and other findings suggest that a form or forms of tau that precede NFT formation may be the toxic species. neurofibrillary tangle Alzheimer disease MitotrackerGreenTM tetramethylrhodamine methyl ester reactive oxygen species Krebs-Ringer HEPES buffer carbonyl cyanide p-trifluoromethoxyphenylhydrazone permeability transition pore mitochondrial PTP cyclosporine A 2,7-dichlorofluorescin diacetate. There is increasing evidence that, in addition to aberrant phosphorylation, caspase cleavage of tau plays a role in the oligomerization and formation of a pathological tau species in AD (7.Gamblin T.C. Chen F. Zambrano A. Abraha A. Lagalwar S. Guillozet A.L. Lu M. Fu Y. Garcia-Sierra F. LaPointe N. Miller R. Berry R.W. Binder L.I. Cryns V.L. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 10032-10037Crossref PubMed Scopus (690) Google Scholar, 8.Rissman R.A. Poon W.W. Blurton-Jones M. Oddo S. Torp R. Vitek M.P. LaFerla F.M. Rohn T.T. Cotman C.W. J. Clin. Invest. 2004; 114: 121-130Crossref PubMed Scopus (469) Google Scholar). Tau is an in vitro substrate for caspase-3 and is readily cleaved at Asp-421, the caspase-3 cleavage site, located on the carboxyl-terminal end of the protein (7.Gamblin T.C. Chen F. Zambrano A. Abraha A. Lagalwar S. Guillozet A.L. Lu M. Fu Y. Garcia-Sierra F. LaPointe N. Miller R. Berry R.W. Binder L.I. Cryns V.L. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 10032-10037Crossref PubMed Scopus (690) Google Scholar, 8.Rissman R.A. Poon W.W. Blurton-Jones M. Oddo S. Torp R. Vitek M.P. LaFerla F.M. Rohn T.T. Cotman C.W. J. Clin. Invest. 2004; 114: 121-130Crossref PubMed Scopus (469) Google Scholar, 9.Fasulo L. Ugolini G. Cattaneo A. J. Alzheimers Dis. 2005; 7: 3-13Crossref PubMed Scopus (55) Google Scholar, 10.Chung C.W. Song Y.H. Kim I.K. Yoon W.J. Ryu B.R. Jo D.G. Woo H.N. Kwon Y.K. Kim H.H. Gwag B.J. Mook-Jung I.H. Jung Y.K. Neurobiol. Dis. 2001; 8: 162-172Crossref PubMed Scopus (190) Google Scholar). This cleavage event results in a highly fibrillogenic tau isoform which in in vitro studies aggregates more readily and to a greater extent than full-length tau and facilitates aggregate formation of full-length tau (7.Gamblin T.C. Chen F. Zambrano A. Abraha A. Lagalwar S. Guillozet A.L. Lu M. Fu Y. Garcia-Sierra F. LaPointe N. Miller R. Berry R.W. Binder L.I. Cryns V.L. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 10032-10037Crossref PubMed Scopus (690) Google Scholar, 8.Rissman R.A. Poon W.W. Blurton-Jones M. Oddo S. Torp R. Vitek M.P. LaFerla F.M. Rohn T.T. Cotman C.W. J. Clin. Invest. 2004; 114: 121-130Crossref PubMed Scopus (469) Google Scholar). Antibodies that specifically recognize Asp-421-truncated tau show that tau cleaved at Asp-421, active caspase-3, and fibrillar tau pathologies co-localize in AD patient brains (7.Gamblin T.C. Chen F. Zambrano A. Abraha A. Lagalwar S. Guillozet A.L. Lu M. Fu Y. Garcia-Sierra F. LaPointe N. Miller R. Berry R.W. Binder L.I. Cryns V.L. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 10032-10037Crossref PubMed Scopus (690) Google Scholar, 8.Rissman R.A. Poon W.W. Blurton-Jones M. Oddo S. Torp R. Vitek M.P. LaFerla F.M. Rohn T.T. Cotman C.W. J. Clin. Invest. 2004; 114: 121-130Crossref PubMed Scopus (469) Google Scholar). In a mouse tauopathy model it was also found that the majority of cells with active caspases also had NFTs (11.Spires-Jones T.L. de Calignon A. Matsui T. Zehr C. Pitstick R. Wu H.Y. Osetek J.D. Jones P.B. Bacskai B.J. Feany M.B. Carlson G.A. Ashe K.H. Lewis J. Hyman B.T. J. Neurosci. 2008; 28: 862-867Crossref PubMed Scopus (116) Google Scholar). Furthermore, experiments in cell culture models provide evidence that Asp-421-cleaved tau is toxic to neurons (9.Fasulo L. Ugolini G. Cattaneo A. J. Alzheimers Dis. 2005; 7: 3-13Crossref PubMed Scopus (55) Google Scholar, 10.Chung C.W. Song Y.H. Kim I.K. Yoon W.J. Ryu B.R. Jo D.G. Woo H.N. Kwon Y.K. Kim H.H. Gwag B.J. Mook-Jung I.H. Jung Y.K. Neurobiol. Dis. 2001; 8: 162-172Crossref PubMed Scopus (190) Google Scholar, 12.Matthews-Roberson T.A. Quintanilla R.A. Ding H. Johnson G.V. Brain Res. 2008; 1234: 206-212Crossref PubMed Scopus (30) Google Scholar). However, the mechanism(s) underlying this toxicity has not been elucidated. Increasing evidence suggests that even before the occurrence of AD-related pathologies or any detectable cognitive decline, there are disruptions in mitochondrial function (13.Blass J.P. Gibson G.E. Hoyer S. J. Alzheimers Dis. 2002; 4: 225-232Crossref PubMed Scopus (79) Google Scholar, 14.Hauptmann S. Scherping I. Drose S. Brandt U. Schulz K.L. Jendrach M. Leuner K. Eckert A. Muller W.E. Neurobiol. Aging. 2008; (in press)PubMed Google Scholar). Mitochondria play a major role in regulating neuronal calcium homeostasis (15.Werth J.L. Thayer S.A. J. Neurosci. 1994; 14: 348-356Crossref PubMed Google Scholar). Mitochondria have a vast capacity to accumulate calcium; this ability is driven through an electrochemical gradient provided mainly by the mitochondrial potential and a low intramitochondrial free calcium concentration (16.Beutner G. Sharma V.K. Giovannucci D.R. Yule D.I. Sheu S.S. J. Biol. Chem. 2001; 276: 21482-21488Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). However, in AD and other neurodegenerative diseases the ability of the mitochondria to buffer increases in cytosolic calcium, produce ATP, and regulate oxidative stress is impaired, which could contribute to neuronal degeneration (17.Lin M.T. Beal M.F. Nature. 2006; 443: 787-795Crossref PubMed Scopus (4704) Google Scholar). Given the fact that both caspase cleavage of tau and mitochondrial dysfunction are early events in the pathogenesis of AD (7.Gamblin T.C. Chen F. Zambrano A. Abraha A. Lagalwar S. Guillozet A.L. Lu M. Fu Y. Garcia-Sierra F. LaPointe N. Miller R. Berry R.W. Binder L.I. Cryns V.L. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 10032-10037Crossref PubMed Scopus (690) Google Scholar, 14.Hauptmann S. Scherping I. Drose S. Brandt U. Schulz K.L. Jendrach M. Leuner K. Eckert A. Muller W.E. Neurobiol. Aging. 2008; (in press)PubMed Google Scholar) and the finding that Asp-421-cleaved tau is toxic in our cell culture model (12.Matthews-Roberson T.A. Quintanilla R.A. Ding H. Johnson G.V. Brain Res. 2008; 1234: 206-212Crossref PubMed Scopus (30) Google Scholar), the focus of this study was to determine whether and how Asp-421-cleaved tau affected mitochondrial function. To study the role that Asp-421-cleaved tau may play in compromising mitochondrial function, we used immortalized cortical neurons that inducibly express either a full-length form of tau (T4) or a tau isoform that has been truncated at Asp-421 (T4C3). The cells expressing T4C3 showed an increase in cell toxicity, as measured by lactate dehydrogenase release, compared with T4-expressing cells (12.Matthews-Roberson T.A. Quintanilla R.A. Ding H. Johnson G.V. Brain Res. 2008; 1234: 206-212Crossref PubMed Scopus (30) Google Scholar). In this study we found that mitochondria in the T4C3-expressing cells presented with an abnormal morphology, characterized by a decrease in mitochondrial length, suggestive of fragmentation. Furthermore, when both cell types were treated with thapsigargin to globally increase intracellular calcium levels, we found that mitochondria in cells expressing T4C3 showed diminished calcium buffering capacity and higher mitochondrial reactive oxygen species (ROS) levels. Mitochondrial fragmentation and mitochondrial membrane integrity damage were completely inhibited in T4C3 cells by pretreating with the calcineurin inhibitors cyclosporine A (CsA) or FK506, suggesting a role for this calcium-dependent phosphatase in these pathogenic processes. Our results indicate that the presence of Asp-421-cleaved tau in neurons may compromise the ability of mitochondria to function normally and ultimately contribute to the mitochondrial impairment and neuronal death observed in the AD brain. Immortalized cortical neurons (18.Bongarzone E.R. Foster L. Byravan S. Casaccia-Bonnefil P. Schonmann V. Campagnoni A.T. J. Neurosci. Res. 1998; 54: 309-319Crossref PubMed Scopus (19) Google Scholar) expressing inducible full-length (T4) or Asp-421-truncated (T4C3) tau were prepared as described previously (19.Krishnamurthy P.K. Johnson G.V. J. Biol. Chem. 2004; 279: 7893-7900Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Cells were cultured in Dulbecco's modified Eagle's medium with high glucose (Mediatech) and supplemented with 5% fetal bovine serum, 0.1% gentamicin, 4 mm glutamine, and 10 units/ml penicillin and 100 units/ml streptomycin at 33 °C. In these studies cells were treated with the tetracycline derivative doxycycline at a concentration of 2 μg/ml for 48 h to induce tau expression. After 48 h the induction media was removed from cells and replaced with Dulbecco's modified Eagle's medium/high glucose containing only 4 mm glutamine and 1% fetal bovine serum with 2 μg/ml doxycycline and the cells were moved to a 39 °C incubator and maintained under these conditions before treatment. Cells were grown on 35-mm dishes and loaded for 30 min (37 °C) with 5 μm Fluo-3 AM and 10 μm Rhod-2 AM in KRH-glucose buffer containing 0.02% pluronic acid. The fluorescence changes determined by Fluo-3 represent the cytoplasmic calcium changes (20.Quintanilla R.A. Muñoz F.J. Metcalfe M.J. Hitschfeld M. Olivares G. Godoy J.A. Inestrosa N.C. J. Biol. Chem. 2005; 280: 11615-11625Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and Rhod-2 fluorescence indicates calcium changes in the mitochondria (21.Zhu L.P. Yu X.D. Ling S. Brown R.A. Kuo T.H. Cell Calcium. 2000; 28: 107-117Crossref PubMed Scopus (84) Google Scholar, 22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23.Collins T.J. Lipp P. Berridge M.J. Bootman M.D. J. Biol. Chem. 2001; 276: 26411-26420Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). To estimate Rhod-2 fluorescence pattern in live mitochondria, we used MitoTracker GreenTM (MTG) to mark the mitochondria (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23.Collins T.J. Lipp P. Berridge M.J. Bootman M.D. J. Biol. Chem. 2001; 276: 26411-26420Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Cells were washed 3 times and left in KRH-glucose buffer for 10 min until cell fluorescence equilibrated. Fluorescence was imaged with a confocal laser scanning microscope (Leica TCS SP2) using a 40× water immersion lens, as described previously (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24.Milakovic T. Quintanilla R.A. Johnson G.V. J. Biol. Chem. 2006; 281: 34785-34795Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Images were acquired using a 488-nm argon laser to excite Fluo-3 fluorescence and a 563-nm He-Ne laser to excite Rhod-2 fluorescence. The signals were collected at 505–530 nm (Fluo-3) and at 590 nm (Rhod-2). Fluorescence background signal was subtracted from cell fluorescence measurements in every experiment. The fluorescence intensity variation was recorded from 10–20 cells on average per experiment. Estimation of fluorescence intensities were presented as the pseudoratio (ΔF/Fo), which was calculated using the formula ΔF/Fo = (F − Fbase)/(Fbase − B), where F is the measured fluorescence intensity of the indicator, Fbase is the fluorescence intensity before the stimulation, and B is the background signal determined from the average of areas adjacent to the cells (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Cells grown on 35-mm dishes were incubated with the fluorescent probe 2.7-DCF (10 μm) and TMRM (100 nm) for 30 min in KRH buffer supplemented with 5 mm glucose. Cells were washed 3 times and left in KRH-glucose buffer for 10 min until cell fluorescence equilibrated. Fluorescence was imaged with a confocal laser scanning microscope (Leica TCS SP2) using a 40× water immersion lens, as described previously (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Images were acquired using a 488-nm argon laser to excite 2.7-DCF fluorescence and a 563 nm He-Ne laser to excite TMRM fluorescence. Calculating fluorescence levels in a co-localized area of 2.7-DCF and TMRM fluorescent signals was used to estimate mitochondrial ROS production. The fluorescence background signal was subtracted from cell fluorescence measurements in every experiment. Images were quantified using Image-Pro Plus 6 software. Results in intensity units were expressed as the average of fluorescence signal (F) minus background fluorescence (Fo) in every image (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Mitochondrial superoxide levels were determined using MitoSOX Red (25.Cassina P. Cassina A. Pehar M. Castellanos R. Gandelman M. de Leon A. Robinson K.M. Mason R.P. Beckman J.S. Barbeito L. Radi R. J. Neurosci. 2008; 28: 4115-4122Crossref PubMed Scopus (242) Google Scholar) in conjunction with mitochondrial marker MTG. Cells grown on 35-mm dishes were incubated with 20 μm MTG and 200 nm MitoSOX Red for 20 min in KRH buffer supplemented with 5 mm glucose. Images were acquired using a 488-nm argon laser to excite MTG and a 563-nm He-Ne laser to excite MitoSOX Red fluorescence. Estimation of mitochondrial superoxide production was quantified using Image-Pro Plus 6 software. Results in intensity units were expressed as the average of fluorescence signal (F) minus background fluorescence (Fo) in every image. Mitochondrial membrane potential was determined using TMRM (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Before thapsigargin treatment, the cells were loaded for 30 min with TMRM (100 nm) in KRH-glucose buffer containing 0.02% pluronic acid, then washed and allowed to equilibrate for 20 min. Analyses were carried out using a confocal laser scanning microscope (Leica SP2). TMRM fluorescence was detected by exciting with a 563-nm He-Ne laser attenuated (30% laser power), and emission was collected at >570 nm. Signals from T4 cells and T4C3 cells treated with thapsigargin were compared using identical settings for laser power and detector sensitivity for each separate experiment. The images were collected with LCS Leica confocal software (Germany) and recorded as the mean TMRM fluorescence signal per live cell. TMRM fluorescence intensity was calculated as described above and is presented as the pseudoratio (ΔF/Fo) (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). To estimate mitochondrial integrity damage in live mitochondria, we used MTG dye (26.Pendergrass W. Wolf N. Poot M. Cytometry A. 2004; 61: 162-169Crossref PubMed Scopus (347) Google Scholar, 27.Kim G.J. Fiskum G.M. Morgan W.F. Cancer Res. 2006; 66: 10377-10383Crossref PubMed Scopus (134) Google Scholar). MTG accumulates in the lipophilic environment of live mitochondria, and it has been shown that the signal is independent of the mitochondrial membrane potential and oxidant status (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 27.Kim G.J. Fiskum G.M. Morgan W.F. Cancer Res. 2006; 66: 10377-10383Crossref PubMed Scopus (134) Google Scholar) (see supplemental data). To corroborate these observations, naïve cells were loaded with MTG and TMRM (mitochondrial potential indicator) for 30 min, and MTG/TMRM fluorescence levels were recorded in cells exposed to 10 μm FCCP for 20 min. Treatment of naïve cells with FCCP resulted in a pronounced decrease in the TMRM fluorescence levels after a few minutes of treatment (see supplementalFig. 1); in contrast, MTG fluorescence levels remained constant during the entire FCCP treatment period (see supplementalFig. 1). For our studies, cells were grown on 35-mm dishes and loaded for 30 min (37 °C) with 20 μm MTG in KRH-glucose buffer containing 0.02% pluronic acid. Fluorescence was imaged with a confocal laser scanning microscope as described previously (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Images were acquired using a 488-nm argon laser to excite MTG, and signals were collected at 505–530 nm. The fluorescence intensity variation was recorded from 5–10 cells on average per experiment. An estimation of MTG fluorescence intensity was calculated and is presented as a pseudoratio (ΔF/Fo). Estimation of mitochondrial length and frequency analysis is based on Rintoul et al. (28.Rintoul G.L. Filiano A.J. Brocard J.B. Kress G.J. Reynolds I.J. J. Neurosci. 2003; 23: 7881-7888Crossref PubMed Google Scholar). The mitochondrial length was calculated using the measured perimeter of identified objects, in live cortical cells preloaded with MTG for 30 min. Confocal and fluorescence images were taken in untreated and treated cells using a 40× water immersion objective with a 4× digital zoom on a SP2 Leica confocal microscope and a Zeiss Axiovert fluorescence microscope using a 63× oil objective. Mitochondrial length quantification was estimated using Image Pro 6 software (Media Cybernetics, MA). All data are expressed as the mean of at least three independent experiments ±S.E. unless otherwise stated. Statistical comparisons between treatment groups were performed using Student's t test. Immortalized cortical neurons were induced to express tau by treatment with, doxycycline (2 μg/ml) for 48 h (12.Matthews-Roberson T.A. Quintanilla R.A. Ding H. Johnson G.V. Brain Res. 2008; 1234: 206-212Crossref PubMed Scopus (30) Google Scholar, 29.Shelton S.B. Krishnamurthy P. Johnson G.V. J. Neurosci. Res. 2004; 76: 110-120Crossref PubMed Scopus (24) Google Scholar). In the absence of doxycycline, inducible cells express an almost undetectable amount of tau, as measured by Western blotting (29.Shelton S.B. Krishnamurthy P. Johnson G.V. J. Neurosci. Res. 2004; 76: 110-120Crossref PubMed Scopus (24) Google Scholar). However, treatment with doxycycline resulted in a robust increase in tau expression to levels approximately equivalent to concentrations seen in rat primary neuronal cortical cultures from E18 rat cortices (12.Matthews-Roberson T.A. Quintanilla R.A. Ding H. Johnson G.V. Brain Res. 2008; 1234: 206-212Crossref PubMed Scopus (30) Google Scholar). Considering that mitochondrial dysfunction occurs in the AD brain (30.Gibson G.E. Sheu K.F. Blass J.P. J. Neural Transm. 1998; 105: 855-870Crossref PubMed Scopus (307) Google Scholar, 31.Hirai K. Aliev G. Nunomura A. Fujioka H. Russell R.L. Atwood C.S. Johnson A.B. Kress Y. Vinters H.V. Tabaton M. Shimohama S. Cash A.D. Siedlak S.L. Harris P.L. Jones P.K. Petersen R.B. Perry G. Smith M.A. J. Neurosci. 2001; 21: 3017-3023Crossref PubMed Google Scholar), we investigated the possibility that T4C3 may sensitize cells to a loss of viability by compromising mitochondrial function. Mitochondria in T4 and T4C3 cells were labeled with the mitochondria-specific marker MTG to examine mitochondrial morphology in situ (Fig. 1). Nontransfected immortalized cortical neurons (referred to in the text as naïve cells) present with a mixture of tubular and rounded mitochondrial morphologies, and the mitochondria tend to be more aggregated (Fig. 1A). In contrast, mitochondria in cells expressing T4 were distributed throughout the cell body, with the expected tubular, rod-like morphology (Fig. 1A), whereas cells expressing T4C3 exhibited more rounded and apparently fragmented mitochondria (Fig. 1A). In fact, greater than 90% of mitochondria in cells expressing T4C3 were less than 2 μm in length (Fig. 1B, see bars). Quantification of the data revealed that mitochondria in cells expressing T4C3 showed more than a 2-fold decrease in mitochondrial length as compared with mitochondria in naïve cells and cells expressing T4 (Fig. 1C). To assess mitochondrial function in these cells, we measured basal mitochondrial ROS production and mitochondrial membrane potential. 2.7-DCF was used to measure ROS production (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 32.Fuenzalida K. Quintanilla R. Ramos P. Piderit D. Fuentealba R.A. Martinez G. Inestrosa N.C. Bronfman M. J. Biol. Chem. 2007; 282: 37006-37015Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) and TMRM was used to measure mitochondrial potential (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24.Milakovic T. Quintanilla R.A. Johnson G.V. J. Biol. Chem. 2006; 281: 34785-34795Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Cells were loaded simultaneously with 2.7-DCF and TMRM and subsequently mounted in a confocal microscope chamber for imaging. Fig. 2A shows representative confocal images of naïve, T4, and T4C3 cells loaded with 2.7-DCF and TMRM in basal conditions. T4C3 cells showed increased levels of mitochondrial ROS production in comparison with naive and T4 cells (Fig. 2A). T4 cells showed low mitochondrial ROS levels, indicating that under basal conditions expression of the T4 isoform did not increase oxidative stress levels in these cells (Fig. 2, A and B). Additionally, we measured basal mitochondrial membrane potential levels in the same cells using TMRM. Those studies showed that mitochondrial potential levels are not significantly altered in any cell type (Fig. 2, A and B). Quantitative analysis of mitochondrial ROS production and mitochondrial membrane potential is presented in Fig. 2B. To verify the effect of T4C3 expression on mitochondrial ROS production, as measured with 2.7-DCF, we used MitoSOX Red to determine mitochondrial superoxide levels in untreated cells. MitoSOX Red dye is used for mitochondrial superoxide determinations (25.Cassina P. Cassina A. Pehar M. Castellanos R. Gandelman M. de Leon A. Robinson K.M. Mason R.P. Beckman J.S. Barbeito L. Radi R. J. Neurosci. 2008; 28: 4115-4122Crossref PubMed Scopus (242) Google Scholar) in conjunction with a mitochondrial marker; in our experiments we used MTG (Fig. 1). T4C3 cells showed higher levels of mitochondrial superoxide as compared with T4 cells (Fig. 2C). Naïve and T4 cells showed similar mitochondrial superoxide levels in basal conditions (data not shown). These results suggest that T4C3 expression increased mitochondrial ROS and superoxide production, events that could impair mitochondrial function in immortalized cortical neurons. Due to the aberrant mitochondrial morphology and high levels of ROS observed in cells expressing T4C3, the calcium buffering ability of mitochondria in these cells was examined. Cells were treated with thapsigargin, which results in a global and transient increase in cytosolic calcium levels (22.Quintanilla R.A. Jin Y.N. Fuenzalida K. Bronfman M. Johnson G.V. J. Biol. Chem. 2008; 283: 25628-25637Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 33.Yu T. Robotham J.L. Yoon Y. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2653-2658Crossref PubMed Scopus (879) Google Scholar). This treatment allowed us to examine the ability of mitochondria in these cell models to effectively uptake and sequester calcium. We found that there was no significant difference in increases in cytosolic calcium levels between cells expressing T4 and T4C3 after 1 μm thapsigargin treatment (Fig. 3A), although the level of cytosolic calcium increase in T4C3 cells did trend higher. When mitochondrial calcium levels were measured, differences between T4 and T4C3 cells were apparent (Fig. 2B). Cells expressing T4 and loaded with Rhod2 AM (a mitochondrial calcium indicator) show
IntroductionPhysical disruption of the extracellular matrix influences the mechanical and chemical environment of intervertebral disc cells. We hypothesise that this can explain degenerative changes such as focal proteoglycan loss, impaired cell-matrix binding, cell clustering, and increased activity of matrix-degrading enzymes.MethodsDisc tissue samples were removed surgically from 11 patients (aged 34–75 yrs) who had a painful but non-herniated disc. Each sample was divided into a pair of specimens (approximately 5mm3), which were cultured at 37°C under 5% CO2. One of each pair was allowed to swell, while the other was restrained by a perspex ring. Live-cell imaging was performed with a wide field microscope for 36 hrs. Specimens were then sectioned at 5 and 30 μm for histology and immunofluorescence using a confocal microscope. Antibodies were used to recognise free integrin receptor α5β1, matrix metalloprotease MMP-1, and denatured collagen types I-III. Proteoglycan content of the medium, analysed usi...
Glycogen synthase kinase 3 (GSK3) is a widely expressed Ser/Thr protein kinase that phosphorylates numerous substrates. This large number of substrates requires precise and specific regulation of GSK3 activity, which is achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Members of the Wnt canonical pathway have been shown to influence GSK3 activity. Through a yeast two-hybrid screen, we identified the Wnt canonical pathway co-receptor protein low density lipoprotein receptor-related protein 6 (LRP6) as a GSK3-binding protein. The interaction between the C terminus of LRP6 and GSK3 was also confirmed by in vitro GST pull-down assays and in situ coimmunoprecipitation assays. In vitro assays using immunoprecipitated proteins demonstrated that the C terminus of LRP6 significantly attenuated the activity of GSK3β. In situ, LRP6 significantly decreased GSK3β-mediated phosphorylation of tau at both primed and unprimed sites. Finally, it was also demonstrated that GSK3β phosphorylates the PPP(S/T)P motifs in the C terminus of LRP6. This is the first identification of a direct interaction between LRP6 and GSK3, which results in an attenuation of GSK3 activity. Glycogen synthase kinase 3 (GSK3) is a widely expressed Ser/Thr protein kinase that phosphorylates numerous substrates. This large number of substrates requires precise and specific regulation of GSK3 activity, which is achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Members of the Wnt canonical pathway have been shown to influence GSK3 activity. Through a yeast two-hybrid screen, we identified the Wnt canonical pathway co-receptor protein low density lipoprotein receptor-related protein 6 (LRP6) as a GSK3-binding protein. The interaction between the C terminus of LRP6 and GSK3 was also confirmed by in vitro GST pull-down assays and in situ coimmunoprecipitation assays. In vitro assays using immunoprecipitated proteins demonstrated that the C terminus of LRP6 significantly attenuated the activity of GSK3β. In situ, LRP6 significantly decreased GSK3β-mediated phosphorylation of tau at both primed and unprimed sites. Finally, it was also demonstrated that GSK3β phosphorylates the PPP(S/T)P motifs in the C terminus of LRP6. This is the first identification of a direct interaction between LRP6 and GSK3, which results in an attenuation of GSK3 activity. Glycogen synthase kinase 3 (GSK3) 2The abbreviations used are: GSK3, glycogen synthase kinase 3; Fz, Frizzled; FRAT, frequently rearranged in advanced T-cell lymphoma; LDL, low density lipoprotein; LRP, LDL receptor-related protein; BSA, bovine serum albumin; CHO, Chinese hamster ovary; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; HA, hemagglutinin. is a widely expressed protein kinase with high expression in the brain, and specifically within neurons (for a review, see Ref. 1Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Crossref PubMed Scopus (1319) Google Scholar). GSK3 is a unique Ser/Thr protein kinase that phosphorylates both primed (target Ser/Thr is 4 amino acids N-terminal to a prephosphorylated Ser/Thr) and unprimed (target Ser/Thr is flanked by a Pro) substrates (for a review, see Ref. 2Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). A screen of a rat brain cDNA library revealed that GSK3 is encoded by two independent genes, GSK3α and GSK3β, with molecular masses of 51 and 47 kDa, respectively (3Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1161) Google Scholar). The two genes display 85% overall sequence identity, which is even higher in the catalytic domain (93%). In the brain, although GSK3α mRNA level is higher than GSK3β, GSK3β protein level is higher (4Lau K.F. Miller C.C. Anderton B.H. Shaw P.C. J. Pept. Res. 1999; 54: 85-91Crossref PubMed Scopus (104) Google Scholar). The poor relationship between transcription and translation in some tissues indicates that these two isoforms are subject to differential regulation, but little is known about the isoform-specific functions. More than 40 proteins have been reported to be phosphorylated by GSK3, including over a dozen transcription factors (reviewed in Ref. 2Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). This large number of substrates illustrates the great potential of GSK3 to affect many cellular functions and suggests that the activity of GSK3 must be carefully regulated by individual mechanisms for each substrate. Although the mechanisms regulating GSK3 are not fully understood, precise control appears to be achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins (reviewed in Ref. 2Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). Protein complexes that contain GSK3 are of major importance in regulating its actions. The best characterized of these complexes is involved in the Wnt canonical pathway, where GSK3-binding proteins control access to the GSK3 substrate, β-catenin, and generate a high degree of specificity in regulating the actions of GSK3. In the absence of Wnt signal, adenomatous polyposis coli, β-catenin, GSK3, casein kinase I (5Amit S. Hatzubai A. Birman Y. Andersen J.S. Ben-Shushan E. Mann M. Ben-Neriah Y. Alkalay I. Genes Dev. 2002; 16: 1066-1076Crossref PubMed Scopus (594) Google Scholar), and other proteins all bind to the scaffold protein axin to form a complex (6Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1105) Google Scholar). In this complex, β-catenin is phosphorylated by casein kinase I and subsequently by GSK3, which allows the β-transducing repeat-containing protein, an F-box protein in the E3 ubiquitin ligase complex, to bind and tag β-catenin for proteasome-mediated degradation (7Hart M. Concordet J.P. Lassot I. Albert I. del los Santos R. Durand H. Perret C. Rubinfeld B. Margottin F. Benarous R. Polakis P. Curr. Biol. 1999; 9: 207-210Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 8Li L. Yuan H. Weaver C.D. Mao J. Farr III, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (359) Google Scholar). When specific Wnts, such as Wnt 3a and Wnt 8, bind to the appropriate Frizzled (Fz) receptors, Dishevelled is activated; this, in concert with the GSK3-binding protein, frequently rearranged in advanced T-cell lymphoma (FRAT), facilitates disruption of the axin-based complex. This decreases the phosphorylation of β-catenin and results in β-catenin accumulation and activation (for a review, see Ref. 9Seidensticker M.J. Behrens J. Biochim. Biophys. Acta. 2000; 1495: 168-182Crossref PubMed Scopus (238) Google Scholar). This is a classical example of how GSK3-binding proteins can regulate the action of GSK3 toward individual substrates. In addition to the Fz receptors, Wnt canonical signaling requires single span transmembrane proteins that belong to a subfamily of low density lipoprotein (LDL) receptor-related proteins (LRPs): vertebrate LRP5 and -6 and their Drosophila ortholog Arrow (10Pinson K.I. Brennan J. Monkley S. Avery B.J. Skarnes W.C. Nature. 2000; 407: 535-538Crossref PubMed Scopus (902) Google Scholar, 11Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1102) Google Scholar, 12Wehrli M. Dougan S.T. Caldwell K. O'Keefe L. Schwartz S. Vaizel-Ohayon D. Schejter E. Tomlinson A. DiNardo S. Nature. 2000; 407: 527-530Crossref PubMed Scopus (726) Google Scholar). Unlike Fz, which is required for multiple Wnt pathways (13Veeman M.T. Axelrod J.D. Moon R.T. Dev. Cell. 2003; 5: 367-377Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar, 14Strutt D. Development. 2003; 130: 4501-4513Crossref PubMed Scopus (213) Google Scholar), Arrow and LRP5/6 appear to be specifically required for Wnt/β-catenin signaling (12Wehrli M. Dougan S.T. Caldwell K. O'Keefe L. Schwartz S. Vaizel-Ohayon D. Schejter E. Tomlinson A. DiNardo S. Nature. 2000; 407: 527-530Crossref PubMed Scopus (726) Google Scholar, 15Semenov M.V. Tamai K. Brott B.K. Kuhl M. Sokol S. He X. Curr. Biol. 2001; 11: 951-961Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar). Although the mechanism for LRP5/6 involvement in Wnt signaling has not been fully elucidated, recent studies suggest that the C terminus of LRP5/6 binds to axin and localizes axin and other bound proteins of the destruction complex to the plasma membrane. This is followed by axin degradation and dispersal of proteins of the destruction complex (16Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 17Mao J. Wang J. Liu B. Pan W. Farr III, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). Furthermore, a study demonstrated that a single PPP(S/T)P motif, which is reiterated five times in the LRP5/6/Arrow intracellular domain, can fully activate the Wnt pathway (16Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Wnt signaling stimulates and requires phosphorylation of the PPP(S/T)P motif (16Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar), although it has not been determined which kinases and/or phosphatases are involved in this modification. Although this is the prevailing model for LRP5/6/Arrow function, it does not explain the observation that the intracellular domain alone of LRP5/6 can activate and potentiate the Wnt 3a-induced LEF-1 signal, although it is not membrane-anchored (18Mi K. Johnson G.V. J. Cell. Biochem. 2005; 95: 328-338Crossref PubMed Scopus (54) Google Scholar). Members of the Wnt canonical pathway have also been shown to influence GSK3 activity toward substrates outside of the pathway, with the microtubule-associated protein tau being a notable example (reviewed in Ref. 2Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). Tau is phosphorylated by GSK3 at both unprimed and primed motifs. It has been demonstrated that primed phosphorylation of tau at Thr231 by GSK3 plays an essential role in decreasing the ability of tau to both bind and stabilize microtubules (19Cho J.H. Johnson G.V. J. Neurochem. 2004; 88: 349-358Crossref PubMed Scopus (208) Google Scholar). In contrast, GSK3-mediated phosphorylation of unprimed sites on tau (e.g. Ser396/404) may be more pathological and contribute to the formation of filamentous inclusions in Alzheimer disease and other neurodegenerative disorders (20Abraha A. Ghoshal N. Gamblin T.C. Cryns V. Berry R.W. Kuret J. Binder L.I. J. Cell Sci. 2000; 113: 3737-3745Crossref PubMed Google Scholar). Previously, our group demonstrated that axin potently inhibits tau phosphorylation by GSK3β, probably by sequestering GSK3β away from tau (21Stoothoff W.H. Bailey C.D. Mi K. Lin S.C. Johnson G.V. J. Neurochem. 2002; 83: 904-913Crossref PubMed Scopus (20) Google Scholar). Dishevelled has also been shown to decrease tau phosphorylation by GSK3β (22Mudher A. Chapman S. Richardson J. Asuni A. Gibb G. Pollard C. Killick R. Iqbal T. Raymond L. Varndell I. Sheppard P. Makoff A. Gower E. Soden P.E. Lewis P. Murphy M. Golde T.E. Rupniak H.T. Anderton B.H. Lovestone S. J. Neurosci. 2001; 21: 4987-4995Crossref PubMed Google Scholar), despite the fact that Dishevelled and GSK3β do not interact (8Li L. Yuan H. Weaver C.D. Mao J. Farr III, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (359) Google Scholar). Interestingly, studies showed that FRAT-1 inhibits GSK3-mediated tau phosphorylation (23Thomas G.M. Frame S. Goedert M. Nathke I. Polakis P. Cohen P. FEBS Lett. 1999; 458: 247-251Crossref PubMed Scopus (205) Google Scholar). Further, antisense-induced knock-down of Dickkopf-1, a negative modulator of the Wnt pathway that functions through binding to the co-receptor LRP6 (24Mao B. Wu W. Li Y. Hoppe D. Stannek P. Glinka A. Niehrs C. Nature. 2001; 411: 321-325Crossref PubMed Scopus (907) Google Scholar, 25Zorn A.M. Curr. Biol. 2001; 11: R592-R595Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), attenuates the increased tau phosphorylation in β-amyloid peptide-treated neurons (26Caricasole A. Copani A. Caraci F. Aronica E. Rozemuller A.J. Caruso A. Storto M. Gaviraghi G. Terstappen G.C. Nicoletti F. J. Neurosci. 2004; 24: 6021-6027Crossref PubMed Scopus (328) Google Scholar). However, whether LRP6 directly influences GSK3 activity has not been elucidated. Here we identify LRP6 as a GSK3-binding protein through a yeast two-hybrid assay. The interaction between LRP6 and GSK3 was confirmed by an in vitro GST pull-down assay and coimmunoprecipitation assays. The interaction of LRP6 with GSK3 significantly attenuated the activity of GSK3, and LRP6 was found to be a GSK3 substrate. Overall, this study suggests that LRP6 plays a unique and important role in and out of Wnt canonical signaling through regulating GSK3 activity. Cell Culture—Chinese hamster ovary (CHO) cells were maintained in Ham's F-12/Dulbecco's modified Eagle's medium (Cellgro) supplemented with 5% fetal bovine serum (Hyclone), 100 units/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), and 2 mm l-glutamine (Invitrogen). Cells were grown in a humidified atmosphere containing 5% CO2 at 37 °C. Constructs and Recombinant Protein Preparation—The tau construct that does not contain exons 2 and 3 (T4) was described previously (27Huber A.H. Nelson W.J. Weis W.I. Cell. 1997; 90: 871-882Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). Human GSK3β-HA was a gift from Dr. J. R. Woodgett. The plasmid encoding glutathione S-transferase (GST) fusion β-catenin in a bacterial expression vector was a gift from Dr. W. I. Weis (27Huber A.H. Nelson W.J. Weis W.I. Cell. 1997; 90: 871-882Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). To make the GSK3α-HA construct, human brain mRNA (Clontech) was used as the template for RT-PCR of GSK3α (Superscript™ one-step reverse transcription-PCR (Invitrogen)). The following primers were used in the reaction: forward, 5′-GCG AAT TCC GTT ATG AGC GGC GGC GGG CCT TCG-3′; reverse, 5′-GCG AAT TCC AGC ACA CTG GCG GCC GTA-3′. The PCR product was digested with EcoRI and subcloned into the same vector as GSK3β-HA. The cytosolic fragment of the C terminus of human LRP6 (LRP6-C3) was described previously (18Mi K. Johnson G.V. J. Cell. Biochem. 2005; 95: 328-338Crossref PubMed Scopus (54) Google Scholar). Using LRP6-C3 in pCMV5A (18Mi K. Johnson G.V. J. Cell. Biochem. 2005; 95: 328-338Crossref PubMed Scopus (54) Google Scholar) as a template, PCR was performed to subclone the LRP6-C3 fragment into corresponding vectors. The following primers were used for each PCR: forward (5′-CGC GGA TCC AGC ATG GGA CCA GCT TCT GTG CCT CTT GGT TAT GTG-3′) and reverse (5′-GCG CGT CGA CTT CTA GGA GGA GTC TGT ACA GGG AGA GGG TGG CGG TGG GT-3′) for subcloning LRP6-C3 into pGEX-6P-2 (Amersham Biosciences) (GST-LRP6-C3); forward (5′-CCG GAA TTC GGG ACC AGC TTC TGT GCC TCT TGG TTA TGT-3′) and reverse (5′-GGG ATC CTC AGG AGG AGT CTG TAC AGG GAG AG-3′) for subcloning LRP6-C3 into pEGFP-C1 (BD Biosciences) (GFP-LRP6-C3). The PCR products were digested and ligated into the corresponding cloning sites in vectors. For subcloning LRP6-C3 into pGBD-C1 vector (Clontech), LRP6-C3 was cut out from LRP6-C3 in pCMV5A by BamHI and SalI and ligated into the corresponding cloning sites in the vector. We also mutated all five PPP(S/T)P motifs in GST-LRP6-C3 to PPPAP to make GST-mut-LRP6-C3. The mutatgenesis reactions were carried out using either the GeneEditor™ in vitro site-directed mutagenesis system from Promega or the QuikChange™ site-directed mutagenesis kit from Stratagene. Plasmid integrity was verified using DNA sequencing analysis. GST-LRP6-C3, GST-mut-LRP6-C3, and GST-β-catenin were purified according to the manufacturer's protocol, with the exception that cleared bacterial lysates were incubated with preblocked Fast Flow Glutathione-Sepharose beads (Amersham Biosciences) for 1 h with rotation at 4 °C. For controls, GST was prepared in the same manner. For functional studies, GST-LRP6-C3 was treated on column with 60 units of Prescission protease (Amersham Biosciences) in cleavage buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, pH 7.0) overnight at 4 °C to remove the GST, and the LRP6-C3 was eluted. Where indicated, the fusion protein was left intact and eluted with glutathione buffer (20 mm Tris, 10 mm reduced glutathione, pH 8). After elution, GST-β-catenin, GST-LRP6-C3, GST-mut-LRP6-C3, and LRP6-C3 were dialyzed into phosphate-buffered saline. To determine the concentration of the purified protein, the samples were diluted in 2× SDS stop buffer (0.25 m Tris-HCl, pH 7.5, 2% SDS, 25 mm dithiothreitol, 5 mm EDTA, 5 mm EGTA, 10% glycerol, and 0.01% bromphenol blue) and electrophoresed on an 8% SDS-polyacrylamide gel, in addition to aliquots containing known amounts of bovine serum albumin (BSA) (Fisher). The gel was Coomassie-stained, destained, and dried. The gel was scanned and quantitated using UNSCANIT software (Silk Scientific, Inc.), and the amount of recombinant protein in the sample was determined as a function of the BSA standards. Yeast Two-hybrid Screening—The MATCHMAKER yeast two-hybrid system and an adult human brain cDNA library were purchased from Clontech. The bait for library screening was the intracellular domain of human LRP6 (residues 1416–1613), which was fused to the GAL4 DNA binding domain by subcloning into the pGBD-C1 vector. The adult human brain cDNA library (Clontech) in the pGAD-C2 vector, which has a transcription activation domain, was used in the screen. Prior to the screening, the LRP6-C3 construct in the pGBD-C1 vector was first transformed into the Y190 yeast hosts (Clontech), and subsequently the yeast Y190 cells carrying LRP6-C3 were transformed with the human brain cDNA library using the lithium acetate/single-stranded carrier DNA/polyethylene glycol protocol (28Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2097) Google Scholar). A small fraction of the transformation reaction was plated on synthetic dropout medium –Leu, –Trp, to estimate the total number of transformants. The remainder was plated on synthetic dropout medium –Leu, –Trp, –Ade to identify ADE2 reporter gene-positive colonies, which were subsequently transferred to synthetic dropout medium –Leu, –Trp, –His plates (Teknova) for further selection. Transient Transfections and Collection—CHO cells were transiently transfected using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's protocol. Forty-eight hours after transfection, cells were harvested in lysis buffer (0.5% Nonidet P-40, 150 mm NaCl, 10 mm Tris-Cl (pH 7.4), 1 mm EGTA, and 1 mm EDTA, 0.1 μm okadaic acid, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml each of aprotinin, leupeptin, and pepstatin). After sonication on ice for 10 s, cellular debris was removed by centrifugation. Protein concentrations of supernatants were then determined by the bicinchoninic acid assay (Pierce). Immunoblotting, Pull-down Assays, and Immunoprecipitation— Cells were collected as described above. After determination of protein concentrations, samples were diluted in 2× SDS buffer and electrophoresed on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked for 1 h with 5% nonfat dry milk in TBST (20 mm Tris-HCl, pH 7.6, 137 mm NaCl, 0.05% Tween 20) and then probed with antibody overnight at 4 °C. Blots were then rinsed with TBST, incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch) for 1 h at room temperature, rinsed with TBST, and developed with enhanced chemiluminescence (Amersham Biosciences). To evaluate the phosphorylation state of tau, the following antibodies were used: AT180 (Pierce/Endogen), which recognizes tau phosphorylated on Thr231 (29Goedert M. Jakes R. Crowther R.A. Cohen P. Vanmechelen E. Vandermeeren M. Cras P. Biochem. J. 1994; 301: 871-877Crossref PubMed Scopus (349) Google Scholar, 30Hoffmann R. Lee V.M. Leight S. Varga I. Otvos Jr., L. Biochemistry. 1997; 36: 8114-8124Crossref PubMed Scopus (151) Google Scholar) (numbering based on the longest human tau isoform (31Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1870) Google Scholar)) and PHF-1 (a gift from Dr. P. Davies), which recognizes tau phosphorylated on Ser396/404(32Greenberg S.G. Davies P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5827-5831Crossref PubMed Scopus (660) Google Scholar, 33Otvos Jr., L. Feiner L. Lang E. Szendrei G.I. Goedert M. Lee V.M. J. Neurosci. Res. 1994; 39: 669-673Crossref PubMed Scopus (409) Google Scholar). Total tau levels were determined using a combination of the phospho-independent monoclonal tau antibodies Tau 5 (a gift from Dr. L. Binder) (34Carmel G. Mager E.M. Binder L.I. Kuret J. J. Biol. Chem. 1996; 271: 32789-32795Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) and 5A6 (35Johnson G.V. Seubert P. Cox T.M. Motter R. Brown J.P. Galasko D. J. Neurochem. 1997; 68: 430-433Crossref PubMed Scopus (140) Google Scholar). Levels of transfected GSK3 and GFP-LRP6-C3 were detected using monoclonal HA (Transduction Laboratories) and monoclonal GFP (Roche Applied Science) antibodies, respectively. The in vitro pull-down assay was carried out as described previously (36Bafico A. Gazit A. Wu-Morgan S.S. Yaniv A. Aaronson S.A. Oncogene. 1998; 16: 2819-2825Crossref PubMed Scopus (71) Google Scholar). Equal amounts of GST- or GST-LRP6-C3-conjugated glutathione beads were incubated for 2 h with lysates from CHO cells transiently transfected with GSK3 and washed eight times with phosphate-buffered saline containing 350 mm NaCl (high salt) and 0.2% Triton X-100 prior to immunodetection by Western blotting. Alternatively, GST- or GST-LRP6-C3-conjugated beads were incubated with 200 ng of recombinant GSK3β (Upstate Biotechnology, Inc., Lake Placid, NY) and washed in the same manner. For immunoprecipitation, 100 μg of the total lysate was diluted to a final concentration of 0.5 mg/ml and subjected to immunoprecipitation at 4 °C with preconjugated monoclonal anti-GFP antibody (Roche Applied Science) M-280 sheep anti-mouse magnetic IgG beads (Dynal Biotech) for 2 h on a rotational shaker. Precipitates were washed at least five times and boiled in 2× SDS stop buffer. The supernatants were collected, resolved on 12% SDS-polyacrylamide gels, and blotted as described above. Five micrograms of total protein of each condition were probed to check expression levels. GSK3 Activity Assays—Cell lysates were incubated for 3 h at 4 °C with magnetic IgG beads, which were precoupled with 1.5 μg of the monoclonal anti-GSK3β antibody (BD Biosciences) (to immunoprecipitate endogenous GSK3β) or with 1 μg of the monoclonal anti-GFP antibody (to immunoprecipitate GFP-LRP6-C3 and the associated GSK3β). After washing IgG beads containing the immunomobilized GSK3β four times with phosphate-buffered saline containing 350 mm NaCl (high salt) and 0.5% Nonidet P-40, the beads were washed two additional times with GSK3 kinase buffer (20 mm Tris-Cl, pH 7.5, 5 mm MgCl2, and 1 mm dithiothreitol). GSK3β activity assays were performed as described using phosphoglycogen synthase peptide 2 or recombinant tau as substrates (37Stoothoff W.H. Cho J.H. McDonald R.P. Johnson G.V. J. Biol. Chem. 2005; 280: 270-276Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) with the exception that GSK3β activity was normalized to GSKβ levels in the sample, and the data were expressed as percentages of control (GFP alone). The same immunoprecipitation was performed to detect phospho-Ser9 GSK3β (Cell Signaling) in each condition. Recombinant in Vitro GSK3β Activity Assays—GST-β-catenin, purified as described above, was bound to washed glutathione beads for 4 h and washed twice with GSK3 kinase buffer. Sixty units of recombinant GSK3β (New England Biolabs) was preincubated with or without LRP6-C3 (cleaved from GST as described above) in the presence of 1 mg/ml BSA in GSK3 kinase buffer for 30 min at 4 °C. Preliminary experiments were conducted to determine optimal conditions for functional assay readouts, and a 40-fold molar excess of LRP6-C3 over GSK3β was used for all experiments. Following preincubation, reaction components were added to final concentrations of 125 μm ATP, 0.182 μCi/μl [γ-32P]ATP, and 1 mg/ml BSA in GSK3 kinase buffer. Washed GST-β-catenin bound to beads was then incubated in the reaction mix for 30 min at 30 °C. After removing the reaction supernatant and washing the beads twice with GSK3 kinase buffer, the beads were boiled with 30 μlof 2× SDS buffer, and the supernatants were resolved on an 8% polyacrylamide gel. The gels were dried and analyzed by phosphorimaging (Storm 840 (Amersham Biosciences) and ImageQuant software). GST-LRP6-C3 and GST-mut-LRP6-C3 in vitro phosphorylation by recombinant GSK3β was performed in a similar manner except that the purified GST-LRP6-C3 or GST-mut-LRP6-C3 was incubated with recombinant GSK3β alone without binding to the glutathione beads and without the addition of the BSA. For comparison, recombinant tau was incubated with recombinant GSK3β under identical conditions. Data Analysis—Data were analyzed using analysis of variance between individual groups. Values were considered significantly different when p was <0.05. Results were expressed as mean ± S.E. The C Terminus of LRP6 Directly Interacts with GSK3—Previous studies have shown that the Wnt canonical pathway co-receptors LRP5 and -6 can bind to axin and recruit it to the membrane, resulting in the degradation of axin and propagation of the Wnt signal (16Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 17Mao J. Wang J. Liu B. Pan W. Farr III, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). Although this is one mechanism by which LRP5/6 facilitates canonical Wnt signaling, our previous study showed that membrane targeting was not essential for the C terminus of LRP5/6 to constitutively activate the Wnt canonical pathway and synergistically increase LEF-1 activation in response to Wnt 3a (18Mi K. Johnson G.V. J. Cell. Biochem. 2005; 95: 328-338Crossref PubMed Scopus (54) Google Scholar). These findings suggested that LRP5/6 can facilitate the propagation of the Wnt signal through mechanisms other than recruitment of axin to the membrane. To determine which other proteins may be involved in the LRP5/6 signaling cascades, a yeast two-hybrid screen was carried out by using the intracellular domain of human LRP6 (LRP6-C3) as the bait, which was described previously (18Mi K. Johnson G.V. J. Cell. Biochem. 2005; 95: 328-338Crossref PubMed Scopus (54) Google Scholar). An adult human brain cDNA library was used in the screen. We screened ∼2.5 million clones and identified four clones as GSK3 by nucleotide sequencing analyses. GSK3 interacted strongly with LRP6-C3 in yeast (Fig. 1). We also identified nine other unique clones, some of which are in the Wnt canonical pathway. However, none of them were axin. To confirm the interaction between LRP6-C3 and GSK3 in vitro, we made a GST-fused LRP6-C3 construct. Purified GST and a GST-LRP6-C3 fusion protein were conjugated to glutathione beads. CHO cells were transfected with GSK3α or GSK3β, and lysates from the transfected cells were incubated with GST- or GST-LRP6-C3-conjugated beads. Both GSK3α and GSK3β were specifically pulled down by LRP6-C3 in vitro (Fig. 2). To rule out an indirect interaction between LRP6-C3 and GSK3, a pull-down assay was performed using recombinant GSK3β and recombinant GST-LRP6-C3 (Fig. 2C), which confirmed a direct protein-protein interaction. Further co-immunoprecipitation assays also revealed that LRP6-C3 interacts directly with both GSK3α and GSK3β (Fig. 3).FIGURE 2The C terminus of LRP6 interacts with both GSK3α and GSKβ in vitro. CHO cells were collected 48 h after transfection with the indicated HA-tagged GSK3 constructs. 500 μg of the GSK3α cell lysates (A) or 200 μg of the GSK3β cell lysate (B) was incubated with GST or GST-LRP6-C3 proteins conjugated to glutathione beads for 2 h. Both GSK3α and GSK3β were specifically pulled down by LRP6-C3. Aliquots of the lysates (3 μg for GSK3α (A) and 4 μg for GSK3β (B)) were run in the first lane of each blot to show input. Blots were probed with an anti-HA antibody. C, GST- or GST-LRP6-C3-conjugated beads were incubated with 200 ng of recombinant GSK3β (rGSK3β), washed, and blotted with a GSK3β antibody. D, Coomassie-stained gel showing the amount of GST and GST-LRP6-C3 protein in the assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3The C te
The microtubule-associated protein tau plays a central role in the pathogenesis of Alzheimer disease (AD) and abnormally accumulates as neurofibrillary tangles; therefore, the pathways by which tau is degraded have been examined extensively. In AD brain tau is abnormally truncated at Asp(421) (tauDeltaC), which increases its fibrillogenic properties and results in compromised neuronal function. Given the fact that the accumulation of tauDeltaC is a pathogenic process in AD, in this study we examined whether full-length tau and tauDeltaC are degraded through similar or different mechanisms. To this end a tetracycline-inducible model was used to show that tauDeltaC was degraded significantly faster than full-length tau (FL-tau). Pharmacological inhibition of the proteasome or autophagy pathways demonstrated that although FL-tau is degraded by the proteasome, tauDeltaC is cleared predominantly by macroautophagy. We also found that tauDeltaC binds C terminus of Hsp70-interacting protein more efficiently than tau. This interaction leads to an increased ubiquitylation of tauDeltaC in a reconstituted in vitro assay, but surprisingly, tau (full-length or truncated) was not ubiquitylated in situ. The finding that tauDeltaC and FL-tau are differentially processed by these degradation systems provides important insights for the development of therapeutic strategies, which are focused on modulating degradation systems to preferentially clear pathological forms of the proteins.
Since the last edition of ''The Effects of Nuclear Weapons'' in 1962 much new information has become available concerning nuclear weapon effects. This has come in part from the series of atmospheric tests, including several at very high altitudes, conducted in the Pacific Ocean area in 1962. In addition, laboratory studies, theoretical calculations, and computer simulations have provided a better understanding of the various effects. A new chapter has been added on the electromagnetic pulse. The chapter titles are as follows: general principles of nuclear explosions; descriptions of nuclear explosions; air blast phenomena in air and surface bursts; air blast loading; structural damage from air blast; shock effects of surface and subsurface bursts; thermal radiation and its effects; initial nuclear radiation; residual nuclear radiation and fallout; radio and radar effects; the electromagnetic pulse and its effects; and biological effects. (LTN)
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