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• W2022060333 abstract "Tau is a substrate of caspases, and caspase-cleaved tau has been detected in Alzheimer's disease brain but not in control brain. Furthermore, in vitro studies have revealed that caspase-cleaved tau is more fibrillogenic than full-length tau. Considering these previous findings, the purpose of this study was to determine how the caspase cleavage of tau affected tau function and aggregation in a cell model system. The effects of glycogen synthase kinase 3β (GSK3β), a well established tau kinase, on these processes also were examined. Tau or tau that had been truncated at Asp-421 to mimic caspase cleavage (Tau-D421) was transfected into cells with or without GSK3β, and phosphorylation, microtubule binding, and tau aggregation were examined. Tau-D421 was not as efficiently phosphorylated by GSK3β as full-length tau. Tau-D421 efficiently bound microtubules, and in contrast to the full-length tau, co-expression with GSK3β did not result in a reduction in the ability of Tau-D421 to bind microtubules. In the absence of GSK3β, neither Tau-D421 nor full-length tau formed Sarkosyl-insoluble inclusions. However, in the presence of GSK3β, Tau-D421, but not full-length tau, was present in the Sarkosyl-insoluble fraction and formed thioflavin-S-positive inclusions in the cell. Nonetheless, co-expression of GSK3β and Tau-D421 did not result in an enhancement of cell death. These data suggest that a combination of phosphorylation events and caspase activation contribute to the tau oligomerization process in Alzheimer's disease, with GSK3β-mediated tau phosphorylation preceding caspase cleavage. Tau is a substrate of caspases, and caspase-cleaved tau has been detected in Alzheimer's disease brain but not in control brain. Furthermore, in vitro studies have revealed that caspase-cleaved tau is more fibrillogenic than full-length tau. Considering these previous findings, the purpose of this study was to determine how the caspase cleavage of tau affected tau function and aggregation in a cell model system. The effects of glycogen synthase kinase 3β (GSK3β), a well established tau kinase, on these processes also were examined. Tau or tau that had been truncated at Asp-421 to mimic caspase cleavage (Tau-D421) was transfected into cells with or without GSK3β, and phosphorylation, microtubule binding, and tau aggregation were examined. Tau-D421 was not as efficiently phosphorylated by GSK3β as full-length tau. Tau-D421 efficiently bound microtubules, and in contrast to the full-length tau, co-expression with GSK3β did not result in a reduction in the ability of Tau-D421 to bind microtubules. In the absence of GSK3β, neither Tau-D421 nor full-length tau formed Sarkosyl-insoluble inclusions. However, in the presence of GSK3β, Tau-D421, but not full-length tau, was present in the Sarkosyl-insoluble fraction and formed thioflavin-S-positive inclusions in the cell. Nonetheless, co-expression of GSK3β and Tau-D421 did not result in an enhancement of cell death. These data suggest that a combination of phosphorylation events and caspase activation contribute to the tau oligomerization process in Alzheimer's disease, with GSK3β-mediated tau phosphorylation preceding caspase cleavage. Alzheimer's disease is a progressive neurodegenerative disorder characterized by neuronal cell loss, extracellular amyloid plaques, and intracellular neurofibrillary tangles. The relationship between these three hallmarks in the progression of Alzheimer's disease is not entirely clear; however, increased levels of Aβ42, which forms the amyloid plaques, is likely an initiating event (1Hardy J. J. Mol. Neurosci. 2003; 20: 203-206Crossref PubMed Scopus (60) Google Scholar, 2Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (10641) Google Scholar). Interestingly, treatment of cells with Aβ can result in the activation of caspases and apoptosis (3Ivins K.J. Thornton P.L. Rohn T.T. Cotman C.W. Neurobiol. Dis. 1999; 6: 440-449Crossref PubMed Scopus (178) Google Scholar, 4Marx J. Science. 2001; 293: 2192-2194Crossref PubMed Scopus (60) Google Scholar, 5Troy C.M. Rabacchi S.A. Xu Z. Maroney A.C. Connors T.J. Shelanski M.L. Greene L.A. J. Neurochem. 2001; 77: 157-164Crossref PubMed Google Scholar), and there is evidence for caspase activation in Alzheimer's disease brain (6Stadelmann C. Deckwerth T.L. Srinivasan A. Bancher C. Bruck W. Jellinger K. Lassmann H. Am. J. Pathol. 1999; 155: 1459-1466Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 7Rohn T.T. Head E. Nesse W.H. Cotman C.W. Cribbs D.H. Neurobiol. Dis. 2001; 8: 1006-1016Crossref PubMed Scopus (138) Google Scholar, 8Rohn T.T. Rissman R.A. Davis M.C. Kim Y.E. Cotman C.W. Head E. Neurobiol. Dis. 2002; 11: 341-354Crossref PubMed Scopus (207) Google Scholar). Although there is not a general consensus concerning the role of apoptosis in Alzheimer's disease (9Roth K.A. J. Neuropathol. Exp. Neurol. 2001; 60: 829-838Crossref PubMed Scopus (158) Google Scholar, 10Selznick L.A. Zheng T.S. Flavell R.A. Rakic P. Roth K.A. J. Neuropathol. Exp. Neurol. 2000; 59: 271-279Crossref PubMed Scopus (93) Google Scholar), increased levels of caspase-cleaved proteins are present in Alzheimer's disease brain (8Rohn T.T. Rissman R.A. Davis M.C. Kim Y.E. Cotman C.W. Head E. Neurobiol. Dis. 2002; 11: 341-354Crossref PubMed Scopus (207) Google Scholar, 11Gamblin 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 (663) Google Scholar, 12Rohn T.T. Head E. Su J.H. Anderson A.J. Bahr B.A. Cotman C.W. Cribbs D.H. Am. J. Pathol. 2001; 158: 189-198Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 13Rossiter J.P. Anderson L.L. Yang F. Cole G.M. Neuropathol. Appl. Neurobiol. 2000; 26: 342-346Crossref PubMed Scopus (39) Google Scholar). In addition to Aβ deposition, the accumulation of polymeric filaments of tau as intracellular neurofibrillary tangles is an essential feature of Alzheimer's disease brain. As a microtubule-associated protein, tau plays an essential role in maintaining microtubule stability; however, in Alzheimer's disease brain tau is aberrantly phosphorylated, and this results in an impairment of the normal functions of tau. Intriguingly, neurons from tau knockout mice are resistant to Aβ-induced toxicity, suggesting that tau plays a fundamental role in the pathogenic events that occur in Alzheimer's disease brain (14Rapoport M. Dawson H.N. Binder L.I. Vitek M.P. Ferreira A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6364-6369Crossref PubMed Scopus (671) Google Scholar). In addition to impairing tau function, the phosphorylation of key sites on tau may also result in an increase in the fibrillogenic properties of tau, which may represent a toxic gain of function (15Abraha 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). Although the protein kinases that phosphorylate tau in vivo have not been unequivocally identified, a protein kinase that is likely to play a key role in regulating the phosphorylation state of tau is glycogen synthase kinase 3β (GSK3β). 1The abbreviations used are: GSK3β, glycogen synthase kinase 3β; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid. 1The abbreviations used are: GSK3β, glycogen synthase kinase 3β; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid. In cell culture models there is clear evidence that tau is a substrate of GSK3β (16Johnson G.V. Hartigan J.A. J. Alzheimers Dis. 1999; 1: 329-351Crossref PubMed Scopus (93) Google Scholar, 17Johnson G.V. Bailey C.D. J. Alzheimers Dis. 2002; 4: 375-398Crossref PubMed Scopus (72) Google Scholar), and in mouse models increased expression of GSK3β results in increased tau phosphorylation (18Spittaels K. Van den Haute C. Van Dorpe J. Geerts H. Mercken M. Bruynseels K. Lasrado R. Vandezande K. Laenen I. Boon T. Van Lint J. Vandenheede J. Moechars D. Loos R. Van Leuven F. J. Biol. Chem. 2000; 275: 41340-41349Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 19Lucas J.J. Hernandez F. Gomez-Ramos P. Moran M.A. Hen R. Avila J. EMBO J. 2001; 20: 27-39Crossref PubMed Scopus (798) Google Scholar). Furthermore, there is evidence for increased activation of GSK3β in Alzheimer's disease brain (20Leroy K. Boutajangout A. Authelet M. Woodgett J.R. Anderton B.H. Brion J.P. Acta Neuropathol. (Berl.). 2002; 103: 91-99Crossref PubMed Scopus (165) Google Scholar, 21Eldar-Finkelman H. Trends Mol. Med. 2002; 8: 126-132Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 22Bhat R.V. Budd S.L. Neurosignals. 2002; 11: 251-261Crossref PubMed Scopus (86) Google Scholar). Recent studies demonstrate that caspase-cleaved tau is present in Alzheimer's disease but not control brain (8Rohn T.T. Rissman R.A. Davis M.C. Kim Y.E. Cotman C.W. Head E. Neurobiol. Dis. 2002; 11: 341-354Crossref PubMed Scopus (207) Google Scholar, 11Gamblin 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 (663) Google Scholar). Additionally in in vitro assays, caspase-cleaved tau (i.e. tau that had been truncated at Asp-421) is more fibrillogenic than full-length tau (11Gamblin 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 (663) Google Scholar). Considering these findings, the focus of the study was on determining how caspase cleavage of tau affects its ability to bind microtubules and aggregate in situ. Furthermore, the modulation of these processes by GSK3β was also examined. In these studies tau with exons 2, 3, and 10 (T4L) and tau without exons 2 and 3 (T4) were truncated at Asp-421 to mimic caspase cleavage (T4L-D421 and T4-D421, respectively). Intriguingly, both T4L-D421 and T4-D421 were not phosphorylated as efficiently by GSK3β as the full-length tau constructs both in situ and in vitro. The full-length and Asp-421-truncated tau constructs interacted with the cytoskeleton and bound microtubules to the same extent. In contrast, co-expression of GSK3β with full-length tau constructs resulted in the expected decrease in tau ability to bind microtubules, whereas the ability ofT4L-D421 and T4-D421 to bind microtubules was not affected by the presence of GSK3β. When either full-length or Asp-421-truncated tau was expressed alone, no tau was detected in the Sarkosyl-insoluble fraction. However, when GSK3β was co-expressed with either T4L-D421 or T4-D421, there was a robust increase in the presence of tau in the Sarkosyl-insoluble fraction, and the tau in this fraction was phosphorylated. Furthermore, inclusions that were tau and thioflavin-S-positive were detected in cells transfected with GSK3β and T4L-D421 or T4-D421. Even in the presence of GSK3β, full-length tau was not detected in the Sarkosyl-insoluble fraction. In this model system expression of T4L-D421 or T4-D421 tau did not result in an increase in cell death. These data demonstrate for the first time that a combination of phosphorylation events and caspase cleavage results in formation of Sarkosyl-insoluble tau aggregates in an in situ model system. Cell Culture—Chinese hamster ovary cells were grown in F-12 medium supplemented with 5% fetal bovine serum (Hyclone), 2 mm l-glutamine (Invitrogen), 10 units/ml penicillin (Invitrogen), and 100 units/ml streptomycin (Invitrogen). Cells were used at a confluency of 50-80% for all experiments. Plasmid Constructs—Preparation of the full-length tau construct containing exons 2, 3, and 10 (T4L) and the tau construct that does not contain exon 2 and 3 (T4) have been described previously (23Cho J.H. Johnson G.V. J. Biol. Chem. 2003; 278: 187-193Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 24Cho J.H. Johnson G.V. J. Neurochem. 2004; 88: 349-358Crossref PubMed Scopus (201) Google Scholar). To generate the T4L-D421 and T4-D421 constructs, T4 and T4L in pcDNA3.1(+) were used as templates, and PCR was performed using Turbo DNA polymerase (Stratagene, La Jolla, CA) with forward primer 5′-CGC GGA TCC GCG ATG GCT GAG CCC CGC CAG GAG TTC-3′ and reverse primer 5′-CTG CTC TAG AGC ATC AGT CTA CCA TGT CGA TGC TGC CGG TGG-3 to remove the last 20 amino acids of tau. Amplified fragments were digested with BamHI and XbaI and ligated into the same sites of pcDNA3.1(+). The integrity of T4L-D421 and T4-D421 was confirmed by sequence analysis. Hemagglutinin-GSK3β-S9A was constructed in pcDNA3.1(-) as described (23Cho J.H. Johnson G.V. J. Biol. Chem. 2003; 278: 187-193Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) and is referred to as GSK3β throughout the text. Transient Transfections—T4L, T4L-D421, T4, or T4-D421 with or without GSK3β was transiently transfected into Chinese hamster ovary cells using FuGENE 6 (Roche Applied Science) transfection reagent according to the manufacturer's protocol. Thirty-three hours after transfection, the cells were washed with ice-cold PBS and then collected and processed as described below for the different assays. Immunoblotting—Cells were collected in lysis buffer (150 mm NaCl, 10 mm Tris-HCl, 1 mm EGTA, 1 mm EDTA, 0.2 mm sodium vanadate, 0.5% Nonidet P-40) containing 1 mm phenylmethylsulfonyl fluoride, 0.1 μm okadaic acid, and a 10 μg/ml concentration each of aprotinin, leupeptin, and pepstatin. Lysates were sonicated on ice and centrifuged, and protein concentrations in the supernatants were determined using the bicinchoninic acid assay (Pierce). Samples were diluted with 2× SDS stop buffer (2% SDS, 5 mm EGTA, 5 mm EDTA, 25 mm dithiothreitol, 10% glycerol, 0.01% bromphenol blue, and 0.25 m Tris-Cl, pH 6.8) followed by incubation in a boiling water bath for 5 min. Equal amounts of protein from each sample were electrophoresed on 10% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with the indicated antibodies. The tau antibodies used in this study were: Tau5 (from Dr. L. Binder) and 5A6, which are phospho-independent tau antibodies (25Johnson G.V. Seubert P. Cox T.M. Motter R. Brown J.P. Galasko D. J. Neurochem. 1997; 68: 430-433Crossref PubMed Scopus (137) Google Scholar, 26Carmel G. Mager E.M. Binder L.I. Kuret J. J. Biol. Chem. 1996; 271: 32789-32795Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar); AT180 (Endogen), which recognizes tau when it is phosphorylated at Thr-231 (27Hoffmann R. Lee V.M. Leight S. Varga I. Otvos Jr., L. Biochemistry. 1997; 36: 8114-8124Crossref PubMed Scopus (150) Google Scholar); PHF1 (from Dr. P. Davies), which recognizes tau phosphorylated at Ser-396/404 (28Otvos Jr., L. Feiner L. Lang E. Szendrei G.I. Goedert M. Lee V.M. J. Neurosci. Res. 1994; 39: 669-673Crossref PubMed Scopus (404) Google Scholar); 12E8 (from Dr. P. Seubert), which recognizes tau phosphorylated at Ser-262 (29Seubert P. Mawal-Dewan M. Barbour R. Jakes R. Goedert M. Johnson G.V. Litersky J.M. Schenk D. Lieberburg I. Lee V.M.-Y. J. Biol. Chem. 1995; 270: 18917-18922Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar); Tau-1 (from Dr. L. Binder), which recognizes a dephosphorylated epitope between residues 189 and 207 (30Szendrei G.I. Lee V.M. Otvos Jr., L. J. Neurosci. Res. 1993; 34: 243-249Crossref PubMed Scopus (191) Google Scholar); and the following polyclonal antibodies from BIOSOURCE, which recognize the following specific phospho-tau epitopes: Ser(P)199, Thr(P)-205, Thr(P)-231, and Ser(P)-422. The monoclonal GSK3β antibody was purchased from Transduction Laboratories, and the monoclonal actin antibody was from Chemicon. After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Labs), the blots were developed using ECL (Amersham Biosciences). In Vitro GSK3β Kinase Assay—Full-length (T4L) and truncated tau (T4L-D421) His-tagged constructs in pGT100/D-TOPO (Invitrogen) were generous gifts from Dr. R. Guttmann. His-tagged T4L and T4L-D421 were expressed in BL21 (DE3) Escherichia coli and purified using nickel nitrilotriacetic acid-agarose (Qiagen) following the manufacturer's protocol. For the kinase reaction, recombinant GSK3β (New England BioLabs) was incubated with the recombinant tau (0.1 μg/μl) in 30 μl of GSK3β buffer (20 mm Tris, pH 7.5, 10 mm MgCl2, 5 mm dithiothreitol, 200 μm ATP, 1.4 μCi of [γ-32P] ATP (Amersham Biosciences) at 30 °C for 5 or 30 min. The reaction was terminated by the addition of 30 μl of 2× SDS stop buffer and incubation in a boiling water bath for 5 min followed by separation on a 10% SDS-polyacrylamide gel. The gels were vacuum-dried, exposed to a phosphoscreen overnight, and quantitated using a PhosphorImager (Amersham Biosciences). Separation into Soluble and Cytoskeletal-insoluble Fractions—Cell were rinsed once with warm PBS, scraped off the plate, collected by centrifugation, resuspended in 50 μl of prewarmed extraction buffer (0.1% (v/v) Triton X-100, 80 mm PIPES/KOH, 1.0 mm MgCl2, 2.0 mm EGTA, 0.1 mm EDTA, and 30% glycerol, pH 6.8) containing protease inhibitors (1.0 mm phenylmethylsulfonyl fluoride, 10 μg/ml each of leupeptin pepstatin, and aprotinin) and a phosphatase inhibitor (0.5 μm okadaic acid) and incubated for 8 min at 37 °C, and the pellet (cytoskeletal-insoluble) and supernatant (soluble) fractions were separated by centrifugation (15 min at 15,000 × g and 25 °C). Samples were diluted with 2× SDS stop buffer and incubated in a boiling water bath for 5 min. Equal amounts of pellet and supernatant factions were separated on 10% SDS-polyacrylamide gels, blotted, and processed for immunodetection with anti-tau and anti-actin antibodies. Microtubule Binding Assay—The microtubule binding assay was carried out as described previously with a few modifications (23Cho J.H. Johnson G.V. J. Biol. Chem. 2003; 278: 187-193Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Cells were collected and resuspended in 80 mm PIPES/KOH (pH 6.8)-containing protease and phosphatase inhibitors (see above). Cell suspensions were briefly sonicated on ice and incubated on ice for 15 min. Samples were brought to 1.5 mm EGTA and centrifuged at 21,000 × g for 30 min at 4 °C. Equal amounts of protein lysate were used in each binding assay. Supernatants were adjusted to 1 mm GTP and 10 μm taxol and incubated with taxol-stabilized microtubules prepared from rat brain (31Davis P.K. Johnson G.V. J. Biol. Chem. 1999; 274: 35686-35692Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) for 10 min at 37 °C. The mixtures were centrifuged through 100 μl of 30% (w/v) sucrose cushions in 80 mm PIPES/KOH containing 1 mm EGTA, 1 mm GTP, and 10 μm taxol at 100,000 × g for 30 min in an Airfuge at room temperature. The supernatant was collected, and the pellet was resuspended in 50 μlof2× SDS stop buffer. The supernatant was diluted with 2× SDS stop buffer, and both the pellet (bound) and supernatant (unbound) fractions were incubated in a boiling water bath for 5 min. Samples were separated by electrophoresis on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with the Tau5/5A6 antibodies. Sarkosyl Fractionation Assay—The assay was carried out as described previously with a few modifications (32DeTure M. Ko L.W. Easson C. Yen S.H. Am. J. Pathol. 2002; 161: 1711-1722Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The cells were scraped off the plate, pelleted by spinning at 180 × g for 10 min, resuspended in Mes buffer (20 mm Mes at pH 6.8, 80 mm NaCl, 1 mm MgCl2, 2 mm EGTA, 10 mm NaH2PO4, 20 mm NaF, 1 mm phenylmethylsulfonyl fluoride, and 10 μm leupeptin), and then homogenized with 30 strokes using a tissue grinder (Thomas Scientific, Swedesboro, NJ). The homogenates were centrifuged for 5 min at 500 × g to remove nuclei. Post-nuclear lysates were incubated at 4 °C for 20 min to depolymerize the microtubules and then centrifuged for 30 min at 200, 000 × g at 4 °C. Supernatants were collected, diluted with 2× SDS stop buffer, and incubated in a boiling water bath for 5 min (soluble fraction). The pellets were resuspended in Mes buffer containing 500 mm NaCl, 10% sucrose, and 1% Sarkosyl (Sigma). The samples were vortexed for 30 min at room temperature, incubated overnight at 4 °C, and centrifuged at 200,000 × g for 30 min at 4 °C. The supernatants were collected in 2× SDS stop buffer (Sarkosyl-soluble fraction), and pellets were resuspended in 2× SDS stop buffer (Sarkosyl-insoluble fraction) and incubated in a boiling water bath for 5 min. Samples were electrophoresed on 10% SDS-polyacrylamide gels and immunoblotted for tau as described above. Immunocytochemistry—These procedures were modified from previously described protocols (24Cho J.H. Johnson G.V. J. Neurochem. 2004; 88: 349-358Crossref PubMed Scopus (201) Google Scholar). The cells were transiently transfected individually with each tau construct in the absence or presence of GSK3β using Fugene-6 (Roche Applied Science) transfection reagent. Forty-eight hours after transfection the cells were rinsed with PBS and fixed at room temperature for 1 h in fixation buffer (2% paraformaldehyde, 0.2% glutaraldehyde, 1 mm MgCl2, 1 mm EGTA, 30%(v/v) glycerol in 70 mm PIPES, pH 6.8). Cells were washed 3 times with PBS then permeabilized with 0.2% Triton X-100 in PBS for 2 min. After rinsing with PBS, cells were incubated in NaBH4 (10 mg/ml in PBS) for 7 min and rinsed with PBS. The cells were blocked with 4% bovine serum albumin in PBS for 30 min to reduce background before staining. The cells were incubated for 1.5 h with a tau rabbit polyclonal antibody (Dako Corporation) diluted in 0.4% bovine serum albumin. After extensive rinsing with PBS, the cells were incubated with Texas Red dye-conjugated goat anti-rabbit IgG (Jackson Laboratories) to visualize tau. Cells were then washed extensively in PBS before being incubated in a thioflavin-S solution (0.005%) (Sigma) for 10 min. The cells were then washed 3 times in 70% ethanol and once in water before mounting (33Sun A. Nguyen X.V. Bing G. J. Histochem. Cytochem. 2002; 50: 463-472Crossref PubMed Scopus (100) Google Scholar). Cells were viewed with a Nikon Diaphot 200 epifluorescence microscope, and images were captured with a Digital spot camera (Diagnostic Instruments), digitally stored, and displayed using the accompanying software. Caspase Cleavage Assay—Chinese hamster ovary cells were transiently transfected with T4L alone or T4L and GSK3β and harvested in lysis buffer followed by incubation at 85 °C for 15 min to inactivate endogenous caspases. Protein concentrations were determined as described above, and 60 μg of total cell lysate was incubated in a final volume of 200 μl in a reaction mixture containing active recombinant caspase-3 (Calbiochem) (200 ng/ml), 20 mm PIPES, 100 mm NaCl, 10 mm dithiothreitol, 1 mm EDTA, 0.1% (w/v) CHAPS, and 10% sucrose, pH 7.2. Incubations were carried out for 0, 5, 30, and 60 min at room temperature, and the reactions were stopped by the addition of 2× stop buffer and incubation for 10 min in a boiling water bath. Aliquots from each sample were separated by electrophoresis on an 8% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with the Tau5/5A6 antibodies. Lactate Dehydrogenase Assay—The release of the intracellular enzyme lactate dehydrogenase into the media was used as a quantitative measurement of cell viability. The media and cell lysate samples were collected 48 h after transfection of the tau constructs and/or GSK3β to measure levels of cell viability. The measurement of lactate dehydrogenase was carried out as described previously (34Davis P.K. Dudek S.M. Johnson G.V. J. Neurochem. 1997; 68: 2338-2347Crossref PubMed Scopus (38) Google Scholar). Truncation of Tau at Asp-421 Tau Attenuates GSK3β-mediated Phosphorylation—To mimic caspase cleavage, tau constructs truncated at Asp-421 (i.e. the last 20 amino acids were deleted) were made from T4L (plus exons 2, 3, and 10) and T4 (minus exons 2 and 3, plus exon 10) (11Gamblin 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 (663) Google Scholar) and are referred to as T4L-D421 and T4-D421, respectively. To determine the expression and GSK3β-mediated phosphorylation of the constructs, cells were transfected with each tau construct alone or in combination with GSK3β. GSK3β-S9A (referred to as GSK3β in the text) was used in all experiments, because its activity cannot be inhibited by phosphorylation on Ser-9 (35Frame S. Cohen P. Biondi R.M. Mol. Cell. 2001; 7: 1321-1327Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). The GSK3β-mediated phosphorylation of T4L or T4 resulted in a significant decrease in electrophoretic mobility in comparison to their mobility when they were expressed alone, indicating that the phosphorylation state was increased (Fig. 1A, Total tau). In contrast to what was observed with the full-length tau constructs, the electrophoretic mobility of T4L-D421 or T4-D421 decreased only slightly in the presence of GSK3β (Fig. 1A, Total tau). The extent of phosphorylation of T4L and T4 constructs by GSK3β at both the PHF1 and AT180 epitopes was substantially greater than that of T4L-D421 or T4-D421 (Fig. 1A). This was not unexpected for the PHF1 epitope (Ser-396/404) (28Otvos Jr., L. Feiner L. Lang E. Szendrei G.I. Goedert M. Lee V.M. J. Neurosci. Res. 1994; 39: 669-673Crossref PubMed Scopus (404) Google Scholar) because it is in relative close proximity to the truncation site (Asp-421), and therefore, the ability of GSK3β to phosphorylate the site and/or the ability of the antibody to bind could be affected. However, it was unexpected for the AT180 epitope (Thr-231) (27Hoffmann R. Lee V.M. Leight S. Varga I. Otvos Jr., L. Biochemistry. 1997; 36: 8114-8124Crossref PubMed Scopus (150) Google Scholar), which is quite distal from the C terminus. In all cases GSK3β was expressed at similar levels (Fig. 1A). To further analyze the differential phosphorylation of the full-length and Asp-421-truncated tau constructs by GSK3β, immunoblot analyses were carried out with additional phospho-tau antibodies. T4L-D421 and T4-D421 were phosphorylated less efficiently than T4L or T4 by GSK3β at Thr-205 and Ser-199 (Fig. 1B). Reactivity with the 12E8 antibody was the same in the absence or presence of GSK3β for all constructs, which was expected as Ser-262 is not phosphorylated by GSK3β (36Godemann R. Biernat J. Mandelkow E. Mandelkow E.M. FEBS Lett. 1999; 454: 157-164Crossref PubMed Scopus (89) Google Scholar) (Fig. 1B). To further evaluate the phosphorylation of tau and the Asp-421-truncated tau constructs by GSK3β, an in vitro phosphorylation assay was carried out. Recombinant T4L and T4L-D421 were incubated with recombinant GSK3β in the presence of [γ-32P]ATP, and the extent of phosphorylation was analyzed. The representative autoradiograph shown in Fig. 1C clearly demonstrates that in vitro T4L was robustly phosphorylated by GSK3β, whereas under identical conditions T4L-D421 was phosphorylated by GSK3β to a much lesser extent. These data show that removal of the C terminus of tau at D421 diminishes the ability of tau to be phosphorylated by GSK3β. Phosphorylation of T4L-D421 and T4-D421 by GSK3β Does Not Result in a Decrease in Cytoskeletal Association—To investigate the effects of phosphorylation of either full-length tau or Asp-421-truncated tau on tau interaction with the cytoskeleton, cells were transfected with each tau construct in the absence or presence of GSK3β. The detergent-insoluble cytoskeletal component was separated from the soluble component to determine the amount of tau in the insoluble fraction relative to the amount of free tau. The insoluble fraction contains the cytoskeleton, which includes microtubules. Expression of all the tau constructs alone resulted in most of the tau being present in the insoluble fraction (Fig. 2), indicating that removal of the last 20 amino acids to mimic caspase cleavage does not alter tau interaction with the cytoskeleton. When full-length tau (T4L and T4) was co-transfected with GSK3β, tau shifted into the soluble fraction, and the amount of tau in the insoluble fraction decreased compared with when tau was expressed alone. In contrast, co-transfection of T4L-D421 or T4-D421 with GSK3β did not increase the amount of tau in the soluble fraction compared with the amount of tau in these fractions when the constructs were expressed alone (Fig. 2). Phosphorylation of T4L-D421 and T4-D421 by GSK3β Does Not Result in a Decrease in Microtubule Binding—To investigate how truncation of tau at Asp-421 affects the ability of tau to binding microtubules, a microtubule binding assay was used. Supernatants from cells transfected with each tau construct alone or in combination with GSK3β were incubated with taxol-stabilized microtubules, and the amount of tau bound to the microtubules in the pellet and the amount of tau that remained unbound were measured. In the absence of GSK3β all the tau constructs showed equivalent microtubule binding. As expected, phosphorylation of full-length tau (T4L or T4) by GSK3β reduced the affinity of tau for microtubules and increased the amount of tau in the supernatant (unbound fraction) (Fig. 3). Interestingly, incubation of the Asp-421-truncated forms, T4L-D421 or T4-D421, with GSK3β did not result in" @default.
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• W2022060333 title "Glycogen Synthase Kinase 3β Induces Caspase-cleaved Tau Aggregation in Situ" @default.
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