Stable Expression in Chinese Hamster Ovary Cells of Mutated Tau Genes Causing Frontotemporal Dementia and Parkinsonism Linked to Chromosome 17 (FTDP-17)
1999; Elsevier BV; Volume: 154; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65420-x
ISSN1525-2191
AutoresNobutaka Matsumura, Tsuneo Yamazaki, Yasuo Ihara,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoExtensive neuronal loss and aggregation of tau as cytoplasmic inclusions in neurons and glial cells in selected cortical and subcortical regions is the most striking characteristic of frontotemporal dementia and parkinsonism linked to chromosome 17, which is caused by exonic or intronic mutations in the tau gene. Here, we examined the effects of four exonic mutations in four-repeat tau using stably transfected Chinese hamster ovary cells. The proportion of polymerized tubulin was the largest in the P301L transfectant. G272V and P301L transfectants showed greater instability of microtubules in the presence of Colcemid than wild-type tau, V337M, or R406W transfectants. Thus no distinct phenotypes were shared by the mutant tau transfectants with regard to microtubule assembly and stability. Unexpectedly, R406W showed low and negligible levels of phosphorylation at Thr 231 and Ser 396, respectively, in the transfectant. This presents a sharp contrast to the observation that tau aggregates in R406W-affected brains are heavily phosphorylated at these two sites. This result suggests that hyperphosphorylation at these sites cannot occur in the tau R406W bound to microtubules, and thus that the hyperphosphorylated species of tau may be generated only after disruption of microtubules. Extensive neuronal loss and aggregation of tau as cytoplasmic inclusions in neurons and glial cells in selected cortical and subcortical regions is the most striking characteristic of frontotemporal dementia and parkinsonism linked to chromosome 17, which is caused by exonic or intronic mutations in the tau gene. Here, we examined the effects of four exonic mutations in four-repeat tau using stably transfected Chinese hamster ovary cells. The proportion of polymerized tubulin was the largest in the P301L transfectant. G272V and P301L transfectants showed greater instability of microtubules in the presence of Colcemid than wild-type tau, V337M, or R406W transfectants. Thus no distinct phenotypes were shared by the mutant tau transfectants with regard to microtubule assembly and stability. Unexpectedly, R406W showed low and negligible levels of phosphorylation at Thr 231 and Ser 396, respectively, in the transfectant. This presents a sharp contrast to the observation that tau aggregates in R406W-affected brains are heavily phosphorylated at these two sites. This result suggests that hyperphosphorylation at these sites cannot occur in the tau R406W bound to microtubules, and thus that the hyperphosphorylated species of tau may be generated only after disruption of microtubules. Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) constitutes a distinct group of neurodegenerative diseases characterized clinically by behavioral disorders and parkinsonism, genetically by autosomal dominant inheritance linked to chromosome 17, and neuropathologically by filamentous tau aggregates in neurons and glial cells, accompanied by neuronal loss.1Foster NL Wilhelmsen K Sima AAF Jones MZ D'Amato CJ Gilman S Spillantini MG Lynch T Mayeux RP Gaskell Jr, PC Hulette CM Pericak-Vance MA Welsh-Bohmer KA Dickson DW Heutik P Kros J van Swieten JC Arwert F Ghetti MB Murrell J Lannfelt L Hutton M Phelps CH Snyder DS Oliver E Ball MJ Cummings JL Miller BL Katzman R Reed L Schelper RL Lanska DJ Brun A Fink JK Khul DE Knopman DS Wszolek Z Miller CA Bird TD Lendon C Elechi C Frontotemporal dementia and parkinsonism linked to chromosome 17: a consensus conference.Ann Neurol. 1997; 41: 706-715Crossref PubMed Scopus (606) Google Scholar, 2Spillantini MG Bird TD Ghetti B Frontotemporal dementia and parkinsonism linked to chromosome 17: a new group of tauopathies.Brain Pathol. 1998; 8: 387-402Crossref PubMed Scopus (388) Google Scholar Further studies using the candidate gene approach demonstrated six missense mutations and four intronic mutations in the tau gene in FTDP-17 families.3Poorkaj P Thomas DB Wijisman E Nemens E Garruto RM Anderson L Andreadis A Wiederholt WC Raskind M Schellenberg GD Tau is a candidate gene for chromosome 17 frontotemporal dementia.Ann Neurol. 1998; 43: 815-825Crossref PubMed Scopus (1246) Google Scholar, 4Hutton M Lendon CL Rizzu P Baker M Froelich S Houlden H Pickering-Brown S Chakraverty S Isaacs A Grover A Hackett J Adamson J Lincoln S Dickson D Davies P Petersen RC Stevens M deGraaff E Wauters E van Baren J Hillebrand M Joosse M Kwon JM Nowotny P Che LK Norton J Morris JC Reed LA Trojanowski J Basun H Lannfelt L Neystat M Fahn S Dark F Tannenberg T Dodd PR Hayward N Kwok JBJ Schofield PR Andreadis A Snowden J Craufurd D Neary D Owen F Oostra BA Hardy J Goate A van Swieten J Mann D Lynch T Heutink P Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17.Nature. 1998; 393: 702-705Crossref PubMed Scopus (2967) Google Scholar, 5Spillantini MG Murrell JR Goedart M Farlow MR Klug A Ghetti B Mutation in the tau gene in familial multiple system tauopathy with presenile dementia.Proc Natl Acad Sci USA. 1998; 95: 7737-7741Crossref PubMed Scopus (1332) Google Scholar, 6Clark LN Poorkaj P Wszolek Z Geschwind DH Nasreddine ZS Miller B Li D Payami H Awert F Markkopoulou K Andreadis A Souza ID Lee VM-Y Reed L Trojanowski JQ Zhulareva V Bird T Schellenberg G Wilhelmsen KC Pathogenic implications of mutations in the tau gene in pallido-ponto-nigral degeneration and related neurodegenerative disorders linked to chromosome 17.Proc Natl Acad Sci USA. 1998; 95: 13103-13107Crossref PubMed Scopus (453) Google Scholar, 7Iijima M Tabira T Poorkaj P Schellenberg GD Trojanowski JQ Lee VM-Y Schmidt ML Takahashi K Nabika T Matsumoto T Yamashita Y Yoshioka S Ishino H A distinct familial presenile dementia with a novel missense mutation in the tau gene.Neuro Report. 1999; 10: 497-501PubMed Google Scholar These findings clearly indicate that these tau mutations are necessary and sufficient for the tau aggregation and neuronal loss observed in FTDP-17. Tau, a microtubule (MT)-associated protein (MAP), binds to tubulin at three or four repeats of 31 or 32 amino acids each, called the MT-binding domain, located in the carboxyl half of tau. In adult human brain, six isoforms containing three or four repeats are generated by alternative mRNA splicing from a single gene. Six known missense mutations in tau in FTDP-17 are located within or close to the MT-binding domain, and the intronic mutations are in the 5′ splice site of exon 10, and thought to be involved in the formation of a stem loop, resulting in an increased proportion of four-repeat tau.4Hutton M Lendon CL Rizzu P Baker M Froelich S Houlden H Pickering-Brown S Chakraverty S Isaacs A Grover A Hackett J Adamson J Lincoln S Dickson D Davies P Petersen RC Stevens M deGraaff E Wauters E van Baren J Hillebrand M Joosse M Kwon JM Nowotny P Che LK Norton J Morris JC Reed LA Trojanowski J Basun H Lannfelt L Neystat M Fahn S Dark F Tannenberg T Dodd PR Hayward N Kwok JBJ Schofield PR Andreadis A Snowden J Craufurd D Neary D Owen F Oostra BA Hardy J Goate A van Swieten J Mann D Lynch T Heutink P Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17.Nature. 1998; 393: 702-705Crossref PubMed Scopus (2967) Google Scholar, 5Spillantini MG Murrell JR Goedart M Farlow MR Klug A Ghetti B Mutation in the tau gene in familial multiple system tauopathy with presenile dementia.Proc Natl Acad Sci USA. 1998; 95: 7737-7741Crossref PubMed Scopus (1332) Google Scholar, 6Clark LN Poorkaj P Wszolek Z Geschwind DH Nasreddine ZS Miller B Li D Payami H Awert F Markkopoulou K Andreadis A Souza ID Lee VM-Y Reed L Trojanowski JQ Zhulareva V Bird T Schellenberg G Wilhelmsen KC Pathogenic implications of mutations in the tau gene in pallido-ponto-nigral degeneration and related neurodegenerative disorders linked to chromosome 17.Proc Natl Acad Sci USA. 1998; 95: 13103-13107Crossref PubMed Scopus (453) Google Scholar Regarding exonic mutations, it is tempting to speculate that these mutations in tau interfere with its interaction with tubulin and thus decrease its ability to promote tubulin assembly.3Poorkaj P Thomas DB Wijisman E Nemens E Garruto RM Anderson L Andreadis A Wiederholt WC Raskind M Schellenberg GD Tau is a candidate gene for chromosome 17 frontotemporal dementia.Ann Neurol. 1998; 43: 815-825Crossref PubMed Scopus (1246) Google Scholar, 4Hutton M Lendon CL Rizzu P Baker M Froelich S Houlden H Pickering-Brown S Chakraverty S Isaacs A Grover A Hackett J Adamson J Lincoln S Dickson D Davies P Petersen RC Stevens M deGraaff E Wauters E van Baren J Hillebrand M Joosse M Kwon JM Nowotny P Che LK Norton J Morris JC Reed LA Trojanowski J Basun H Lannfelt L Neystat M Fahn S Dark F Tannenberg T Dodd PR Hayward N Kwok JBJ Schofield PR Andreadis A Snowden J Craufurd D Neary D Owen F Oostra BA Hardy J Goate A van Swieten J Mann D Lynch T Heutink P Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17.Nature. 1998; 393: 702-705Crossref PubMed Scopus (2967) Google Scholar Along this line of investigation, two groups recently reported independently that the exonic mutations cause significant defects in MT assembly in the cell-free system.8Hasegawa M Smith MJ Goedert M Tau proteins with FTDP-17 mutations have a reduced ability to promote microtubule assembly.FEBS Lett. 1998; 437: 207-210Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 9Hong M Zhukareva V Vogelsberg-Ragaglia V Wszolek Z Reed L Miller BI Geschwind DH Bird TD Mckeel D Goate A Morris JC Wilhelmsen KC Schellenberg GD Trojanowski JQ Lee VM-Y Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17.Science. 1998; 282: 1914-1917Crossref PubMed Scopus (832) Google Scholar However, acute depletion of tau was reported to have no effects on MT dynamics in cultured neurons10Tint I Slaughter T Fischer I Black MM Acute inactivation of tau has no effect on dynamics of microtubules in growing axons of cultured sympathetic neurons.J Neurosci. 1998; 18: 8660-8673Crossref PubMed Google Scholar and, further, tau-knockout mice showed remarkably subtle effects on neurogenesis and neuronal organization.11Harada A Oguchi K Okabe S Kuno J Terada S Ohshima T Sato-Yoshitake R Takei Y Noda T Hirokawa N Altered microtubule organization in small-calibre axons of mice lacking tau protein.Nature. 1994; 369: 488-491Crossref PubMed Scopus (600) Google Scholar These results suggest that the tau mutation effects on MT assembly found in the cell-free system may not always be problematic within the cells or in vivo. Accordingly, it is too early to speculate about the pathogenesis of FTDP-17 based on the results from the cell-free system alone. Furthermore, the effects of phosphorylation, which is known to affect the ability of tau to promote MT assembly, cannot be assessed by those cell-free experiments using Escherichia coli-produced tau.8Hasegawa M Smith MJ Goedert M Tau proteins with FTDP-17 mutations have a reduced ability to promote microtubule assembly.FEBS Lett. 1998; 437: 207-210Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 9Hong M Zhukareva V Vogelsberg-Ragaglia V Wszolek Z Reed L Miller BI Geschwind DH Bird TD Mckeel D Goate A Morris JC Wilhelmsen KC Schellenberg GD Trojanowski JQ Lee VM-Y Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17.Science. 1998; 282: 1914-1917Crossref PubMed Scopus (832) Google Scholar Thus, we have established Chinese hamster ovary (CHO) cells stably transfected with wild-type and exon-mutated tau cDNA to investigate the effects of the mutations within the cell. The following antibodies were used: rat monoclonal antibody to tyrosinated α-tubulin, YL1/2 (Harlan Sela-Lab, Hillcrest, UK); mouse monoclonal antibody to α-tubulin, N356 (Amersham Pharmacia Biotech, Airlington Heights, IL); mouse monoclonal antibody to hamster lysosome-associated membrane protein 2 (LAMP2), UH3 (Developmental Studies Hybridoma Bank, Iowa City, IA); and mouse monoclonal and rabbit polyclonal antibodies to tau, tau 1 (Roche Diagnostics, Tokyo),12Liu W-K Moore WT Williams RT Hall FL Yen S-H Application of synthetic phospho- and unphospho-peptides to identify phosphorylation sites in a subregion of the tau molecule, which is modified in Alzheimer's disease.J Neurosci Res. 1993; 34: 371-376Crossref PubMed Scopus (62) Google Scholar, 13Szendrei GI Lee VM-Y Otvos Jr, L Recognition of the minimal epitope of monoclonal antibody tau-1 depends upon the presence of phosphate group but not its location.J Neurosci Res. 1993; 34: 243-249Crossref PubMed Scopus (195) Google Scholar AT8 (Innogenetics, Kapelanielaan, Belgium),14Goedert M Jakes R Vanmechelen E Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205.Neurosci Lett. 1995; 189: 167-169Crossref PubMed Scopus (485) Google Scholar 5E2,15Kosik KS Orecchio LD Binder L Trojanowski JQ Lee VM Lee G Epitopes that span the tau molecule are shared with paired helical filaments.Neuron. 1988; 1: 817-825Abstract Full Text PDF PubMed Scopus (440) Google Scholar M4 and C5,16Hasegawa M Watanabe A Takio K Suzuki M Arai T Titani K Ihara Y Characterization of two distinct monoclonal antibodies to paired helical filaments (PHF): further evidence for fetal-type phosphorylation of the tau in PHF.J Neurochem. 1993; 60: 2068-2077Crossref PubMed Scopus (105) Google Scholar, 17Hasegawa M Jakes R Crowther RA Lee VM-Y Ihara Y Goedert M Characterization of mA6 AP422, a novel phosphorylation-dependent monoclonalantibody against tau protein.FEBS Lett. 1996; 384: 25-30Abstract Full Text PDF PubMed Scopus (150) Google Scholar PHF 1,18Otvos Jr, L Feiner L Lang E Szendrei GI Goedert M Lee VM Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404.J Neurosci Res. 1994; 39: 669-673Crossref PubMed Scopus (411) Google Scholar and AP422.19Morishima-Kawashima M Hasegawa M Takio K Suzuki M Titani K Ihara Y Proline-directed and non-proline-directed phosphorylation of PHF-tau.J Biol Chem. 1995; 270: 823-829Crossref PubMed Scopus (533) Google Scholar MitoTracker Red was purchased from Molecular Probes, Inc. (Eugene, OR). Unique EcoRI and BglII sites were introduced into the 5′ and 3′ noncoding regions, respectively, of four-repeat (0N4R) tau cDNAs without exons 2 and 3, using polymerase chain reaction. The EcoRI-BglII fragment was isolated from the amplified product and ligated with pCXN20Niwa H Yamamura K Miyazaki J Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 192-200Google Scholar digested with the same endonucleases. The resultant recombinant plasmid, pCXN-4Rtau-wild-type, was used for further studies. To generate tau cDNA harboring mutations (G272V, P301L, V337M, and R406W, numbered according to the longest human tau isoform),21Goedert M Spillantini MG Jakes R Rutherford D Crowther RA Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease.Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1912) Google Scholar the polymerase chain reaction/oligonucleotide primer-directed mutagenesis strategy was used. The entire nucleotide sequence was determined by the dideoxynucleotide termination method using a DNA sequencer (model 4000L; LI-Cor, Lincoln, NE). The resultant amplified fragment was digested with the appropriate endonucleases and then inserted into the same sites of pCXN. The derivatives were pCXN-4Rtau G272V, P301L, V337M, and R406W. Vector alone, vectors containing 4R wild-type tau, and mutant tau were transfected into CHO cells using Lipofectamine (Life Technologies, Rockville, MD) according to the manufacturer's instructions. Clones that survived in G418 (Wako Pure Chemical, Tokyo; 0.4 mg/ml) were isolated and maintained at 37°C in 5% CO2 in Ham's F-12 medium (Life Technologies) supplemented with 10% fetal bovine serum containing G418 (0.1 mg/ml). Cultured cells grown in 10-cm dishes were scraped into ice-cold Tris-buffered saline (50 mmol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl) with various protease inhibitors as described before.22Gu T Oyama F Ihara Y τ is widely expressed in rat tissues.J Neurochem. 1996; 67: 1235-1244Crossref PubMed Scopus (173) Google Scholar The homogenates were solubilized with sodium dodecyl sulfate and the protein concentration in each homogenate was determined by bicinchoninic acid protein assay (Pierce, Rockford, IL). For quantitative blotting, the homogenates containing the same protein amounts were spun at 5000 rpm for 5 minutes and resultant supernatants were treated by heat. The heat-stable fractions containing tau were concentrated with saturated ammonium sulfate.22Gu T Oyama F Ihara Y τ is widely expressed in rat tissues.J Neurochem. 1996; 67: 1235-1244Crossref PubMed Scopus (173) Google Scholar The alkaline phosphatase treatment was performed essentially as described elsewhere.22Gu T Oyama F Ihara Y τ is widely expressed in rat tissues.J Neurochem. 1996; 67: 1235-1244Crossref PubMed Scopus (173) Google Scholar Aliquots of protein were subjected to Western blotting with tau 1 or N356. The expression levels of tau and tubulin in each of the transfectants were quantitated using a GS700 Imaging Densitometer (Bio-Rad, Hercules, CA) using authentic tau or tubulin within a linear range, and particular cell sublines containing similar levels of tau (< mean ± 100%) were selected for the following studies. Transfected cells from each confluent 15-cm culture dish were fractionated as previously described.23Brandt R L 130 gar J Lee G Interaction of tau with the neutral plasma membrane mediated by tau's amino-terminal projection domain.J Cell Biol. 1995; 131: 1327-1340Crossref PubMed Scopus (538) Google Scholar Separation of polymerized tubulin and free tubulin was performed according to the protocol designed by Merrick et al.24Merrick SE Trojanowski JQ Lee VM Selective destruction of stable microtubules and axons by inhibitors of protein serine/threonine phosphatases in cultured human neurons.J Neurosci. 1997; 17: 5726-5737Crossref PubMed Google Scholar Tau, bound or unbound to MT in cultured cells, was extracted as described.25Bramblett GT Goedert M Jakes R Merrick SE Trojanowski JQ Lee VM-Y Abnormal tau phosphorylation at Ser396 in Alzheimer's disease recapitulates development and contributes to reduce microtubule binding.Neuron. 1993; 10: 1089-1099Abstract Full Text PDF PubMed Scopus (770) Google Scholar Cells were fixed with cold methanol or 4% paraformaldehyde in MT-stabilizing buffer (80 mmol/L PIPES, pH 6.9, 1 mmol/L MgCl2, 1 mmol/L EGTA), followed by incubation in 0.3% Triton X-100 in phosphate-buffered saline for 5 minutes and incubation with antibodies essentially as described.26Lee G Rook SL Expression of tau protein in non-neuronal cells: microtubule binding and stabilization.J Cell Sci. 1992; 102: 227-237Crossref PubMed Google Scholar For the secondary antibodies, donkey anti-mouse IgG conjugated with rhodamine or fluorescein and donkey anti-rat IgG conjugated with rhodamine were used (Jackson ImmunoResearch Lab, West Grove, PA). Anti-mouse and anti-rat antibodies were preabsorbed with rat and mouse nonimmune serum, respectively, to remove cross-reactivity. To visualize mitochondria, MitoTracker Red was added at a concentration of 300 nmol/L 30 minutes before fixation. In some experiments, cells were treated with 0.1 μg/ml Colcemid (Wako Pure Chemical) for 2 hours or 20 μmol/L cytochalasin B (Wako Pure Chemical) for 20 minutes. Labeled cells were examined under a Zeiss Axioskop microscope (Carl Zeiss Co., Tokyo). First, we examined by immunocytochemistry the intracellular distribution of tau in transfected CHO cells that do not normally express tau. When fixed with paraformaldehyde, the immunoreactivity of tau 1 was distributed diffusely in the cytoplasm, suggesting that a significant proportion of tau is unbound to MTs (Figure 1, a and b), an observation consistent with a previous report.25Bramblett GT Goedert M Jakes R Merrick SE Trojanowski JQ Lee VM-Y Abnormal tau phosphorylation at Ser396 in Alzheimer's disease recapitulates development and contributes to reduce microtubule binding.Neuron. 1993; 10: 1089-1099Abstract Full Text PDF PubMed Scopus (770) Google Scholar To see the tau bound to MTs, the cells were fixed with cold methanol to wash away cytosolic tau.26Lee G Rook SL Expression of tau protein in non-neuronal cells: microtubule binding and stabilization.J Cell Sci. 1992; 102: 227-237Crossref PubMed Google Scholar With this fixation protocol, tau 1 immunoreactivity was almost completely colocalized with MTs (Figure 1, c and d). In many cells, MT bundles were observable after tau transfection (Figure 1, a–h) 27Kanai Y Takemura R Oshima T Mori H Ihara Y Yanagisawa M Masaki T Hirokawa N Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA.J Cell Biol. 1989; 109: 1173-1184Crossref PubMed Scopus (298) Google Scholar but no distinct difference in the incidence and localization of MT bundles was found among transfectants (Figure 1, c and e-h). As expected, mock-transfected cells were completely negative for tau (data not shown). One of the physiological roles of tau within the cell is thought to be the stabilization of MTs.27Kanai Y Takemura R Oshima T Mori H Ihara Y Yanagisawa M Masaki T Hirokawa N Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA.J Cell Biol. 1989; 109: 1173-1184Crossref PubMed Scopus (298) Google Scholar It is possible that mutant tau molecules stabilize MTs to a lesser extent in the cell. To examine the possibility, each transfectant was treated with Colcemid, a MT-depolymerizing drug.28Barlow S Gonzalez-Garay ML West RR Olmsted JB Cabral F Stable expression of heterologus MT-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in MT organization.J Cell Biol. 1994; 126: 1017-1029Crossref PubMed Scopus (72) Google Scholar Treatment with 0.1 mg/ml Colcemid for 2 hours completely depolymerized MTs in mock-transfected cells (Figure 1m). With the same treatment, a certain population of wild-type tau, tauV337M, and R406W (hereafter referred to as WT, V337M, and R406W, respectively) transfectants still showed MT staining (Figure 1, n, q, and r), whereas such MT staining was very rare in tauG272V and P301L (hereafter referred to as G272V and P301L, respectively) transfectants (Figure 1, o and p). To quantitate these results, the number of MT-positive cells per 1000 cells was counted under a fluorescence microscope. The proportion of MT-positive cells was 46% for WT, 48% for V337M, 37% for R406W, 14% for G272V, and 6.3% for P301L transfectants. Thus, G272V and P301L transfectants showed significantly greater vulnerability to Colcemide than WT transfectant (P < 0.01; Student's t-test). This experiment was repeated four times with similar results. It was recently reported that overexpression of tau affects kinesin-dependent trafficking of intracellular organelles.29Ebneth A Godemann R Stamer K Illenberger S Trinczek B Mandelkow E-M Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease.J Cell Biol. 1998; 143: 777-794Crossref PubMed Scopus (674) Google Scholar To examine whether the mutations alter the intracellular distribution of organelles, mitochondria and lysosomes were labeled by MitoTracker Red and UH3 (anti-LAMP2 antibody), respectively. In mock-transfected cells, both organelles were distributed throughout the cytoplasm (Figure 1, i and k). In contrast, in tau-transfected cells, mitochondria were confined to the perinuclear area (Figure 1j), while lysosomes were scattered throughout the cytoplasm (Figure 1l). These results were consistent with the previously reported result,29Ebneth A Godemann R Stamer K Illenberger S Trinczek B Mandelkow E-M Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease.J Cell Biol. 1998; 143: 777-794Crossref PubMed Scopus (674) Google Scholar but again, no difference was observed between wild-type and mutant transfectants. It is well known that disruption of the subcortical microfilament network induces process formation in cultured cells.30Edson K Weisshaar B Matus A Actin depolymerisation induces process formation on MAP2-ransfected non-neuronal cells.Development. 1993; 117: 689-700PubMed Google Scholar Because tau also binds to actin in several cell lines,31Cross D Vial C Maccioni RB A tau-like protein interacts with stress fibers, and microtubules in human, and rodent cell lines.J Cell Sci. 1993; 105: 51-60PubMed Google Scholar we examined whether tau mutants had any effect on process formation. Treatment of the transfected CHO cells with cytochalasin B induced the outgrowth of MT-containing processes from the cell surface. Although mock-transfected cells tended to give off a greater number of processes than WT and mutant tau-transfected cells, there were no significant differences in the number and length of processes among the transfectants (data not shown). Quantitation of polymerized tubulin in each transfectant provided rather conflicting data. The expression levels of tubulin in tau transfectants appeared to be slightly increased compared to that in mock transfectants (Figure 2, Figure 3). The proportion of polymerized tubulin was significantly decreased in WT, V337M, and R406W transfectants compared to that in mock transfectants (P < 0.05, Mann-Whitney U test) (Figure 2). Furthermore, tubulin in G272V and P301L transfectants was polymerized to a greater extent than that in WT transfectants (P < 0.05, Mann-Whitney U test). These results apparently contradict the results on the ability of the mutants to promote MT assembly in the cell-free system.8Hasegawa M Smith MJ Goedert M Tau proteins with FTDP-17 mutations have a reduced ability to promote microtubule assembly.FEBS Lett. 1998; 437: 207-210Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 9Hong M Zhukareva V Vogelsberg-Ragaglia V Wszolek Z Reed L Miller BI Geschwind DH Bird TD Mckeel D Goate A Morris JC Wilhelmsen KC Schellenberg GD Trojanowski JQ Lee VM-Y Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17.Science. 1998; 282: 1914-1917Crossref PubMed Scopus (832) Google Scholar Consistent with these results, tau appeared to be much more abundant in the supernatant of each transfectant homogenate (Figure 2 and legend). This probably agrees with the above-described immunocytochemical observations of tau transfectants showing diffuse cytoplasmic staining for tau when fixed with paraformaldehyde.Figure 3Tubulin and tau in the stable transfectants. The same amount of protein was subjected to Western blotting with N356 (A), tau 1 (B and C), 5E2 (D), AT8 (E), M4 (F), C5 (G), or PHF1 (H). A: The levels of α-tubulin in all transfectants were slightly increased as compared with that in the mock transfectant. B: After alkaline phosphatase treatment, the mobility of all tau species corresponded to that of the fastest one indicated by the lowest bar on the right. C: Without alkaline phosphatase treatment, there are three reactive bands on the blot. In WT, G272V, P301L, and V337M-transfected cells, the top band was the most intense, followed by the second band, and the lowest band was very faint. In contrast, in the R406W transfectant, the lowest band was strongest. D: Labeling similar to that in C was observed with 5E2. E: No immunoreactivity for AT8 was found. F: WT, G272, P301, and V337M were phosphorylated at Thr 231 only to a slight extent, whereas this site in R406W was phosphorylated to a lesser extent. Note that only the top band is labeled for WT and all of the mutant species except R406W. G: C5 labeled all tau species but R406W, which showed almost undetectable reactivity. Note that only the top band was labeled. H: PHF 1 strongly labeled WT, G272, P301, and V337M, but labeled R406W only very weakly.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It was reported that a single tau cDNA transfected in L-cells provided multiple tau 1-immunoreactive bands on the Western blot, presumably generated by various degrees of phosphorylation.27Kanai Y Takemura R Oshima T Mori H Ihara Y Yanagisawa M Masaki T Hirokawa N Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA.J Cell Biol. 1989; 109: 1173-1184Crossref PubMed Scopus (298) Google Scholar In our hands, three bands were detected by tau 1 when samples were not treated with alkaline phosphatase (Figure 2). The top band was stronger in intensity than the lower two bands in WT, G272V, P301L, and V337M-transfected cells. However, in R406W transfectants, the intensity of the lowest band was much stronger than that of the upper two bands (Figure 2). After dephosphorylation, the mobilities of tau in all transfectants corresponded to that of the lowest band (Figure 2B). Thus, the upper two bands and the lowest band represent differentially phosphorylated and nonphosphorylated species of tau, respectively. The three bands were probed with various phosphorylation-dependent and phosphorylation-independent antibodies including tau 1,12Liu W-K Moore WT Williams RT Hall FL Yen S-H Application of synthetic phospho- and unphospho-peptides to identify phosphorylation sites in a subregion of the tau molecule, which is modified in Alzheimer's disease.J Neurosci Res. 1993; 34:
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