Neurochemical Research, Vol. 25, No. 1, 2000, pp. 43–50

430364-3190/00/0100–0043$18.00/0 © 2000 Plenum Publishing Corporation

Tau Dephosphorylation at Tau-1 Site Correlates with itsAssociation to Cell Membrane*

Montserrat Arrasate,1 Mar Pérez,1 and Jesús Avila1,2

(Accepted April 22, 1999)

It has been considered that tau protein is mainly a cytoplasmic protein since it is a microtubuleassociated protein. However, it has also been suggested that tau could be located in the cell nu-cleus and membrane. In our work, the cellular distribution of tau has been studied by immuno-fluorescence and western blot analysis, after subcellular fractionation in neuroblastoma cellsand in tau-transfected non neural cells using, mainly, two types of tau antibodies; antibody 7.51(that recognizes tau independent of its phosphorylation level); and antibody Tau-1 (that recog-nizes tau only in its dephosphorylated form). Also, tau was expressed in COS-1 cells to test forthe features involved in the sorting of tau to different cell localizations. Our results show thattau associated to cell membrane has a lower phosphorylation level in its proline-rich region.Additionally, in differentiated neuroblastoma cells, tau phosphorylation, at that region, de-creases and the amount of tau associated to cell membrane increases.

KEY WORDS: Tau; phosphorylation; neuron differentiation; membrane.

1 Centro de Biología Molecular “Severo Ochoa”, UniversidadAutónoma de Madrid, 28049-Cantoblanco, Madrid, Spain.

2 Address reprint requests to: J. Avila, Centro de Biología Molecu-lar Severo Ochoa, Universidad Autónoma de Madrid, 28049 Can-toblanco, Madrid, Spain. Fax: 34-91-397 47 99, E-mail: javila@cbm.uam.es

* Special issue dedicated to Dr. Héctor S. Barra.

The primary structure of tau molecule (19) canbe divided in two regions. One one of them containsthe very acidic amino-terminus sequences togetherwith a central part, rich in proline and in phosphory-table residues; and a second region containing the basictubulin binding motifs (19–21) and the carboxy termi-nus, also containing phosphorytable residues closed toprolines (22).

Tau can be present in three cell localizations,membrane, cytoplasm and nucleus (23–26). The regioncontaining the amino-terminus appears to be needed forthe interaction with the neural plasma membrane (26).Tau present in the cytoplasm mainly binds to micro-tubules through the region containing the tubulin bind-ing motifs (19,20). Nothing is known about tau regioninvolved in its nuclear localization.

INTRODUCTION

Tau is a microtubule associated protein that in vitrois a promoter of protein polymerization (1) and in vivofavors the stabilization of microtubule network (2),neurite sprouting (3) and the formation of microtubulebundles (4) that could facilitate the microtubule or-ganization in small caliber axons (5).

Additionally, the analysis of tau protein is of greatinterest due to its aberrant aggregation in some neuronspresent in the brain of Alzheimer’s disease patients,assembling the structures known as paired helical fil-aments that can aggregate yielding the neurofibrillarytangles (6–18).

Abbreviations. ab, antibody; U2snRNP, small nuclear ribonucleo-protein particle; PMSF, phenylmethylsulfonyl fluoride; ER, endo-plasmic reticulum; OA, okadaic acid; DMEM, Dulbecco’s modifiedEagle’s medium; COS-1, CV-1 origen-SV40 transfected cells; AD,Alzheimer’s disease; TBD, tubulin binding domain.

The cytoplasmic localization of tau bound to micro-tubule could be modulated by the level of proteinphosphorylation at the regions adjacent to the micro-tubule binding domain (27,28), but little is knownabout the influence of tau phosphorylation on its inter-action with plasma membrane or its nuclear localiza-tion. Although in the second case it has been reportedthat nuclear tau could be present in phosphorylatedform (24).

In this work, we have tested if there is any corre-lation between the phosphorylation level of tau and itsassociation to cell membrane. Our results indicate thattau associated to membrane, is underphosphorylatedat its proline rich region. Also, the phosphorylationlevel of tau decreases upon differentiation of neuro-blastoma cells.

EXPERIMENTAL PROCEDURE

Materials. Tau monoclonal antibodies 7.51 (29) and Tau-1(30) were kind gifts of Dr. C. Wischik (MRC, Cambridge, UK) andDr. L. Binder (MGC, Il, USA), respectively. Tau antibody AT-8 waspurchased from Innogenetics, Belgium. Tubulin antibody raisedagainst a tubulin was from Amersham. Polyclonal antibody (ab196)against tubulin will be described elsewhere. Antibodies raisedagainst the nuclear protein U2snRNP and the membrane proteinribophorin II were kindly donated by Dr. J. Ortín (CNB, Madrid,Spain).

Cell Culture. Human neuroblastoma SH-SY5Y cells (31) weregrown in DMEM supplemented with 10% fetal bovine serum and2mM glutamine plus 0’01% piruvate in a humidified atmosphere with7% CO2. For differentiation, cells were plated and then were grownin Neurobasal-B27 medium (GIBCO), supplemented with 10 µMdibutyril cAMP and 1 mM glutamine for 4 days.

COS-1 cells (32) were grown and transfected with cDNA tau aspreviously described (33).

In some cultures 2 µM okadaic acid was added as inhibitor forendogenous phosphatases (34).

Immunofluorescence Analysis to Study the Localization of TauProtein. Neuroblastoma cells or COS-1 transfected cells were fixedwith 2% paraformaldehyde and 0.1% glutaraldehyde or permeabi-lized with 0.02% saponin in a buffer containing 80 mM PIPES pH6.8, 1 mM MgCl2, 1mM EGTA, 30% glycerol and 1mM GTP,washed with that buffer and fixed as indicated above. Immunofluo-rescence analyses using antibodies against tau were performed aspreviously described (33).

Construction of Plasmid pSGT-2N.To obtain the plasmidpSGT-2N the polymerase chain reaction (PCR) was used usingpSGT-42 as a template, and the oligonucleotide A6 (GCG GATCCA TAT GGC TGA GCC C) which includes the BamHI site for aproper cloning and the oligonucleotide A12 (CCG ATT CTT AGGGCA CGG GGG) which includes the EcoRI site. The fragment gen-erated was digested with BamHI and EcoRI and ligated into BamHIand EcoRI digested pSG5. The plasmid was analysed by restrictionanalysis to test for a proper orientation and correct size of the in-serts. The estimated size for the expressed 2N tau fragment is 30 kd,although it has an electrophoretically mobility corresponding to aprotein a 40 kd.

Expression and Purification of Transfected Protein.The humantau cDNA clones were expressed in COS-1 cells as previously de-scribed (34). Cytoplasmic extracts of transfected cells were obtainedby gentle homogenization of the cells in buffer A (0.1M MES pH 6.4,0.5mM MgCl2, 2mM EGTA) containing protease inhibitors (1mMPMSF, 10 µg/ml aprotinin, 10 µg/ml pepstatine, 10 µg/ml leupeptine).On the other hand, a subcellular fractionation was performed by plac-ing the cells in a hypotonic buffer (0,25 M sucrose, 20 mM HEPESpH 7.4, 2 mM EGTA, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/mlleupeptine, 10 µg/ml pepstatine) and lysing them by passing the cellsuspension twenty times through a syringe (0.6 mm × 25 mm). Thehomogenate was first centrifuged at 750 g 5 min to isolate the nuclearfraction as a pellet. In some experiments, for a further purification,nuclei were sedimented through a 2M sucrose cushion. This nuclearfraction was resuspended in a magnesium containing buffer (20 mMHepes pH 7.4, 2 mM EGTA, 2 mM MgCl2) whereas the supernatantwas centrifuged at 4°C again at 31,000 g for 1 hour to isolate themembrane fraction as a pellet and the cytoplasmic fraction as thesupernatant. In some experiments a higher speed centrifugation, 2.4105g for 1 hour, was achieved to isolate a plasma membrane frac-tion. Similar results were obtained with this and the lower (low cen-trifugation speed) membrane fraction. The different fractions werecharacterized by electrophoresis and western blot (35).

Westerns Blot Analysis.Protein characterization by gel elec-trophoresis and Western blot was carried out as previously de-scribed (35). For quantitative purpose the western blot was per-formed testing the same amount of protein from different subcellularfractions with, in parallel, known amounts of recombinant tau. Allthe samples were blotted, the reaction of the antibody was deter-mined by densitometry of the reacting proteins and the values ob-tained were compared with those of recombinant tau protein. Theaverage of three different experiments are indicated in the quanti-tative analyses.

RESULTS

As a control to determine the suitability of thesubcellular fractionation method, the localization of acytoplasmic protein, tubulin; that of a nuclear protein,U2 snRNP, and a that of membrane protein, ribophorinII, were tested. Fig. 1 shows that tubulin is mainlypresent in the cytoplasm, whereas the nuclear proteinis in the nuclear fraction. The membrane protein ispresent in the cell membrane fraction and, also, in thenuclear fraction. This could be due to the associationof the endoplasmic reticulum (ER) to nuclei and to thefact that ribophorin II is present in ER.

Tau Distribution in Subcellular Fractions ofNeuroblastoma.The previous procedure was used toanalyze tau distribution in subcellular fractions inneuroblastoma cells. First, the total amount of tau inneuroblastoma cells was measured by testing the re-action of the protein from cell homogenates with twotau antibodies (ab 7.51 and ab Tau-1) and by compar-ing that reaction with that obtained by the reaction ofthose antibodies with increasing amounts of recom-binant tau. In that analysis, an amount of 1.5 ± 0.4 µg

44 Arrasate, Pérez, and Avila

tau/mg protein was calculated when ab 7.51 was usedand a value of 0.4 ± 0.2 µg tau/mg protein was cal-culated when ab Tau-1 was tested. The second valueis similar to that described by Tanaka et al. (36) inneuroblastoma cells.

When the subcellular fractionation was performed,it was found (Figure 2) that tau is present in membrane,cytoplasm and nuclear fractions. The Western blotshows the appearance of two proteins (around 64 kDand 60 kD), as determined by running in parallel knownmolecular weight markers, reacting with ab 7.51, innuclear and membrane fractions. Only the 60 kD pro-tein was observed in the cytoplasmic fraction. In some,but not in all preparations, a third protein with an elec-trophoretic mobility of 100 kD was also observed.

When ab Tau-1 was used, the reaction was mainlyobserved with the protein showing the fastest mobil-ity and proteolytic fragments, probably generated bythe higher susceptibility of dephosphorylated tau toproteases (37). Quantitative analyses were performedwith two antibodies raised against tau (ab 7.51 and abTau-1). The relative amounts of tau present in the dif-ferent subcellular fractions from undifferentiated neu-roblastoma (ND) cells were calculated. From the totaltau present in the cell, 24 ± 5% of it was found in thenucleus, 65 ± 10% in cytoplasm and 11 ± 5% in cellmembrane, using ab 7.51. When ab Tau-1 was tested,the percentage of tau protein found in the membranefraction was 47% respect to the total tau.

The previous results were obtained in undifferen-tiated neuroblastoma cells. When those cells were sub-jected to differentiation by adding dibutyril cAMP thefollowing results were obtained: the amount of taumeasured by ab 7.51 in whole extracts is now 2.7 ±0.8 µg tau/mg protein and 1.3 ± 0.3 µg tau/mg proteinwhen ab Tau-1 was used. An increase of tau proteinupon neuroblastoma differentiation has been also re-ported by other authors (38).

In differentiated neuroblastoma cells when thesubcellular fractionation was performed, the followingvalues were obtained for the localization of tau whenit was measured by using ab 7.51; 17 ± 2% was foundin the cell nucleus, 52 ± 5% in the cytoplasm and 31 ±3% in the membrane. The value obtained with abTau-1 for membrane fraction was 69% of the totaltau. Also, the characterization of the tau isoformspresent in those fractions was achieved by Westernblot (Fig. 2, D lines). Again, it was observed the ap-

Tau Dephosphorylation at Tau-1 Site 45

Fig. 1. Specificity of subcellular fractionation. In order to test forthe specificity of the subcellular fractionation, the localization ofknown cytoplasmic protein tubulin (Tb), that of a nuclear protein(U2snRNP) and a that of membrane protein (ribophorin) were analyzedin the nuclear (N), cytoplasmic (C) and membrane (M) fractions ofCOS-1 cells.

Fig. 2. Subcellular fractionation of neuroblastoma cells. Tau protein, present in the whole homogenate (H) in nuclear (N), cytoplasmic (C) andmembrane (M) fractions from undifferentiated (ND) and differentiated (D) neuroblastoma cells, was characterized by using ab 7.51 and ab Tau-1.

pearance of two proteins reacting with ab 7.51, andmainly one reacting with ab tau-1. Also, it was found,as previously indicated, a significant increase, com-pared to undifferentiated cells, in the reaction of taulocated in the cell membrane with ab Tau-1. Addition-ally, Tau-1 not only reacts with the 60 kD tau proteinbut also with proteolytic fragments of this protein (seeabove). In summary, it was found a higher proportionof tau, dephosphorylated at its proline rich region, inthe protein fraction associated to membrane. The pro-portion of that tau population increases during neuro-blastoma differentiation.

Localization of Tau in Neuroblastoma Cells.Two approaches were used to test for the localizationof tau in the cells by immunofluorescence analyses.The immunofluorescence analyses were performedusing ab 7.51 and ab Tau-1 on fixed cells to preservethe cell membrane or in cells treated with saponin toremove the soluble (non particulate) proteins. Indeed,saponin treatment removes cytosolic proteins but notthose associated to the membrane. Then, these pro-teins could be further characterized (39). Fig. 3shows that ab 7.51 reacts with antigens present in thecytoplasm. Also, Tau-1 could react with nuclear anti-gens as it has been indicated in other reports forhuman neuroblastoma cultures (23). Additionally, areaction of these antibodies with cell membrane pro-teins could be suggested as previously reported (26).These results suggest that tau could be present in cellmembrane, cytoplasm and nucleus, and that the tauregion reacting with ab 7.51 could be masked in cellnucleus.

Immunofluorescence analysis of differentiatedneuroblastoma cells shows that ab 7.51 and Tau-1mainly react with cytoplasm protein and with that pres-ent in extended neurites of the cells, in untreated andsaponin treated cells. Also, an increase in the reactionwith Tau-1 antibodies respect to undifferentiated cellswas observed (Figure 3).

Localization of Tau in Non Neural Cells.To testfor the basis of tau localization in different subcellu-lar fractions, COS-1 cells were transfected with taucDNA, and the localization of the expressed tau wasanalysed by immunofluorescence and Western blot.Fig. 4 shows, again, that tau is located in the differ-ent cellular compartments, as detected by immuno-fluorescence using ab Tau-1.

Additionally, Western blot analysis of nontrans-fected (c) or transfected cells, cultured in the absence orpresence of okadaic acid, (Fig. 5) indicates the presenceof tau in the three subcellular fractions. Fig. 5 showsthat ab 7.51 reacts with the nuclear, cytoplasmic and

membrane proteins. Also, a positive reaction was foundfor ab Tau-1. Only, a reaction of the cytoplasmic pro-teins was observed with ab AT-8 (an antibody comple-mentary to ab Tau-1) when phosphatases were inhibitedby the addition of okadaic acid (OA). In this case, no re-action with the membrane protein fraction was detected.In the presence of OA was observed a decrease in theelectrophoretic mobility for some tau isoforms when ab7.51 was used, in the nuclear and cytoplasmic, but notin the membrane fraction. Similar results were observedwith those tau cDNAs lacking or containing exons 2, 3or 10 (data not shown). These results indicate that thepresence or the absence of any of these exons is not thecause for a different subcellular localization of tau. Fi-

46 Arrasate, Pérez, and Avila

Fig. 3. Localization of tau in neuroblastoma cells. A) Cultured SH-SY5Y neuroblastoma cells were grown and fixed with 2% para-formaldehyde and 0.1% glutaraldehyde (a, c). In some cases (b, d)the cells were also permeabilized with saponin to extract the solubleprotein. The reaction of neuroblastoma with ab 7.51 (a, b), and ab Tau-1 (c, d) is shown. B) Cultured neuroblastoma cells wereinduced to differentiate in the presence of dibutyril cAMP and fixed(a, c) or, additionally, extracted with saponin (b, d). The reactionwith ab 7.51 (a, b), and ab Tau-1 (c, d) is shown.

nally, ab Tau-1 reacts with the faster electrophoreticmobility protein and with some proteolytic fragments ofit (see above, Fig. 2). The reaction with ab PHF-1 wasalso analyzed, giving the same result than that of AT.8(data not shown) (40); in the presence of OA, it wasobserved a weak reaction in the nuclear fraction andstrong reaction in the cytoplasm.

In tau transfected-non neural cells, the amount ofexpressed tau protein was also calculated. The pro-portion of transfected cells, determined by immuno-fluorescence analysis, was 22 ± 4% (33). In those cul-tures, the amount of tau, measured by its reactionwith ab 7.51, was of 0.3 ± 0.1% of total protein.

Thus, taking together both results it can be calculatedthat in those cells tau is about 1,5% of the total pro-tein of transfected cells. Thus, a huge amount of tauis expressed compared with that present in neuro-blastoma cells. Since, the expression of a larger amountof protein could help its aggregation, we have testedthe proportion of the expressed protein in nucleus,cytoplasm and membrane fractions. The results ob-tained indicate that there is: 38 ± 12% in nuclear frac-tion, 40 ± 10% in cytoplasm and 22 ± 6% in themembrane fraction.

In presence of OA, the amount measured by ab7.51 of tau in the different cell fractions changes to42 ±11% in the nuclear fraction, 49 ± 13% in the cyto-plasm and only 7 ± 2% in the membrane fraction. Itsuggests that tau associated to membrane fraction

Tau Dephosphorylation at Tau-1 Site 47

Fig. 5. Subcellular fractionation of tau-transfected COS-1 cells.COS-1 cells were transfected with Tau cDNA (expressing thelongest isoform, tau 42), using non transfected cells as a control (C).Then, cells were fractionated into nuclear (N), cytoplasmic (C) andmembrane (M) fractions and the presence of tau in these fractionswas characterized by western blot using abs 7.51, Tau-1 and AT-8.In some experiments, okadaic acid (OA) (a phosphatase inhibitor)was added to the culture to test for the reaction of tau antibodies inthese conditions.

Fig. 6. A tau fragment containing the proline-rich region, is presentin the membrane fraction. A) Schematic diagram of the largest tauisotype (tau 42) and tau fragment containing the first 251 aminoacidsof that molecule. In the diagram, the presence of exon 2 and 3, andthat of the tubulin binding repeats (TBD), is indicated by open box,whereas the proline rich region is shown as closed boxes. Also, thelocalization of the epitope for ab Tau-1 is indicated. B) COS-1 cells,were transfected with the cDNA that express the terminal half of tauand the transfected cells were fractionated to isolate the membranefraction. The presence of tau fragment (tau 2N) in that fraction wascharacterized by Western blotting, in untreated or OA-treated cells,using ab Tau-1. As a control to mesure the level of a membraneprotein in parallel Western blot the amount of ribophorin was tested.

Fig. 4. Localization of tau in tau-transfected COS-1 cells. COS- 1cells were transfected with the cDNA expressing the whole taumolecule and the localization of tau and tubulin was analyzed by double immunofluorescence using ab Tau-1 (A), and ab 196 (a polyclonal antibody raised against tubulin) (B).

has, preferentially, a low level of phosphorylation inregions like the proline rich region.

A Tau Fragment Containing the Proline Rich Re-gion Binds to the Membrane Fraction.The cDNAcodifying the aminoterminal half of tau protein (thatcontains the proline rich region) was expresed (upontransfection) in untreated and okadaic acid treated COS-1 cells. The reaction of the expresed protein with ab tau-1 was measured and it is shown in Fig. 6. This figureshows that the expressed protein is located in the cellmembrane fraction and that such reaction decreases inOA-treated cells. This result supports the data of Fig. 5,indicating that the aminoterminal tau fragment (con-taining the proline rich region) behaves like the wholetau molecule respect to its association to membrane.

DISCUSSION

In this work it has been analysed the proportionof tau present in the different subcellular (nucleus,cytoplasm and membrane) fractions of neuroblastomacells. The total amount of tau and also its proportionin phosphorylated form at the proline-rich region inneuroblastoma cells, has been calculated. In the lattercase the proportion found was similar to that indicatedin a previous report (36). Also, the electrophoretic be-haviour of the neuroblastoma tau isoforms analyzedby western blot resembled those indicated in otherworks (36,38,41).

The analysis of the subcellular localization of tauwas carried out in neuroblastoma cells and in tau trans-fected COS-1 cells. In the first type of cells it has beendescribed that mainly two isotypes of tau containingonly three tubulin binding motifs are endogenouslyexpressed (41). Thus, the appearance of additional iso-forms, by fractionation in gel electrophoresis, shouldbe the consequence of a postranslational modification,such as phosphorylation, of the protein. That modifi-cation occurs in both undifferentiated and differenti-ated cells as recognized by the different mobilities ofunmodified or modified tau, or by the reaction of theprotein with antibodies that specifically recognizestau, depending on its phosphorylation state.

In a similar way, in tau transfected COS-1 cellssingle tau isotypes (or tau fragments) are expressed (34)and the appearance of more than one tau bands is theconsequence of the protein phosphorylation.

It has been suggested that tau is, mainly, a cyto-plasmic protein because it is a microtubule associatedprotein. However, in this and in previous studies ithas been shown that tau is not only present in the cy-

toplasm but also in the nucleus and cell membrane(23–41). The characteristics of tau protein present innucleus, cytoplasm and membrane have been ana-lyzed and it has been found that the presence or the ab-sence of exons 2, 3 or 10 does not affect the subcellu-lar localization of tau.

The association of tau to the cell membrane ap-pears to be regulated by the phosphorylation of theprotein at its proline-rich region. It has been observedthat the protein associated to cell membranes containsa large tau population where the proline-rich region isin unmodified form. Also, the addition of okadaic acid,that prevents dephosphorylation at that region (42),results in a decrease in the amount of tau associated tomembrane.

The association of tau to the cell membranecould be stablished through other protein, like spec-trin (43), phospholipase C-γ (44), fyn (45), or throughits interaction with lipids (46). In the first case theproline-rich region could be involved in a protein-protein interaction and this interaction could play arole in some processes of signal transduction (44,45),a process that could be modified by the phosphoryla-tion in that proline-rich region. In the proline rich tauregion there are two sequences that are similar to twoclasses of consensus SH3-binding proteins, PTPPTRand RTPPKSP (47). These sequences could be in-volved in the association of tau with a membrane pro-tein. More recently, Lee et al. (45) has shown that theinteraction with a membrane protein such as fyn mayoccur through the tau sequence TPPAP, and that insuch interaction, a tyrosine phosphorylation in tau(the closest one is tyrosine 197) could play a role. Itshould be analysed if that tyrosine phosphorylationaffects to the modification in the proline rich regionrecognized by ab Tau-1. Additionaly, changes in tauphosphorylation and in cell localization (e.g. near themembrane surface), that may affect to the conforma-tion and functional characteristics of tau protein, hasbeen recently suggested (48).

Taking together all the results shown in this work,a model about tau localization could be postulated. Itsuggests that tau, after being synthetized in the cyto-plasm, could bind to microtubules and remain in thecytoplasm or, alternatively, could be located to thecell membrane, mainly if its phosphorylation level islow in regions such us the proline rich region. Theselocalizations could occur in neuroblastoma or in tau-transfected non-neural cells and phosphorylation oftau could play a role in its subcellular localization. Inthis way, it has been widely indicated that tau proteinis a phosphoprotein containing multiple phosphorylated

48 Arrasate, Pérez, and Avila

residues that are mainly modified in the protein pres-ent in the brain of Alzheimer’s disease patients (22).It has been indicated that some of the phosphorylatedresidues (P-Thr 231 and P-Ser 262) are involved inpreventing the interaction of tau with microtubules(49). However, those serines (198–202) recognized byab Tau-1 are not involved in the interaction of tauwith microtubules, and we propose that its modifica-tion may play a role in the association of tau to mem-branes, a function that could be also important in dis-orders such as AD.

In this way, we have found that during neuro-blastoma cells differentiation, the level of phosphory-lation of tau decreases and it may facilitate an increasein the proportion of the cell membrane located protein.This feature could be related to the gradient of tau de-phosphorylation, at its proline rich region, that takesplace towards the distal part of the growing axon (50).Further experiments should be performed to know ifthere is a correlation between these events.

ACKNOWLEDGMENTS

We thank to Drs. R. Armas and E. Montejo for their commentsand criticisms, to improve this work. We also thank Dr. J. Ortín fromthe Centro Nacional de Biotecnología, Madrid, Spain, for providingus the antibodies that recognizes U2snRNP and riboforin II. Thiswork was supported by the Spanish CICYT, Fundación La Caixa,Comunidad de Madrid, and Fundación Ferrer. Also, an institutionalgrant of Fundación Ramón Areces is acknowledged.

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Tau Dephosphorylation at Tau-1 Site 49

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50 Arrasate, Pérez, and Avila