Identification and Localization of a tau Peptide to Paired Helical Filaments of Alzheimer Disease

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Identification and Localization of a tau Peptide to Paired Helical Filaments of Alzheimer Disease
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  Proc. Natl. Acad. Sci. USA Vol. 86, pp. 5646-5650, July 1989 Neurobiology Identification and localization ofa T peptide to paired helical filaments of Alzheimer disease  Alzheimer neurofibrillary tangles/amino acid sequence/protein phosphorylation/immunocytochemistry/microtubule-associated proteins) KHALID IQBAL*t, INGE GRUNDKE IQBAL*, ALAN J. SMITHS, LALU GEORGE*, YUNN-CHYN TUNG*, AND TANWEER ZAIDI* *New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314; and *Department of Biological Chemistry,School of Medicine, University of California, Davis, CA 95616 Communicated by Dominick P. Purpura, April 14, 1989 (received for review March 3, 1989) ABSTRACT Amino acid sequencing of a CNBr digest ofthe r protein isolated from bovine brainrevealed an amino acid sequence of 17 residues, Pro-Gly-Leu-Lys-Glu-Ser-Pro-Leu- Gln-Ile-Gly-Ala-Ala-Pro-Gly-Leu-Lys, which we call peptide I, with heterogeneity at position 11 of glycine (peptide Ia) and proline(peptide Ib); peptide I showednohomology with thepreviously reported cDNA-derived mouse and human 7 se- quences. Antisera raised to synthetic peptides corresponding to peptidesIa and lb labeled all the bovine 7 polypeptides recognized by other monoclonal and polyclonal antibodies to bovine 7. Antisera to peptidelb did not label any mouse 7 polypeptides; however,an anti-Ia antiserum labeled two of the four mouse T polypeptides.Antisera to both peptides labeled paired helical filaments (P1F) as neurofibrillary tangles, plaque neurites, and neuropil threads in Alz heimer isease brain and PHF polypeptides onimmunoblots. Immunotaining with anti-Ia antisera of PHF intissuesections and PHF polypeptides, butnot bovine x, on immunoblots wasmarkedly increased when pretreated with alkalinephosphatase. These studies suggest that  i) the amino acid sequences of some isoforms of 7 peptide mightbe different from that predicted from cDNAs,  ii) a 7 peptide that is absent in the predicted sequences is present in PHF in Alzheimer disease, and  iii) T in PHF is abnormally phosphorylated. Microtubule-associated protein X is a family of closely related polypeptides witha molecular weight range of 55,000-68,000 on sodium dodecyl sulfate/polyacrylamide gel electrophore- sis (SDS/PAGE) (1, 2). These polypeptides also differ in theirisoelectric points but have similar peptide maps and amino acid compositions  2). r stimulates microtubule assembly and coassembles with tubulin into microtubules in vitro. T poly- peptidesmicroinjected into cells increasetubulin polymer- ization and decrease the rate ofmicrotubuledepolymeriza- tion, suggesting their rolein assembly of microtubules in vivo  3). Recent studies  4, 5) have shown that there are atleast four molecular species of bovine T, some of which,dependingon the degree ofphosphorylation, have different electrophoretic mobilities on SDS/PAGE. T, in a phosphorylated form, has been shown to be present in paired helical filaments (PHF), the aberrant fibrils making the intraneuronal neurofibrillary tangles in Alzheimer disease (6-8).In addition to the neu- ronal cell body, the PHF also accumulate in the neurites of the neuritic(senile) plaques and in the neuropil as  neuropil threads (9) in affected brains. ThecDNA-derived sequences of mouse and human 7 havebeen reported (10, 11). In addition a fragment of human T has been shown to be intimately associatedwith PHF (12). In this paper we describethe presenceofa unique T peptide not present in either cDNA sequence and the localization of this unique 7 peptide to PHF in Alzheimer disease. Preliminary communications on the amino acid sequencing of the CNBr digest of bovine X have been reported (13, 14). MATERIALS AND METHODS Isolation of 7. 7 was isolated from bovine brain, obtained from a local slaughter house, by the method of Grundke-Iqbal et al. (15). Microtubule proteins obtained by three cycles of assembly/disassembly (16) were heat-treated at pH 2.7, and r from theheat-stable microtubule-associated proteins were then extracted in 2.5% perchloricacid (17, 18) and dialyzed against 2.5 mM Tris (pH 7.6). No protein bands other than 7 bands were detected on Coomassie blue-stained SDS/ PAGE. Amino AcidSequencing. Amino acid sequence 1-17 of bovine T peptide I (seestructure I) was obtained by auto- mated Edman sequencing (13) ofa CNBr digest after one cycle of manual Edman degradation andsubsequent blocking witho-phthalaldehyde. The CNBr digestion was performed on 100 Ag of purified T, and the sequence was recovered at the 700-pmol level. Residues in parentheses at positions -1 and -2 (seestructure I) werededucedfrom mixed sequence information by comparison with the cDNA and were included for the purposeof designing a synthetic peptide for produc- tion of antibodies. SDS/PAGE. SDS/PAGE of bovine and mouse brainmi- crotubules andAlzheimer PHF for subsequent immunoblots (Western blots) was carried out by using theTris-glycine system (19) and a 5-15% polyacrylamide gradient.Synthesisof Peptides. Two peptides, Ia and lb collectively called peptide I, corresponding to amino acidresidues ofa CNBr fragment of X peptide differing only in one residue (13, 14), were synthesized commercially (Biosearch). Cysteine was added at the carboxyl termini in amideform to conjugate to keyhole limpet hemocyanin (KLH). The peptide Ia had the following sequence: (Met)-(Ala)-Pro-Gly-Leu-Lys-Glu- Ser-Pro-Leu-Gln-Ile-Gly-Ala-Ala-Pro-Gly-Leu-Lys, with residues numbered as in structure I. In peptide lb,residue 11 was prolineinstead of glycine. Production of Antisera to the KLH-Conjugated Synthetic Peptides Ia and lb. The amount of total protein in the KLH-conjugated peptides was determined by amino acidanalysis. Two New Zealand White rabbits, weighing about 2-2.5 kg each, were immunized with each peptide as de-scribed (20). Initially each rabbit received an emulsion of 3 Abbreviations: KLH keyhole limpet hemocyanin; PHF, paired helical filaments. tTo whom reprintrequests should beaddressed at: New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314. 5646 The publication costs of thisarticle were defrayed in part by pagecharge payment. This article must therefore be herebymarked  advertisement in accordance with 18 U.S.C. §1734 solely to indicate this fact.  Proc. Natl. Acad. Sci. USA 86 (1989) 5647 mg of peptide-KLH conjugate in complete Freund's adju-vant. After 40 days, the rabbits received in intervals of 2 weekstwo boosters, each containing 0.13 mg of peptide- KLH conjugate emulsified in incompleteFreund's adjuvant administeredintramuscularly and 0.7 mg of unconjugated peptide in alumina Cy administered intravenously. Three weeks later, a thirdbooster, containing 1.0 mg of unconju- gatedpeptide in aluminaCy, was administered intrave- nously. Eight days after the final booster,the animals were sacrificed.Isolation and in Vitro Dephosphorylation of PHF. PHF were isolated from frozenautopsied brains of patients with Alz- heimer disease (21). The PHF (1 mg/ml) were incubated at 370C for 16 hr with alkaline phosphatase (0.1 mg/ml) (Sigma type VII-S, from Sigma) in 50 mM MgCl2/10 mM phenyl-methylsulfonyl fluoride/0.02 sodium azide/2 ,ug of leupep- tin per ml/100 mM Tris, pH 8.8. After incubation the sample was centrifuged througha cushion of 0.5 M sucrose at 200,000 xg for 10 min in a Beckman TL-100 ultracentrifuge. The PHF pellet was resuspended in water and used for immuno- absorption. Reactivity ofPeptideAntisera with   and with Alzheimer Neurofibrillary Tangles. Immunoreaction of each antiserum was tested onWestern blots of the SDS/PAGE of isolated PHF, bovine T, and mouse brain microtubules as described  15). The reactivity of antisera was also examinedon paraffin sections of hippocampus fromAlzheimer diseased brain as described (15). Immunoabsorption. Forimmunoabsorption, the antisera were incubated at 1/10th of their final dilution withvarious amounts of each antigen at 22°C for 1 hr and at 4°C for18 hr (20). Antibodies to r. Antibodies to whole bovine T used in this study were a monoclonal antibody r-1 (22) and a rabbit antiserum92e (17). RESULTS Amino Acid Sequencing of Bovine r. Amino acid sequencing of the CNBr digest of bovine rafter phenylisothiocyanate treatment revealed several peptides, one of which had proline as the second residue (see refs. 13 and 14). By taking advantage of this observation, a CNBr digest of T was subjected to one manual cycle of Edman degradation, fol- lowed by blockage with o-phthalaldehyde of all amino termini except the one starting with proline. Amino acid sequencing of the o-phthalaldehyde-treated CNBr digest of bovine T revealed the presenceof a previously unknown peptide:  Met)- Ala)-Pro-Gly-Leu-Lys-Glu-Ser-Pro-Leu-Gln-Ile-Gljy- 17 Ala-Ala-Pro-Gly-Leu-Lys. I This fragment from bovine T (13, 14), which we call peptide I, hasno homology with the cDNA-predicted sequencesof mouse (10) and human (11) T. Two other peptides identified from the CNBr digest of bovine r are highly homologous to the cDNA-predicted sequences (13). A computer search from data bank failed toreveal anyhomologiesbetween peptide I andany other known amino acid sequence. Production of Antisera to r Peptide I and TheirReaction with r and PHF Polypeptides. Each of the four rabbits immunized had produced antibodies to the r peptides. On immunoblots ofbovine microtubules, all four peptide anti- sera labeledthe r polypeptides identically to polyclonal andmonoclonal antibodies against the total bovine T (Fig. 1; see also Fig. 3). However, on blots of mouse microtubules,only I   I m m I LL LL 2 I   D aQ I m0 .. a. i 9 ~0..; v   .. So I  W . *er>>2>I _o I~~~~ 200- t 92.5- 68 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~t W 43- 25.7- tau-1 a.Ia FIG. 1. Western blot of PHF and invitro assembled microtubules from mouse (lane M-MT),human (lanes H-MT), and bovine (lanes B-MT) brain. Immunolabeling withantiserum102c to peptideIa was at 1:1000 antiserum dilution(lanes a.Ia) andmonoclonal antibody T-1  ref. 22)to T was at 1:50,000ascitesdilution (lanes tau-1). Bound antibodies were visualized by using peroxidase-antiperoxidase and avidin-biotin complex techniques, respectively. Electrotransfers werefrom SDS/polyacrylamide gels (5-15% acrylamide, 8 x 6 x 0.75 cm). *   indicatesblots pretreated with alkaline phosphatase at 86 ,Lg/ml in 0.1 M Tris, pH 8.0/1 mM phenylmethylsulfonyl fluoridefor 3 hr at 37°C prior to immunostaining. Molecular size standards are indicated in kDa: myosin (200), phosphorylaseb (92.5), bovineserumalbumin (68), ovalbumin (43), and a-chymotrypsinogen (25.7). The otherthree antisera to peptide I-i.e., 101c to peptide Ia and 103c and 104c to peptide lb (not shown in this figure)-also reacted withbovine and human rand r in PHF, but the pretreatment with alkaline phosphatase onlyincreased the reactivity with peptideIa antisera. antiserum 102c against peptide Ia reacted with two of the slowest moving T species (Figs. 2 and 3). The three other r peptide antiseradid not react with any of the mouse r polypeptides. No significant change in immunostaining of murine or bovine T polypeptides occurred when the blots were treated with alkaline phosphatase prior to application of the antisera (Figs. 1 and 2). Reactionof the peptide antisera with PHF polypeptides on immunoblots was weak. How- ever, on blots pretreated with alkaline phosphatase, the staining of the PHF polypeptides corresponding to the slow- est moving T polypeptides was increased with theantisera to peptide Ia (Figs. 1 and 2) but not with theantisera to peptide Ib (figure not shown). No enhancement of the immunostain- ing was observed when the blot was treated with alkaline phosphatase in the presence of 25 mM pyrophosphate, an inhibitor of alkaline phosphatase (figure not shown). The r polypeptides in PHF immunolabeled with the anti-peptide I antisera migrated slower than the polypeptidesrecognized with the T-1 antibody on SDS/PAGE (Figs. 1 and 2). Localization of Peptide I to PHF and the Effect of Dephos- phorylation on Immunostaining. On tissue sections of Alz- heimer hippocampus, all four antisera strongly labeled intra- cellular neurofibrillary tangles, neuropil threads, and occa- sional plaque neurites, which are locations where PHF accumulate in the brain of patients with Alzheimer disease(Fig. 4). Stainingof theextracellular tangles or  tomb- stones, the putative end stages of the tangles (23) or of Neurobiology: Iqbal et al.  Proc. Natl. Acad. Sci. USA 86 (1989) PHF M-MT PHF f. 4 a   ~~~~50P  1 7 - . / *. I i ... AL . .e   4 e~ 0   _ tau-- a.Ia FIG. 2. Western blot of PHF and mouse microtubules (lanes M-MT). Immunostaining was with monoclonal antibody -1  ref. 22) to T(lane tau-1) and antiserum 102c to peptide Ta (lanes a.Ta) with(* ) and without pretreatment of the blot with alkaline phosphatase (see Fig. 1 for experimental details). Molecular size standards are indicated in kDa as in Fig. 1. The other threeantisera to peptide I-i.e., 101c to peptide Ta and 103c and 104c to peptide Tb (not shown inthis figure)-also reacted with in PHF but did not label any of the mouse T polypeptides even when pretreated with alkaline phosphatase. as b lop plaque core amyloid, was not observed with any of the antisera. In the case of antisera lOic and 102c to peptide Ia, pretreatment of thesections with alkaline phosphatase prior to immunostaining considerablyincreased both staining in- tensity and the number of immunostained tangles and neu- ropil threads. This change was most dramatic in plaques, most of which became immunopositive only after dephos- phorylation. No change in the staining pattern was observed when the tissue was incubated with alkaline phosphatase in .: ... m; 200-   M 6 8 : i: Anow 43- a.tau a.Ia a.Ib FIG. 3. Comparison of immunolabeling of T in bovine and mouse microtubules (lanes B-MT and M-MT respectively) by antiserum 92e  ref. 17) to bovine T (lanesa.tau) and antisera 102c and 104c to peptides Ta and Tb (lanes a.Ta and a.Tb, respectively). See Fig. 1 for experimental details. Molecular size standards are indicated in kDa as in Fig. 1. C -r FIG. 4. Immunocytochemical staining of paraffin sections of hippocampus from an Alzheimer disease case with antisera to peptide Ta and the effect of dephosphorylation. Antisera at 1:1000 dilution were incubatedwith the tissue sections, and the bound antibodies were visualized by the peroxidase-antiperoxidase tech- nique. (a and c) Immunostainingof a tissue section that hadbeenincubatedwith130 kg alkaline phosphatase per ml for2.5 hr at 320C prior to labeling with anti-peptide Ia antiserum 102c. Neurofibrillary tangles (arrow in c), plaque neurites (arrowheads in c) and fine neuropilthreads (double arrows in c) are labeled by the antibody. (b) Significantly fewer numbers of tangles and plaques in the same area as in a are stained by antiserum102c when the preincubation with alkaline phosphatase was in the presence of25 mM pyrophosphate, an inhibitor of this enzyme. Antiserum 104c to peptide Tb stained almost as many tangles and plaques as did antiserum102c in a (not shown in this figure); plaque neurites, but not plaque amyloid cores, were stained with the peptide antisera. (a and b, x 100; c, x500.) the presence of 2 mM each of EDTA and EGTA or 25 mM pyrophosphate, which are phosphatase inhibitors. In the case of antisera 103c and 104c to peptide Tb, pretreatment of the sections with alkaline phosphataseonlyminimally changed thestaining pattern and intensity. The immunostaining of PHF was eliminated when the peptide I antisera were preabsorbed with 10-50 ng of uncon- jugated peptide per ml of dilutedantisera (Table 1). In the case of the antisera to peptide Ia, the immunostaining was removed equally well by absorptionwithpeptide Ia or Tb. In the case of the antisera to peptide Tb, the staining was obliterated with peptide Tb but not with peptide Ia with levels as high as 500 ng. Absorption of antisera 102c (peptide Ta) and 200- 92.5- 68-43- 0 A J 50p 5648 Neurobiology: Iqbal et al.  I   w v0-1   A .,  NW,. emkw*, 1w   lxw&.  Proc. Natl. Acad. Sci. USA 86 (1989) 5649 Table 1. Absorption of tangle-staining antibodies Antigen, 1g/ml of diluted antiserumAntigenused Anti-peptide Ia Anti-peptide lb Anti-T for absorption 101c 102c103c104c92e PeptideIa0.05 0.05 >>0.5 >0.5 >5 Peptide Tb 0.05 0.05 0.05 0.01 >>5 Bovine MT 50 5020506 Mouse MT >>100 >100 >>100>>100 6 PHF ND 2* ND 2* 1 Immunostaining ofAlzheimer neurofibrillarytangles by antisera to peptides Ia, Tb, and.' was preabsorptionwith synthetic T peptides Ta, Tb, microtubules (MT), and PHF. >> No change in tanglesstaining seen when preabsorbed with the amount of antigen shown;>, definite reduction in stainingintensity seen when preab- sorbed with the amount of antigen shown; *, treated with alkaline phosphatase; ND, not determined. 104c (peptide Tb) with alkaline phosphatase-treated PHF, likewise, removed the tangle staining. When bovine micro- tubules wereused as asource of T peptide for absorption, the immunostaining of PHF by all four antisera was eliminated with50 ug of antigen per ml of diluted antisera, whereas absorption with 100 ,ug of mouse microtubules per nml only slightly reduced the immunostaining by antiserum 102c (pep- tide Ta) andhadno effect in the case of the other three antisera. In contrast, the staining of tangles and neurites by anantiserum toisolated bovine T (serum 92e) was completely eliminated with as little as 6 ,ug of bovine or mouse micro- tubules or 1 ,g of PHF per ml of diluted antiserum. DISCUSSION Although T in brain microtubules was discovered asearlyas 1975 (24), the biochemistry of this family of polypeptides is now only beginning to be understood. The association of a polypeptide, now known to be T, with Alzheimer PHF was observed in 1974 (25). However, it was not definitelyiden- tified until several years later (6-8, 12,15, 20,21, 26-28). Discovery of T in PHF in Alzheimer disease not only has revived and tremendously increased interest inthis otherwise little-studied protein but also hasprovided some indication as to the possible function of T in the brain (6, 29). The present study  i) identifies a previously unknown T peptide and  ii) shows immunochemically the presenceof this peptide in normal human T and in PHF in Alzheimer disease. A comparison of the amino acid sequence of peptide I with the previouslyreported cDNA-predicted sequences of mu- rine (10) and human (11) T showsno homology. Two other CNBr peptidesof bovine T have been found to be highly homologous to the cDNA-derived sequences (13). The ab- senceof peptide I in the cDNA-predicted sequences suggests that the latter represent only certain isoformsof T. The different molecular species of T might bedue to differential splicing of the mRNA as proposed in the earlier reports (10,11). Immunostaining of bovine T polypeptides with antibodies to the synthetic peptides corresponding to Ta and Tb shows the presence of these peptides in the parent protein. Inter- estingly, all molecular species of bovine Trare immunostained by both antibodies to peptides Ta and Tb, suggesting that these peptides are present in most if not all molecular species of T from bovine brain. Likewise, all of these bands arestained by both monoclonaland polyclonal antibodies to T. In contrast, only one of the two peptide Ta antibodies recognizes the larger two of the four molecular speciesof murine T seen on Coomassie blue-stained gels. However, all of those bands were labeled prominently by the monoclonal and polyclonalantibodies to bovine T. Neither of the two antibodies to peptide Tb label anymurine Xpolypeptides. These immuno- labeling findingsare to be expected from thepredicted amino acid sequence of murine X (10)inthat the latter does not contain peptide T. These studies raise an intriguingpossibility that the twomurine rcDNA clones reported byLee et al. (10) correspond to the two smaller molecular speciesofXthat lack peptide I and, hence are negative on immunolabeling by peptide I antibodies. Peptide Ta ora part of its sequence appears to be present in the two larger molecular species of murine X that are labeled with the peptideTa antibodies and that probably do not correspond to the two reported X cDNAs. Peptide Tb, which differs from peptide Ta by a single amino acid at residue 11 and whose antibodies do not label any murine Xrspecies, might representa speciesdifference between calf and mouse. The PHF polypeptides have been identified immunochem- ically to beabnormallyphosphorylatedXpolypeptides  6). The present study also suggests that X in PHF is phospho- rylated because the immunostaining of these polypeptides in PHF with peptideTa antisera is markedly increased on treatment with alkaline phosphatase. This phosphorylation of Xpolypeptides in PHF appears to be different from phos- phorylation of bovine and murine Xin that the reactivity of only PHF polypeptides on Western blots with peptide Ta antibodies, is increased when pretreated with the phospha- tase. This increase in the accessibility of the antigenic deter- minant(s) to peptide Taantibodies with alkaline phosphatase appears to be due to the phosphatase action and not to any proteolysis, since this effect is specifically inhibited in the presence of phosphatase inhibitors. The epitopes recognized by the antisera to peptides Ta and Tb appear to be different from that seen by the monoclonal antibody -1 to bovineT.Unlike -1, which labels all of the four murine T polypeptides, the antisera to peptide I react with, at most, two of the polypeptides. Furthermore, unlike r-1, the antibodies to peptide Ta recognizeonly the slow moving T polypeptides in PHF. The phosphatase-sensitive reactivity of antisera to peptide Ta thus providesevidence, independent from that reported previously with monoclonal antibody T-1  6, 8), forthe abnormal phosphorylation of T in PHF. Phosphorylation of T in neuronal cell bodies and dendrites in normal rat brain perfused with fixatives contain- ing phosphate buffer has also beenobserved (30). However, the phosphorylated Tpeptide accumulates in filaments only in Alzheimer brain and not in normal brain. The number of the phosphate groups added and/or the site s) of phosphoryla- tion of Tin Alzheimer disease might be different from thatin the normal brain. T promotes microtubule assembly (24), and the phosphorylation of T depresses this assembly  31). Fur- thermore, the amount of cytosolic Tin Alzheimer brain invitro is about half of that in normal brain (32). Therefore, the accumulation of Tin the form of PHF in theaffected neurons might deplete the necessary amounts of functional Tin the affected neuron to maintain microtubules and thereby the axoplasmic flow and neurotransmission. The inability to achieve invitro assembly of microtubules from Alzheimer disease brain(29) is consistent with this possibility. Note Added in Proof. The amino acid sequences of peptides Ta and lb are highly homologous to amino acid residues 28-46 of the cDNA- predicted sequence of bovine T (33). The immunostaining of PHF on tissue sections andof PHF polypeptides on Western blots with antisera to peptides Ta and Tb confirm our previous findings  6, 15) showing the. presence of T polypeptides in their entirety ornear entirety in PHF. Our findings predict thatthe cDNA-derived se- quences of human r (11, 34) reported to date do not represent all isoforms of T seen in PHF. We thank Dr. L. I Binder (University of Alabama,Birmingham) for a generous supply of monoclonal antibody v-1 toT polypeptides and for critical reading of the manuscript, the Biomedical Photog-raphy Unit (Institute for Basic Research, Staten Island, NY) for the Ne'urobiology: Iqbal et al.  Proc. Natl. Acad. Sci. USA 86 (1989) preparation of the figures, and Marie Cappolaand Concetta Vene- ziano for typingthe manuscript.This work was supported inpart by New York State Office ofMental Retardation andDevelopmental Disabilities, National Institutes of Health Grants AG 05892, NS 18105, and AG/NS 04220, and a grant from the Alzheimer's Disease Research Program of the American Health Assistance Foundation (Rockville, MD). 1. Cleveland, D. W., Hwo, S.-Y.   Kirschner, M. W. (1977) J. Mol. Biol.116, 207-225. 2. Cleveland, D. W., Hwo, S.-Y.   Kirschner, M. W. (1977) J. Mol. Biol. 116, 227-247. 3. Drubin, D. G.   Kirschner, M. W. (1986) J. Cell Biol.103, 2739-2746. 4. Baudier, J.   Cole, D. R. (1987) J. Biol. 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