U.S. patent application number 12/185319 was filed with the patent office on 2009-06-25 for novel tools for the diagnosis and treatment of alzheimer's disease.
This patent application is currently assigned to MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNGDER WISSENSCHAFTEN, E.V.. Invention is credited to Jacek Biernat, Gerard Drewes, Birgit Lichtenberg-Kraag, Eckhard Mandelkow, Eva-Marie Mandelkow, Barbara Steiner.
Application Number | 20090162336 12/185319 |
Document ID | / |
Family ID | 26129111 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090162336 |
Kind Code |
A1 |
Mandelkow; Eva-Marie ; et
al. |
June 25, 2009 |
Novel Tools for the Diagnosis and Treatment of Alzheimer's
Disease
Abstract
The invention relates to epitopes of the tau protein which are
specifically occurring in a phosphorylated state in tau protein
from Alzheimer paired helical filaments, to protein kinases which
are responsible for the phosphorylation of the amino acids of the
tau protein giving rise to said epitopes, and to antibodies
specific for said epitopes. The invention further relates to
pharmaceutical compositions for the treatment or prevention of
Alzheimer's disease, to diagnostic compositions and methods for the
detection of Alzheimer's disease and to the use of said epitopes
for the generation of antibodies specifically detecting Alzheimer
tau protein. Additionally, the invention relates to methods for
testing drugs effective in dissolving Alzheimer paired helical
filaments or preventing the formation thereof.
Inventors: |
Mandelkow; Eva-Marie;
(Hamburg, DE) ; Mandelkow; Eckhard; (Hamburg,
DE) ; Lichtenberg-Kraag; Birgit; (Barenklau, DE)
; Biernat; Jacek; (Hamburg, DE) ; Drewes;
Gerard; (Hamburg, DE) ; Steiner; Barbara;
(Ludwigshafen, DE) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 SOUTH WACKER DRIVE, 6300 SEARS TOWER
CHICAGO
IL
60606-6357
US
|
Assignee: |
MAX-PLANCK-GESELLSCHAFT ZUR
FORDERUNGDER WISSENSCHAFTEN, E.V.
Munchen
DE
|
Family ID: |
26129111 |
Appl. No.: |
12/185319 |
Filed: |
August 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09640737 |
Aug 17, 2000 |
7408027 |
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12185319 |
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08244603 |
Nov 28, 1994 |
6200768 |
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PCT/EP92/02829 |
Dec 7, 1992 |
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09640737 |
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Current U.S.
Class: |
514/1.1 ; 435/18;
435/194; 435/68.1; 435/7.1; 530/300; 530/387.9 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 9/12 20130101; C07K 16/40 20130101; G01N 2333/4709 20130101;
C07K 14/4711 20130101; Y10S 435/961 20130101; G01N 33/6896
20130101; C12N 9/16 20130101; A61K 39/00 20130101; G01N 2800/2821
20130101; C12N 9/1205 20130101; A61P 25/28 20180101; C07K 16/18
20130101; Y10S 436/811 20130101 |
Class at
Publication: |
424/94.6 ;
530/300; 435/68.1; 435/194; 514/2; 530/387.9; 435/18; 435/7.1 |
International
Class: |
A61K 38/46 20060101
A61K038/46; C07K 2/00 20060101 C07K002/00; C12P 21/02 20060101
C12P021/02; C12N 9/12 20060101 C12N009/12; A61K 38/00 20060101
A61K038/00; C07K 16/18 20060101 C07K016/18; C12Q 1/34 20060101
C12Q001/34; G01N 33/53 20060101 G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 6, 1991 |
DE |
DE91120974.0 |
Nov 16, 1992 |
DE |
DE92119551.7 |
Claims
1. A combination of epitopes of the tau protein which are
specifically occurring in a phosphorylated state in tau protein
from Alzheimer paired helical filaments, said combination including
the phosphorylatable serine residues 46, 199, 202, 235, 262, 293,
324, 356, 396, 404 and/or 422 and/or the phosphorylatable threonine
residues 50, 69, 111, 153, 175, 181, 205, 212, 217 and/or 231, with
the proviso that said combination is not the combination Ser 202,
Ser 235, Ser 404, Thr 205.
2. The combination according to claim 1 which contains an epitope
comprising the amino acid sequence TABLE-US-00004 KESPLQ, YSSPGSP,
PGSPGT, YSSPGSPGTPGS, PKSPSS, YKSPVVS, GDTSPRH, MVDSPQL; PLQTPTE,
LKESPLQTPTED, AKSTPTA, IGDTPSL, KIATPRGA, PAKTPPA, APKTPPS,
PAKTPPAPKTPPS, SPGTPGS, RSRTPSL, SLPTPPT, RSRTPSLPTPPT, VVRTPPK,
VVRTPPKSPSSA, KIGSTENLK, KCGSKDNIK, KCGSLGNIH, or KIGSLDNITH.
3. Use of a protein kinase for specifically converting tau protein
to Alzheimer tau protein by phosphorylation of the amino acid motif
ser-pro or thr-pro, said protein kinase having the following
biochemical properties: (a) It phosphorylates ser-pro and thr-pro
motifs in tau protein; (b) it has an M.sub.r of 42 kD; (c) it is
activated by ATP and has a K.sub.m of 1.5 mM; (d) it is activated
by tyrosine phosphorylation; (e) it is recognized by an anti-MAP
kinase antibody; and (f) it is deactivated by phosphatase PP2a.
4. Use according to claim 3, wherein said protein kinase is
obtainable by (a) homogenizing porcine brain in 10 mM Tris-HCl, pH
7.2, 5 mM EGTA, 2 mM DTT and a cocktail of protease inhibitors
(leupeptin, aprotinin, pepstatin A, .alpha.2-macroglobulin, PMSF);
(b) centrifugating the homogenate at 100,000.times.g for 30 minutes
at 4.degree. C.; (c) removing the supernatant after centrifugation;
(d) precipitating the crude protein by ammonium sulfate
precipitation; (e) desalting the crude preparation by gel
filtration; (f) activating the crude enzyme by incubation in
activation buffer; (g) further purifying the crude preparation by
ion exchange chromatography; and (h) identifying the enzyme by
Western blotting.
5. A protein kinase which is capable of specifically converting tau
protein to Alzheimer tau protein by phosphorylating IGS and/or CGS
motifs (Serines 262, 293, 324, 356) in the repeat region of tau
protein.
6. The protein kinase according to claim 5 which is obtainable by
carrying out the following procedures: (A) subjecting mammalian
brain extract to ion exchange chromatography on Mono Q (Pharmacia);
(B) testing the fractions eluted for phosphorylation of tau protein
and influence on binding to microtubules; (C) further purifying the
active fractions by gel chromatography; (D) subjecting the fraction
eluting at about 35 kDal to ion exchange chromatography on mono Q;
and (E) collecting the major peak eluting between 200 and 250 mM
NaCl; and has the following characteristics: (a) it binds to mono Q
but not to Mono S; (b) it has an acidic pI; (c) it shows a major
band (>95%) at 35 kDal and a minor band (<5%) at 41 kDal on
silver-stained gels; (d) it incorporates a phosphate amount of 3.2
Pi into htau34, as described in FIG. 19 and defined in Neuron 3
(1989), 519-526, 3.4 Pi into htau40, as described in FIGS. 18 and
19 and defined in Neuron 3 (1989), 519-526, 3.3 Pi into htau23, as
defined in Neuron 3 (1989), 519-526 and 2.8 Pi into mutant htau23
(Ser262.fwdarw.Ala); and (e) it phosphorylates serine residues 262,
293, 324 and 356 of tau protein;
7. The protein kinase according to claim 5 which is obtainable by
carrying out the following steps: (A) preparation of high spin
supernatant of extract from mammalian brain; (B) subjecting the
brain extract to chromatography on ion exchange Q-Sepharose
(Pharmacia); (C) testing the fractions and flowthrough for
phosphorylation of tau protein and influence on binding to
microtubules; (D) chromatography of flowthrough on S-Sepharose,
wherein the kinase activity elutes at 250 mM NaCl; (E)
chromatography on heparin agarose, wherein the kinase activity
elutes at 250 mM NaCl; (F) gel filtration, wherein the kinase
activity elutes at 70 kDal; and (G) chromatography on Mono Q,
wherein the kinase activity elutes at 150 mM NaCl; and has the
following characteristics: (a) it does not bind to Q-Sepharose but
to S-Sepharose; (b) it has an alkaline pI; (c) it shows a major
band around 70 kDal on SDS gels; (d) it incorporates 3-4 phosphates
into htau34, as described in FIG. 19 and defined in Neuron 3
(1989), p. 519-526, htau40, as described in FIGS. 18 and 19 and
defined in Neuron 3 (1989), p. 519-526, htau23, as defined in
Neuron 3 (1989), p. 519-526, and the construct K19 (i.e., the
four-repeat microtubule binding region); (e) it does not
phosphorylate a mutant of K19 where Ser 262, 293, 324, and 356 are
mutated into Ala; and (f) it phosphorylates Ser 262, 293, 324, and
356 or tau protein.
8. The protein kinase according to claim 5, which is a 70 kDal
kinase and phosphorylates the two IGS motifs and the two CGS motifs
of tau protein (Serines 262, 293, 324, 356) and may be obtained as
follows: (A) preparation of high spin supernatant of brain extract;
(B) chromatography on Q-Sepharose; (C) chromatography of
flowthrough on S-Sepharose, wherein the kinase activity elutes at
250 mM NaCl: (D) chromatography on heparin agarose, wherein the
kinase activity elutes at 250 mM NaCl; (E) gel filtration, wherein
the kinase activity elutes at 70 kDal; (F) chromatography on Mono
Q, wherein the kinase activity elutes at 150 mM NaCl.
9. Use of a protein kinase which specifically phosphorylates
serines 46, 199, 202, 235, 262, 293, 324, 356, 396, 404, 422,
and/or threonines 50, 69, 111, 153, 175, 181, 205, 212, 217, 231 of
the tau protein for specifically converting tau protein to
Alzheimer's tau protein.
10. Use according to claim 3, 4 or 9, wherein said protein kinase
is glycogen synthase kinase-3 (isoform .alpha., 51 kD and/or
.beta., 45 kD) or cdk2-cyclin A (33 kD) or MAP kinase.
11. Use according to any one of claims 3, 4, 9 or 10, wherein said
protein kinase is a protein kinase from human brain, porcine brain,
or another source.
12. A pharmaceutical composition containing a specific inhibitor
for the protein kinase as defined in any one of claims 3 to 11,
optionally in combination with a pharmaceutically acceptable
carrier and/or diluent for use in the treatment of Alzheimer's
disease.
13. The pharmaceutical composition according to claim 12 which
contains as the specific inhibitor a combination of oligo- or
polypeptides comprising an epitope according to claim 1 or 2.
14. An antibody which specifically recognizes an epitope contained
in the combination according to claim 1 or 2.
15. An antibody which specifically recognizes the protein kinase
according to any one of claims 5 to 8.
16. The antibody according to claim 14 or 15 which is a monoclonal
antibody.
17. A diagnostic composition for the detection and/or monitoring of
Alzheimer's disease comprising: a combination of epitopes according
to claim 1 or 2; a kinase as defined in any one of claims 3 to 11;
an antibody according to claim 14 or 16; and/or an antibody
according to claim 15 or 16.
18. A method for the in vitro diagnosis of the onset of Alzheimer
disease comprising assaying a cerebrospinal fluid isolate of a
patient or carrying out a biopsy of nerve tissue and testing said
tissue for the presence of a phosphorylated serine residue in
position 262 of tau protein.
19. A method for the in vitro diagnosis and/or monitoring of
Alzheimer's disease comprising assaying a cerebrospinal fluid
isolate of a patient or carrying out a biopsy of nerve tissue and
testing said tissue for the presence of a phosphorylated Alzheimer
tau protein containing a combination of epitopes according to claim
1 or 2; for the presence of a protein kinase as defined in any one
of claims 3 to 11; or for the presence of phosphatases PP2a, PP1
and/or calcineurin.
20. The method according to claim 18 or 19, wherein the Alzheimer
tau protein and the phosphorylation of serine residue 262 of tau
protein, respectively, are detected by using an antibody according
to claim 14 or 15.
21. The method according to claim 18 or 19, wherein the protein
kinase is detected by using an oligo or polypeptide comprising an
epitope contained in the combination according to claim 1 or 2
and/or by using an antibody as defined in claim 16.
22. A method for the in vitro conversion of the tau protein into
Alzheimer tau protein wherein normal tau protein is treated with a
protein kinase as defined in any one of claims 3 to 11 under
conditions which allow the phosphorylation of said normal tau
protein.
23. Use of an epitope contained in the combination according to
claim 1 or 2 for the generation of Alzheimer tau protein specific
antibodies or antibodies to a tau protein specific for the onset of
Alzheimer disease.
24. A pharmaceutical composition for use in the treatment or
prevention of Alzheimer's disease comprising an inhibitor of the
formation of Alzheimer paired helical filaments from tau protein
dimers.
25. An in vitro method for testing drugs effective in dissolving
Alzheimer paired helical filaments comprising the following steps:
(a) allowing the formation of Alzheimer paired helical filaments
from polypeptides comprising tau derived sequences under
appropriate conditions; (b) incubating the Alzheimer paired helical
filaments with the drug to be tested; and (c) examining the result
of the incubation of step (b) with respect to the dissolution of
the Alzheimer-like paired helical filaments.
26. The method according to claim 25, wherein the conditions of
step (a) comprise an environment of 0.3 to 0.5 M Tris-HCl and pH
5.0 to 5.5 without additional salts.
27. An in vitro method for testing drugs effective in the
prevention or reduction of the formation of Alzheimer paired
helical filaments comprising the following steps: (a) incubating
the drug to be testing with polypeptides comprising tau-derived
sequences under conditions which allow the formation of Alzheimer
paired helical filaments in the absence of said drug; and (b)
examining the result of the incubation of step (a) with respect to
the presence or absence of Alzheimer paired helical filaments in
the incubation mixture.
28. The method according to any one of claims 25 to 27 wherein said
polypeptides comprise essentially the repeats from the C-terminal
part of the protein only.
29. The method according to any one of claims 25 to 28 wherein said
polypeptides are K11 and/or K12.
30. A method for testing drugs effective in dissolving Alzheimer
paired helical filaments comprising the following steps: (a)
introducing a functional gene encoding a MAP kinase under the
control of suitable regulatory regions into a cell expressing or
overexpressing tau protein; (b) allowing the formation of
phosphorylated tau protein and of Alzheimer paired helical
filaments; (c) isolating said Alzheimer paired helical filaments;
(d) applying the drug to be tested to said paired helical filaments
under appropriate conditions, and (e) examining the effect of said
drug on said paired helical filaments.
31. The method according to claim 31, wherein said cell expressing
tau protein is a neuroblastoma, chromocytoma or primary nerve
cell.
32. Pharmaceutical composition for the treatment of Alzheimer
disease comprising a PP2a and/or PP1 and/or calcineurin phosphatase
as the active or one of the active ingredients.
Description
[0001] The invention relates to epitopes of the tau protein which
are specifically occurring in a phosphorylated state in tau protein
from Alzheimer paired helical filaments, to protein kinases which
are responsible for the phosphorylation of the amino acids of the
tau protein giving rise to said epitopes, and to antibodies
specific for said epitopes. The invention further relates to
pharmaceutical compositions for the treatment or prevention of
Alzheimer's disease, to diagnostic compositions and methods for the
detection of Alzheimer's disease and to the use of said epitopes
for the generation of antibodies specifically detecting Alzheimer
tau protein. Additionally, the invention relates to methods for
testing drugs effective in dissolving Alzheimer paired helical
filaments or preventing the formation thereof.
[0002] The brains of Alzheimer patients contain two characteristic
types of protein deposits, the plaques and the tangles. These
structures have been of peak importance in Alzheimer research
during the last few years (for a recent review see Goedert et al.,
Current Opinion in Neurobiology 1 (1991), 441 to 447). A prominent
component of the tangles are the paired helical filaments (PHFs).
It seems now clear that the PHFs are largely made up of the
microtubule-associated protein tau which is normally attached to
the neuronal microtubule network and, furthermore, particularly
enriched in the axons.
[0003] There are six isoforms of tau in human brain that arise from
alternative splicing of a single gene. All these isoforms also
occur in PHFs (Goedert et al., Neuron 3 (1989), 519-526). The main
biochemical differences between normal and Alzheimer PHF tau
protein known so far may be summarized as follows: [0004] (1) PHF
tau protein is, in contrast to normal tau protein, highly insoluble
which makes a biochemical analysis difficult; [0005] (2) PHF tau
protein reacts with certain antibodies in a phosphorylation
dependent manner, suggesting a special phosphorylation status
(Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA 83 (1986),
4913-4917, Nukina et al., Proc. Natl. Acad. Sci. USA 84 (1987),
3415-3419); [0006] (3) PHF tau protein has a lower electrophoretic
mobility in SDS gels, suggesting a higher M.sub.r value which may
be related to its phosphorylation pattern (Steiner et al., EMBO J.
9 (1990), 3539-3544); [0007] (4) PHF tau protein forms paired
helical filaments with a characteristic 78 nm crossover repeat
(Crowther and Wischik, EMBO J. 4 (1985), 3661-3665).
[0008] Tau protein purified from brain has very little secondary
structure (as judged by CD spectroscopy), and a sedimentation
constant of 2.6 S, pointing to a highly asymmetric shape (Cleveland
et al., J. Mol. Biol. 1161 (1977), 227-247, in agreement with
electron microscopic data (Hirokawa et al., J. Cell. Biol. 107
(1988), 1449-1459. The C-terminal half contains 3 or 4 internal
repeats which are involved in microtubule binding and promoting
their assembly (hence "assembly domain"). This domain can be
phosphorylated by several protein kinases (Steiner et al., EMBO J.
9 (1990), 3539-3544), a point that may be significant in view of
the abnormal phosphorylation of Alzheimer tau (see, e.g.
Grundke-Iqbal et al., ibid.). Moreover, the repeat region also lies
in the core of Alzheimer paired helical filaments (see, e.g.
Goedert et al., ibid.; Jakes et al. EMBO J. 10 (1991),
2725-2729).
[0009] It has been hypothesized that PHF tau protein has a lower
affinity for microtubules compared to normal tau proteins since a
similar effect has been found when normal tau is phosphorylated in
vitro by some kinases (Lindwall and Cole, J. Biol. Chem. 259
(1984), 5301-5305). Lack or reduced binding to microtubules might
therefore be a result of abnormal phosphorylation of the tau
protein. This abnormal state might lead to microtubule disassembly
and interfere with vital neuronal processes, such as rapid axonal
transport. The abnormally phosphorylated tau proteins might then
aggregate into PHFs. As a consequence thereof the neurons would
eventually die thus setting the stage for the generation of the
Alzheimer's disease.
[0010] Up to now, it was not known which protein kinases are
responsible for the abnormal phosphorylation. Ishiguro et al.
(Neuroscience Letters 128, (1991), 195-198) have isolated a kinase
fraction from bovine brain extracts which contain a protein kinase
recognizing the serine/threonine proline motif. This kinase
phosphorylated residues Ser 144, Thr 147, Ser 177 and Ser 315 of
the tau protein. These residues differed from the ones reported by
others (Lee et al., Science 251 (1991), 675-678). Therefore, it
remains unclear which protein kinase and which target amino acid
residue(s) are involved in the generation of Alzheimer's disease,
if at all.
[0011] It is, moreover, of utmost importance for the diagnosis of
Alzheimer's disease, in particular at an early stage of the disease
process, to develop antibodies which are specifically directed to
epitopes on the protein which are characteristic of the Alzheimer
state. A monoclonal antibody, TAU1, has been isolated which is
capable of distinguishing between phosphorylated and
non-phosphorylated forms of the tau protein (see, e.g., Lee et al.,
ibid.). However, this antibody specifically recognizes
dephosphorylated tau protein which is seemingly not associated with
the Alzheimer state. Another antibody, Alz 50 (Ksiezak-Reding et
al., J. Biol. Chem. 263 (1988), 7943-7947) reacts with PHFs as well
as with tau protein. Sternberger et al., Proc. Natl. Acad. Sci. USA
82 (1985), 4774-4776, have isolated an antibody, SMI 34, which
recognizes a phosphorylated epitope common to Alzheimer tau protein
and neurofilament protein. Finally, Lee et al. (ibid.) made
antibodies directed to a phosphorylated peptide comprising the KSPV
motif in the C-terminal region of the tau protein. All these
antibodies known in the art have the disadvantage that for none of
them it is known whether they recognize an epitope which is
uniquely characteristic for the Alzheimer's disease state.
[0012] Furthermore, no reliable data on the fine structure of
Alzheimer paired helical filaments, nor on the mode or regulation
of their formation from tau proteins is available so far. For the
prevention of the formation of PHFs it would be highly advantageous
if the mode of assembly of PHFs from tau protein and the regulatory
mechanisms underlying said assembly were known.
[0013] Thus, the technical problem underlying the present invention
was to provide a phosphorylated epitope characteristic for the
Alzheimer tau protein, a kinase activity which specifically
catalyzes this phosphorylation, pharmaceutical compositions
comprising inhibitors to said kinases, antibodies for recognizing
said epitopes, diagnostic compositions containing said epitopes,
methods involving kinases and/or antibodies for the in vitro
diagnosis of Alzheimer's disease, methods for the in vitro
conversion of normal tau protein into Alzheimer tau protein and
methods for testing drugs effective in dissolving Alzheimer PHFs or
preventing the formation thereof.
[0014] The solution to the above technical problem is achieved by
providing the embodiments characterized in the claims.
[0015] Accordingly, the present invention relates to an epitope of
the tau protein which is specifically occurring in a phosphorylated
state in tau protein from Alzheimer paired helical filaments.
[0016] The term "phosphorylated state in tau proteins from
Alzheimer paired helical filaments" refers to a state of the tau
protein where tau shows an upward M.sub.r shift, has a reduced
binding to microtubules and is phosphorylated at ser or thr
followed by pro, or certain serines in the repeat region (see
below).
[0017] Note: Amino acids are denoted by the one-letter or
three-letter code; see e.g. Lehninger, Biochemistry, 2nd edition,
Worth Publishers, New York, 1975, page 72.
[0018] There may be one or more epitopes of the tau protein which
specifically occur in a phosphorylated state in Alzheimer paired
helical filaments. These epitopes may, moreover, be phosphorylated
by a single or different enzymes displaying phosphorylating
activity.
[0019] In a preferred embodiment of the present invention, said
epitopes are specifically phosphorylated by a protein kinase from
mammalian brain having the following biochemical properties: [0020]
(a) it phosphorylates ser-pro and thr-pro motifs in tau protein;
[0021] (b) it has an M.sub.r of 42 kD; [0022] (c) it is activated
by ATP and has a K.sub.m of 1.5 mM; [0023] (d) it is activated by
tyrosine phosphorylation; [0024] (e) it is recognized by an
anti-MAP kinase antibody; and [0025] (f) it is deactivated by
phosphatase PP2a.
[0026] The term "ser-pro and thr-pro motifs" as used herein refers
to a phosphorylatable ser or thr residue followed by a pro residue.
These types of sites are phosphorylated by the isoforms of MAP
kinase, GSK-3, and cdk2 (see below).
[0027] The term "anti-MAP kinase antibody" refers to an antibody
which specifically recognizes a mitogen activated protein kinase
(MAP kinase). This kinase probably belongs to a family of closely
related enzymes which have been referred to in the art by different
names, e.g. MAP2 (microtubule-associated protein 2, see e.g. de
Miguel et al., DNA and Cell Biology 10 (1991), 505-514) kinase, MBP
(myelin basic protein) kinase or ERK1 (for a review, see Hunter,
Meth. Enzym. 200 (1991), 1-37). MAP kinase is similar with respect
to its biochemical properties to functionally similar enzymes from
a variety of sources (Hunter, ibid.).
[0028] In another preferred embodiment of the present invention
said epitope includes the phosphorylatable serine residues 46, 199,
202, 235, 396, 404 and/or 422 and/or the phosphorylatable threonine
residues 50, 69, 111, 153, 175, 181, 205, 212, 217 and/or 231; see
FIG. 1a.
[0029] The numbering of the amino acids was done in line with the
largest human tau isoform, htau 40, see Goedert et al. (1989
ibid.).
[0030] In a particularly preferred embodiment said epitope includes
the phosphorylatable serine residue of amino acid position 262.
This is phosphorylated by the brain extract and the KD and 70 KD
kinases prepared from it; see below. In accordance with the present
invention it has been shown that phosphorylation of said residue
significantly interferes with binding of tau protein to
microtubuli. This epitope may be used for diagnostic in vitro
methods to test for the onset of Alzheimer disease.
[0031] In another particularly preferred embodiment said epitope
includes the phosphorylatable serine residues 262, 293, 324 and
409.
[0032] Accordingly, another object of the invention is to provide a
method for testing the onset of Alzheimer disease by assaying the
phosphorylation status of serine in position 262 and the other
Ser-Pro or Thr-Pro motifs named above. This may e.g. be done by
incubating a sample of cerebrospinal fluid of a patient or a sample
of nerve tissue after biopsy with a monoclonal or polyclonal
antibody capable of distinguishing between a phosphorylated and a
non-phosphorylated serine 262 comprising epitope.
[0033] The epitopes of the invention may comprise one or more of
the residues enumerated above. Moreover, the epitopes of the
present invention may comprise only one or more phosphorylated
serine residues, one or more phosphorylated threonine residues or a
combination thereof. The actual composition of the epitope may be
determined by methods which are known in the art. It is also clear
to the person skilled in the art that other amino acids of the
protein may contribute to the epitope which is recognized by an
antibody directed against the sites of tau protein which are
phosphorylated by MAP kinase.
[0034] In a further preferred embodiment of the present invention,
said epitope comprises the amino acid sequences
TABLE-US-00001 KESPLQ, YSSPGSP, PGSPGT, YSSPGSPGTPGS, PKSPSS,
YKSPVVS, GDTSPRH, MVDSPQL; PLQTPTE, LKESPLQTPTED, AKSTPTA, IGDTPSL,
KIATPRGA, PAKTPPA, APKTPPS, PAKTPPAPKTPPS, SPGTPGS, RSRTPSL,
SLPTPPT, RSRTPSLPTPPT, VVRTPPK, VVRTPPKSPSSA, KIGSTENLK, KCGSKDNIK,
KCGSLGNIH, KIGSLDNITH.
[0035] Again, it is to be understood that not all of the amino
acids of the peptide necessarily contribute to the specific site
actually recognized by the antibody.
[0036] Another object of the present invention is to provide a
protein kinase which is capable of specifically converting tau
protein to Alzheimer tau protein by phosphorylation of the amino
acid motif ser-pro or thr-pro.
[0037] Preferably, said protein kinase belongs to the class of MAP
kinases. These kinases can be used for various purposes, e.g. for
the in vitro conversion of tau protein into Alzheimer tau protein.
The Alzheimer tau protein thus obtainable may be used to study e.g.
substances which are capable of inhibiting its formation or the
formation of PHFs. Moreover, they may be used for the development
of drugs capable of dissolving said PHFs or for converting
Alzheimer tau protein into normal tau protein. It is also
conceivable that a system based on the ability of the protein
kinase of the invention to convert normal into Alzheimer tau
protein will provide a well defined in vitro system for Alzheimer's
disease.
[0038] In a preferred embodiment of the invention, said protein
kinase has the following biochemical properties: [0039] (a) it
phosphorylates ser-pro and thr-pro motifs in tau protein; [0040]
(b) it has an M.sub.r of 42 kD; [0041] (c) it is activated by ATP
and has a K.sub.m of 1.5 mM; [0042] (d) it is activated by tyrosine
phosphorylation; [0043] (e) it is recognized by an anti-MAP kinase
antibody; and [0044] (f) it is deactivated by phosphatase PP2a.
[0045] The term "M.sub.r" is defined as the relative molecular
weight determined by SDS gel electrophoresis.
[0046] In still another preferred embodiment of the invention, said
protein kinase is obtainable by carrying out the following steps:
[0047] (a) homogenizing porcine brain in 10 mM Tris-HCl, pH 7.2, mM
EGTA, 2 mM DTT and a cocktail of protease inhibitors (leupeptin,
aprotinin, pepstatin A, .alpha.2-macroglobulin, PMSF (phenyl methyl
sulphonyl fluoride)); [0048] (b) centrifugating the homogenate at
100,000.times.g for 30 minutes at 4.degree. C.; [0049] (c) removing
the supernatant after centrifugation; [0050] (d) precipitating the
crude protein by ammonium sulfate precipitation; [0051] (e)
desalting the crude preparation by gel filtration; [0052] (f)
activating the crude enzyme by incubation in activation buffer;
[0053] (g) further purifying the crude preparation by ion exchange
chromatography; and [0054] (h) identifying the enzyme by Western
blotting.
[0055] The term "activation buffer" is defined as a buffer
comprising 25 mM Tris, 2 mM EGTA, 2 mM DDT, 40 mM
p-nitrophenylphosphate, 10 .mu.M okadaic acid, 2 mM MgATP, and
protease inhibitors.
[0056] Another preferred embodiment of the present invention
relates to a protein kinase which is capable of specifically
converting tau protein to Alzheimer tau protein by phosphorylating
IGS and/or CGS motifs in the repeat region of tau protein.
[0057] In a further preferred embodiment of the kinase of the
invention, said kinase is obtainable by carrying out the following
steps: [0058] (A) Subjecting mammalian brain extract to ion
exchange chromatography on Mono Q (Pharmacia); [0059] (B) testing
the fractions eluted for binding to microtubules and
phosphorylation of the protein; [0060] (C) further purifying the
fractions binding to microtubules and capable of phosphorylating
tau protein by gel chromatography; [0061] (D) subjecting the
fraction eluting at about 35 kDal to ion exchange chromatography on
Mono Q;
[0062] (E) collecting the major peak eluting between 200 and 250 mM
NaCl;
and has the following characteristics: [0063] (a) it binds to Mono
Q but not to Mono S; [0064] (b) it has an acidic pI; [0065] (c) it
shows a major band (>95%) at 35 kDal and a minor band (<5%)
at 41 kDal on silver-stained gels; [0066] (d) it incorporates a
phosphate amount of 3.2 Pi into htau34, 3.4 Pi into htau40, 3.3 Pi
into htau23 and 2.8 Pi into mutant htau23 (Ser262.fwdarw.Ala); and
[0067] (e) it phosphorylates serine residues 262, 293, 324 and 409
of tau protein.
[0068] Said brain extract may e.g. be human or bovine brain
extract.
[0069] In still another preferred embodiment, the kinase of the
present invention is obtainable by the following steps: [0070] (A)
preparation of high spin supernatant of extract from mammalian
brain; [0071] (B) subjecting the brain extract to chromatography on
ion exchange Q-Sepharose (Pharmacia); [0072] (C) testing the
fractions and flowthrough for phosphorylation of tau protein and
influence on binding to microtubules; [0073] (D) chromatography of
flowthrough on S-Sepharose, wherein the kinase activity elutes at
250 mM NaCl; [0074] (E) chromatography on heparin agarose, wherein
the kinase activity elutes at 250 mM NaCl; [0075] (F) gel
filtration, wherein the kinase activity elutes at 70 kDal; [0076]
(G) chromatography on Mono Q, wherein the kinase activity elutes at
150 mM NaCl; and has the following characteristics: [0077] (a) it
does not bind to Q-Sepharose but to S-Sepharose; [0078] (b) it has
an alkaline pI; [0079] (c) it shows a major band around 70 kDal on
SDS gels; [0080] (d) it incorporates 3-4 phosphates into htau34,
htau40, htau23, and the construct K19 (i.e., the four-repeat
microtubule binding region); [0081] (e) it does not phosphorylate a
mutant of K19 where Ser 262, 293, 324, and 409 are mutated into
Ala; and [0082] (f) it phosphorylates Ser 262, 293, 324, and 409 or
tau protein.
[0083] In another preferred embodiment of the invention, the 70
kDal kinase which phosphorylates the two IGS motifs and the two CGS
motifs of tau protein (Serines 262, 293, 324, 409) may be obtained
as follows: [0084] (A) Preparation of high spin supernatant of
brain extract; [0085] (B) chromatography on Q-Sepharose; [0086] (C)
chromatography of flowthrough on S-Sepharose, wherein the kinase
activity elutes at 250 mM NaCl; [0087] (D) chromatography on
heparin agarose, wherein the kinase activity elutes at 250 mM NaCl;
[0088] (E) gel filtration, wherein the kinase activity elutes at 70
kDal; [0089] (F) chromatography on Mono Q, wherein the kinase
activity elutes at 150 mM NaCl.
[0090] (See FIG. 45)
[0091] The brain extract in step A may be e.g. human or another
mammalian brain extract.
[0092] The purification steps noted above are conventional ones
known in the art as described throughout this specification.
[0093] Thus, preparation of the brain extract was carried out as
described in Example 11, whereas binding studies between tau and
taxol-stabilized microtubules may be done as described in Example
(6).
[0094] Furthermore, assays of tau-phosphorylation such as in-gel
assays may be carried out as described in detail in Example 11.
[0095] Chromatography on Mono Q may be carried out as described in
Example 11.
[0096] With respect to the actual conditions used for obtaining
said kinase, a person skilled in the art will be able to deviate
from the protocol outlined above and still obtain the kinase of the
invention. Such a deviation may, e.g., concern the composition of
the protease inhibitor cocktail of step (a): It is conceivable to
use different inhibitors under the proviso that the kinase activity
is not diminished or destroyed.
[0097] In a most preferred embodiment the present invention relates
to a protein kinase which specifically phosphorylates serine
residues 46, 199, 202, 235, 262, 396, 404, 422 and threonine
residues 50, 69, 111, 153, 175, 181, 205, 212, 217, 231 of the tau
protein.
[0098] In another most preferred embodiment, said kinase
phosphorylates serine residue 262.
[0099] A further preferred embodiment relates to a protein kinase
which is glycogen synthase kinase-3, that is, isoform .alpha., 51
kD or .beta. (45 kD) and/or cdk2-cyclin A (33 kD).
[0100] In another preferred embodiment of the present invention,
said kinase is a protein kinase from human brain, porcine brain, or
another source.
[0101] Another object of the invention is to provide pharmaceutical
compositions containing a specific inhibitor for the protein kinase
of the invention, optionally in combination with a pharmaceutically
acceptable carrier and/or diluent.
[0102] The term "specific inhibitor for the protein kinase" refers
to substances which specifically inhibit the enzymatic action of
the protein kinase of the present invention. Inhibitors to enzymes
such as protein kinases and their mode of action are well known in
the art. For example, such an inhibitor may bind to the catalytic
domain of the enzyme thus rendering it incapable of converting its
substrate.
[0103] Examples of such inhibitors are peptide inhibitors and
deactivating phosphatases such as PP2a.
[0104] Another example is the deactivation of kinases by their
phosphatases, e.g., PP-2a in the case of MAP kinase.
[0105] Said pharmaceutical composition may be administered to a
patient in need thereof by a route and in a dosage which is deemed
appropriate by the physician familiar with the case.
[0106] Pharmaceutically acceptable carriers and/or diluents are
well known in the art and may be formulated according to the route
of administration or the special disease status of the patient.
[0107] In a preferred embodiment the present invention relates to a
pharmaceutical composition for use in the treatment of Alzheimer's
disease.
[0108] Again, said pharmaceutical composition may be administered
to a patient in need thereof by route and in a dosage which is
deemed appropriate by the physician handling the case.
[0109] In another preferred embodiment of the present invention,
said pharmaceutical composition contains as the specific inhibitor
at least one oligo- or polypeptide comprising an epitope of the
invention.
[0110] The term "oligo- or polypeptide comprising an epitope of the
invention" refers to peptides which in their two- or
three-dimensional structure reconstitute the epitope of the
invention which is specifically recognized by an antibody directed
thereto. Moreover, said oligo- or polypeptides may solely consist
of the amino acids representing said epitope(s) or they may
comprise additional amino acids. The construction of such oligo- or
polypeptides is well known in the art.
[0111] Another object of the invention is an antibody which
specifically recognizes an epitope of the invention.
[0112] Said antibody may be a serum derived or a monoclonal
antibody. The production of both monoclonal and polyclonal
antibodies to a desired epitope is well known in the art (see, e.g.
Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, 1988). Furthermore, said
antibody may be a natural or an antibody derived by genetic
engineering, such as a chimeric antibody derived by techniques
which are well understood in the art. Moreover, said antibody also
refers to a fragment of an antibody which has retained its capacity
to bind the specific epitope, such as a Fab fragment.
[0113] In a preferred embodiment, the antibody of the present
invention recognizes the protein kinase of the present
invention.
[0114] The term "recognizes the protein kinase of the present
invention" as used herein means that the antibody does not or
insignificantly cross-reacts with other substances such as
different protein kinases present in the same biological
environment. Moreover, it means that the antibody does not or
insignificantly cross-reacts with different protein kinases when
tested in in vitro systems.
[0115] In another preferred embodiment, the antibody of the present
invention is a monoclonal antibody.
[0116] Another object of the invention is to provide diagnostic
compositions for the detection and/or monitoring of Alzheimer's
disease comprising [0117] an epitope of the invention; [0118] a
kinase of the invention; and/or [0119] an antibody of the
invention.
[0120] The diagnostic composition of the invention may comprise for
example an antibody of the invention which specifically recognizes
one of the kinases of the invention or an enhanced level of said
kinases in a sample to be tested. In another embodiment, said
diagnostic composition may comprise an antibody of the invention
directed to one of the epitopes of the invention. Thus, an
Alzheimer correlated disease state of a sample may be detected by
treating said sample with an antibody recognizing the epitope of
the invention. The antibody-epitope (hapten) complex may be
visualized using a second antibody directed to the antibody of the
invention and being labelled according to methods known in the art
(see, e.g., Harlow and Lane, ibid.).
[0121] In still another embodiment of the present invention, said
diagnostic composition may consist of an epitope of the invention
and an antibody of the invention. Treatment of a sample with said
antibody may give rise to conclusions with regard to the disease
state of the corresponding patent, if the binding of said antibody
to said sample is brought in relation to binding of said antibody
to said epitope of the invention used as a reference sample.
[0122] In still another embodiment, the diagnostic composition may
comprise an epitope of the invention, a kinase of the invention and
an antibody of the invention. Kinase activity may be monitored with
respect to phosphorylation of the sample as compared to the
phosphorylation of the epitope of the invention. From the
quantitated kinase activity the phosphorylation state of the tau
protein contained in said sample and therefore the disease state of
the patient may be deduced. The kinase activity may e.g. be deduced
by including a substrate analog in the same reaction, which is
visually detectable upon enzymatic conversion. Such substrate
analogs are widely used in the art. Alternatively, the amount of
phosphorylated tau protein in the sample may be detected after
treatment with the kinase of the invention by employing an antibody
of the invention directed to the phosphorylated epitope and using
the amount of antibody-epitope complex provided by the diagnostic
composition as an internal standard, or by determining the amount
of phosphate incorporated into tau protein by the kinase, e.g. by
radioactive tracer methods which are well known in the art.
[0123] The person skilled in the art is in the position to design
other test systems which combine any of the above objects of the
invention. It is to be understood that all conceivable combinations
fall within the scope of protection of the present invention.
[0124] Another object of the invention is to provide a method for
the in vitro diagnosis and/or monitoring of Alzheimer's disease
comprising assaying a cerebrospinal fluid isolate of a patient or
carrying out a biopsy of nerve tissue [0125] for the presence of a
phosphorylated Alzheimer tau protein containing an epitope of the
invention; [0126] for the presence of a protein kinase of the
invention; or [0127] for the presence of phosphatases PP2a, PP1
and/or calcineurin.
[0128] The "cerebrospinal fluid isolate of a patient" is obtained
by standard medical procedures.
[0129] An example for a nerve tissue suitable for said biopsy is
the olfactory epithelium. The person skilled in the art may carry
out said method employing e.g. the diagnostic tools illustrated in
connection with the diagnostic compositions, supra.
[0130] In a preferred method of the present invention, the
Alzheimer tau protein and the phosphorylation of serine residue 262
of tau protein, respectively, is detected by using an antibody of
the invention.
[0131] Said antibody preferably is an antibody directed to an
epitope of the invention.
[0132] In another preferred embodiment of the invention, the
protein kinase is detected by using an oligo- or polypeptide
comprising an epitope of the invention and/or by using an antibody
of the invention.
[0133] Still another object of the invention is to provide a method
for the in vitro conversion of normal tau protein into Alzheimer
tau protein wherein normal tau protein is treated with a protein
kinase of the present invention under conditions which allow the
phosphorylation of said normal tau protein.
[0134] The term "Alzheimer tau protein" refers to tau protein that
is abnormally phosphorylated (e.g. at ser-pro or thr-pro motifs)
and recognized by Alzheimer-specific antibodies.
[0135] The term "conditions which allow the phosphorylation of said
normal tau protein" refers to conditions allowing the activity,
preferably the optimal activity, of protein kinase. This activity
results in phosphorylation of the substrate at the ser-pro and/or
thr-pro motifs. The phosphorylated substrate may then be recognized
by Alzheimer-specific antibodies.
[0136] Normal tau protein may be derived from natural or
recombinant sources. It is, for the purpose of carrying out the
method of the present invention, however, expedient to use
recombinant material.
[0137] The method of the present invention provides sufficient
amounts of Alzheimer tau protein for a variety of purposes: With
the method of the present invention an in vitro model for the study
of the generation of the Alzheimer state of proteins may be
established (see above). Moreover, inhibitors may be tested which
prevent the conversion of normal to Alzheimer tau protein. These
"inhibitors" may be specific for the epitope to be phosphorylated
by e.g. blocking the epitope or may be directed to various domains
on the protein kinase, as long as they prevent or disturb its
biological activity. Another type of inhibition is the antagonistic
action of phosphatases on tau or its kinases. Furthermore, the
Alzheimer tau protein generated by the method of the present
invention may be employed in binding studies to microtubule
structures thus contributing to the elucidation of the molecular
basis underlying Alzheimer's disease.
[0138] The person skilled in the art knows how to employ the method
of the present invention for a variety of different purposes which
all fall under the scope of protection of the present
invention.
[0139] The present invention relates, moreover, to the use of an
epitope of the invention for the generation of Alzheimer tau
protein specific antibodies or antibodies to a tau protein specific
for the onset of Alzheimer disease.
[0140] The methods for obtaining said antibodies are well known in
the art; thus, the generation of polyclonal or monoclonal
antibodies may be conducted using standard methods (see, e.g.,
Harlow and Lane, ibid.). If an oligo- or polypeptide is used for
the generation of antibodies it is desirable to couple the peptide
comprising the epitope to a suitable carrier molecule capable of
inducing or enhancing the immune response to said epitope, such as
bovine serum albumin or keyhole limpet hemocyanin. The methods of
coupling hapten (comprising or being identical to the epitope) and
carrier are also well known in the art (Harlow and Lane, ibid.). It
is also to be understood any animal suitable to generate the
desired antibodies may be used therefor.
[0141] In another aspect, the present invention relates to a
pharmaceutical composition for use in the treatment or prevention
of Alzheimer's disease comprising an inhibitor of the formation of
Alzheimer paired helical filaments from tau protein diners.
[0142] In accordance with the present invention, it was found that
tau proteins form antiparallel dimers via assembly of their repeat
units located in the C-terminal domain of the protein. Whereas
dimerization of tau proteins appears to be a physiological process,
the formation of higher order structures such as PHFs seems to be
due to deregulation in the assembly process. Consequently, PHFs are
formed from a number of tau dimers wherein the cross-linking of
dimers may occur via intermolecular disulfide bridging.
[0143] Deregulation of the assembly process with subsequent
formation of PHFs from tau dimers appears to be due to abnormal
phosphorylation of tau proteins because, as has been found in
accordance with the present invention, truncated tau proteins
consisting merely of the repeat units are able to form PHFs,
whereas tau proteins or tau-like proteins comprising the N-terminus
and C-terminus as well are unable to do so.
[0144] An inhibitor useful in the composition of the present
invention is therefore any inhibitor capable of inhibiting the
formation of PHFs from tau dimers regardless of the molecular
mechanism it interferes with. Such an inhibitor may be, for
example, an inhibitor to a protein kinase responsible for abnormal
phosphorylation of tau proteins as a compound interfering with the
formation of intermolecular cross-links or association of tau
dimers.
[0145] A further object of the present invention is to provide a
method for testing drugs effective in dissolving Alzheimer paired
helical filaments comprising the following steps: [0146] (a)
allowing the formation of Alzheimer paired helical filaments from
polypeptides comprising tau-derived sequences under appropriate
conditions; [0147] (b) incubating the Alzheimer paired helical
filaments with the drug to be tested; and [0148] (c) examining the
result of the incubation of step (b) with respect to the
dissolution of the Alzheimer-like paired helical filaments.
[0149] The term "effective in dissolving Alzheimer paired helical
filaments" as used herein is intended to also include partially
dissolved PHFs. For the object of the present invention it is
sufficient that the drug to be tested is effective in the reduction
of the size or the break-up of PHFs, thus fulfilling a
supplementary function in therapy, although a total dissolution by
the drug is preferred.
[0150] The term "polypeptides comprising tau derived sequences"
refers to any polypeptide which comprises sequences from tau
protein capable of forming PHFs regardless of the length of said
sequences or of mutations, deletions, insertions or heterologous
sequences as long as the function of said polypeptides to form PHFs
remains intact.
[0151] The term "appropriate conditions" in connection with the
formation of Alzheimer PHFs refers to any condition which allows
said formation. Said conditions may include the availability of a
MAP kinase if natural tau protein is used.
[0152] In a preferred embodiment, the conditions applied in step
(a) of said method comprise an environment of 0.3 to 0.5 M Tris-HCl
and pH 5.0 to 5.5 without additional salts.
[0153] Still another object of the invention is to provide a method
for testing drugs effective in the prevention or reduction of the
formation of Alzheimer paired helical filaments comprising the
following steps: [0154] (a) incubating the drug to be tested with
polypeptides comprising tau-derived sequences under conditions
which allow the formation of Alzheimer paired helical filaments in
the absence of said drug; and [0155] (b) examining the result of
the incubation of step (a) with respect to the presence or absence
of Alzheimer paired helical filaments in the incubation
mixture.
[0156] The term "conditions which allow the formation of Alzheimer
paired helical filaments in the absence of said drug" refers to any
condition which allows the formation of PHFs provided said drug is
not included in the incubation mixture. A preferred example of such
a condition is an environment of 0.3 to 0.5 M Tris-HCl and pH 5.0
to 5.5 without additional salts.
[0157] The term "presence or absence of Alzheimer paired helical
filaments" as used herein is intended to include results wherein
only a limited amount of PHFs has been formed as compared to
control experiments where no such drug has been used.
[0158] In a preferred embodiment in the above methods, said
polypeptides comprise essentially the repeats from the C-terminal
part of the tau protein only.
[0159] In accordance with the present invention, it was found that
the repeats comprised in the C-terminal domain of the tau protein
are responsible for dimerization of the protein under physiological
conditions and subsequent oligomerization leading to Alzheimer-like
paired helical filaments. The term "Alzheimer-like paired helical
filaments" is used here as opposed to "Alzheimer paired helical
filament" solely to indicate that non-repeat unit parts of the tau
protein normally present in PHFs are absent from PHFs generated by
said polypeptides.
[0160] Accordingly, the polypeptides comprising essentially the
repeat units only provide an ideal in vitro system to study PHF
formation and studies on the fine structure of PHFs.
[0161] In a particularly preferred embodiment, said polypeptides
are comprising mainly the repeat regions of tau, such as K11 and/or
K12.
[0162] K11 and K12 are ideally suited for the above testing
purposes because they are essentially comprised of repeat units
from the tau protein only.
[0163] For the method of the invention, K11 and K12 may be used
alone or in combination.
[0164] In a further aspect, the present invention relates to a
method for testing drugs effective in dissolving Alzheimer paired
helical filaments comprising the following steps: [0165] (a)
introducing a functional gene encoding a MAP kinase under the
control of suitable regulatory regions into a cell expressing or
overexpressing tau protein; [0166] (b) allowing the formation of
phosphorylated tau protein and of Alzheimer paired helical
filaments; [0167] (c) isolating said Alzheimer paired helical
filaments; [0168] (d) applying the drug to be tested to said paired
helical filaments under appropriate conditions; and [0169] (e)
examining the effect of said drug on said paired helical
filaments.
[0170] The term "cell expressing tau protein" as used in step (a),
supra, refers to cells which endogenously express tau or which have
the capacity to express tau and into which a functional tau gene
has been introduced. In the latter case the person skilled in the
art is aware of the fact that the sequence of the introduction of
the genes encoding the MAP-kinase and tau is irrelevant for the
purpose of the method of the invention.
[0171] The term "under appropriate conditions" in step (c), supra,
refers to conditions which allow the drug to be effective in
dissolving PHFs and are particularly optimal conditions.
[0172] Said method is particularly advantageous, since the system
involved which is based on the use of continuously growing cell
lines providing a close image of the in vitro situation provide an
ample supply of phosphorylated tau protein.
[0173] In a preferred embodiment said cell expressing tau protein
is a neuroblastoma or chromocytoma cell or a primary culture of
nerve cells.
[0174] Such cells or cell lines are well known in the art.
Preferred examples are the neuroblastoma cell lines N21 and
PC12.
[0175] These cell lines are particularly preferred because they
express tau endogenously.
[0176] A further object of the invention is a pharmaceutical
composition for the treatment of Alzheimer disease comprising a
PP2a and/or PP-1 and/or calcineurin phosphatase as the active or
one of the active ingredients.
[0177] The Figures show:
[0178] FIG. 1a: Amino acid sequence of tau (isoform htau40, Goedert
et al., 1989). The motifs SP, TP, IGS and CGS are highlighted.
[0179] FIG. 1b: (a) SDS gel of tau isoforms, (b) immunoblots of (a)
and PHF tau with the AT8 antibody. (a) SDS gel. Lane 1, marker
proteins. Lane 2: Tau from bovine brain, showing several isoforms
in a mixed state of phosphorylation. Lane 3, bovine brain tau after
dephosphorylation with alkaline phosphatase. Note that all isoforms
shift to a lower M.sub.r. Lanes 4 and 5: Tau from normal human
brain, before and after dephosphorylation. Lanes 6-11: bacterially
expressed human tau isoforms htau23, 24, 37, 34, 39, 40 (see
Goedert et al., 1989, ibid.). These isoforms have either three or
four internal repeats of 31 residues each in the C-terminal half
(three: htau23, 37, 39; four: htau24, 34, 40). Near the N-terminus
there can be zero, one, or two inserts of 29 residues (zero:
htau23, 24; one: htau37, 34; two: htau39, 40). [0180] (b)
Immunoblots with the AT8 antibody. Lane 1, PHF tau, showing 4-6
isoforms in the range of 60-70 kD; all of them react strongly with
AT8. Lanes 2-11, same preparations as in (a); none of the bovine or
normal human tau isoforms show any reaction.
[0181] FIG. 2: Phosphorylation of bacterially expressed human tau
isoforms with the kinase from brain. (a) SDS gels, (b) immunoblots
with AT8. [0182] (a) Lanes 1 and 2, SDS gel of htau23 before and
after extract phosphorylation (note the upward shift in M.sub.r).
Lanes 3-10 show analogous pairs for other isoforms (htau24, 34, 39,
40). [0183] (b) Immunoblots of (a) with AT8 antibody. It reacts
with all tau isoforms after phosphorylation (even lanes; including
htau37, not shown here).
[0184] FIG. 3: Diagram of constructs K3M, K10, K19, and K17. K19
(99 residues) contains the sequence Q244-E372 of htau23 plus an
N-terminal methionine. This comprises three of the repeats (repeat
1, 3, and 4; repeat 2 is absent in htau23). K10 (168 residues) is
similar, except that it extends to the C-terminus of htau23 (L441).
K17 (145 residues) contains the sequence S198-E372 (assembly domain
starting at the chymotryptic cleavage site, up to end of fourth
repeat, but without the second repeat, plus an N-terminal
methionine). K3M (335 residues) contains the N-terminal 154
residues of bovine tau4, plus the sequence R221-L441 of htau23
(without second repeat). The location of peptide S198-T220 is
indicated in K17. By comparison of the constructs the epitope of
AT8 must be in this region (see FIG. 4).
[0185] FIG. 4: Phosphorylation of htau40 and constructs K10, K17,
K3M, and K19. [0186] (a) SDS gel. Odd lanes, htau40, K10, K17, and
K3M before phosphorylation, even lanes, after phosphorylation. Note
the upward shift of the bands after phosphorylation. In lane 4
there are two bands because K10 is not completely phosphorylated.
[0187] (b) Immunoblot of (a) with AT8. The anti-body reacts only
with htau40 (lane 2) and K17 (lane 6), both in the phosphorylated
state, but not with K10 (lane 4) or K3M (lane 8), although these
constructs are also phosphorylated and show an M.sub.r shift.
[0188] (c) Construct K19 before and after incubation with the
kinase. Lanes 1 and 2, SDS gel; there is no M.sub.r shift and no
phosphorylation, confirmed by autoradiography (not shown). Lanes 3
and 4, immunoblot with AT8, showing no reaction. This confirms that
the epitope is not in the repeat region.
[0189] FIG. 5: Diagram of tryptic peptide S195-R209. The 15 residue
peptide (containing 5 serines and 1 threonine) was labeled with two
radioactive phosphates at S199 and S202, as determined by
sequencing.
[0190] FIG. 6: Phosphorylation and antibody reactions of the
D-mutant of htau23 (S199 and S202 changed into D). Lanes 1 and 2,
SDS gel of htau23 before and after extract phosphorylation; lanes 3
and 4, D-mutant before and after extract phosphorylation. Note that
the D-mutant runs slightly higher than htau23 (lanes 1, 3), but
after phosphorylation both proteins have the same position in the
gel (lanes 2, 4). [0191] Lanes 5-8, immunoblots of lanes 1-4 with
AT8. The antibody reacts only with extract phosphorylated htau23
(lane 6), but neither with the unphosphorylated form (lane 5) nor
with the D-mutant (lanes 7, 8), although it was phosphorylated as
seen by the additional shift and autoradiography (not shown).
[0192] Lanes 9-12, immunoblots of lanes 1-4 with TAU1. This
antibody reacts only with htau23 before phosphorylation (lane 9),
but not with the phosphorylated form (lane 10) nor with the
D-mutant (lanes 11, 12). The aspartic acid apparently mimics a
phosphorylated serine and thus masks the epitope. The minor
reaction of htau23 with TAU1 in lane 10 shows that the protein is
not completely phosphorylated.
[0193] FIG. 7: Time course of phosphorylation of bacterially
expressed human isoform htau23 with the brain kinase activity and
corresponding autoradiogram. [0194] (a) SDS-PAGE of htau23 after
incubation with the kinase between 0 and 24 hours, as indicated.
The unphosphorylated protein is a single band of M.sub.r0=48 kD
(lane 1). Lanes 3-14 show that phosphorylation leads to a
progressive shift to higher M.sub.r with well defined intermediate
stages. The even lanes (numbered 4, 6, etc. below FIG. 1b) are
observed in the presence of 10 .mu.M okadaic acid (OA) (labeled "+"
below FIG. 1a). The odd lanes (3, 5, etc. labeled "-") are without
okadaic acid. The first stage takes about 2 hours (shift to a new
M.sub.r1=52 kD), the second is finished around 10 hours
(M.sub.r2=54 kD), the third is finished around time 24 hours
(M.sub.r3=56 kD); no further shift is observed during the
subsequent 24 hours. Lane 2 shows a mutant that is not of
significance in this context. [0195] (b) Autoradiogram of (a). The
quantitation of the phosphate incorporated (mol P.sub.i/mol
protein) in this experiment was as follows (-OA/+OA): 30 min
(0.5/1.0), 60 min (0.7/1.4), 120 min (1.0/2.0), 10 hours (2.0/3.0),
24 hours (3.2/4.0).
[0196] FIG. 8: (a) SDS gel showing the time course of
phosphorylation of htau23 similar to that of FIG. 1a, but with 10
.mu.M okadaic acid throughout; (b) immunoblot of (a) with the
monoclonal antibody SMI34. The antibody recognizes the protein only
in the second and third stage of phosphorylation, but not in the
first.
[0197] FIG. 9: Binding of tau isoforms to microtubules before and
after phosphorylation. [0198] (a) SDS gel of a binding experiment,
illustrated for the case of the tau isoform htau40 (whose band is
clearly separated from that of tubulin (T) so that both components
can be shown simultaneously, without having to remove tubulin by a
boiling step). The top line indicates pellets (P) or supernatants
(S), with or without phosphorylation for 24 hours (+ or -P.sub.i).
Lanes 1-4, 20 .mu.M tau protein (total concentration),
phosphorylated (lanes 1, 2) or not (lanes 3, 4). The comparison of
lanes 1 and 2 shows that most of the phosphorylated protein is free
(S), while only a small fraction is bound to the microtubules (P).
Lanes 3 and 4 show that in the unphosphorylated state about half of
the protein is bound, the other half free (note also that the
phosphorylated protein bands, lanes 1, 2, are higher in the gel
than the unphosphorylated ones, lanes 3, 4, similar to FIG. 1).
Lanes 5-8, similar experiment with 15 .mu.M htau40. Lanes 9, 10
show the case of 10 .mu.M phosphorylated protein. Lanes 11-15 are
for density calibration with known amounts of htau40 (15, 10, 7.5,
5, and 2.5 .mu.M, resp.). [0199] (b) Binding curves of htau23 and
(c) htau34 to microtubules before (circles) and after 24 hour
phosphorylation (triangles); these curves were derived from SDS
gels similar to that of FIG. 3a. Polymerized tubulin is 30 .mu.M.
Fitted dissociation constants K.sub.d and stoichiometries are as
indicated. In each case the most dramatic effect is on the number
of binding sites which decrease about three-fold upon
phosphorylation, from around 0.5 (i.e. one tau for every two
tubulin dimers) down to about 0.16 (one tau for six tubulin
dimers). Note that the binding of unphosphorylated 4-repeat
isoforms (such as htau34) is particularly tight (K.sub.d round 1-2
.mu.M).
[0200] FIG. 10: Diagram of htau40, showing the location of the 7
ser-pro motifs phosphorylated by the kinase activity. The boxes
labeled 1-4 are the internal repeats involved in microtubule
binding; the second is absent in some isoforms (e.g. htau23). The
two shaded boxes near the N-terminus are inserts absent in htau23
and htau24 so that these molecules have only 6 ser-pro motifs. The
following radioactive tryptic peptides were found: [0201] 24-49:
KDQGGYTHHQDQEGDTDAGLKES.PLQ [0202] 191-209: SGDRSGYSS.PGS.PGTPGSR
[0203] 231-240: TPPKS.PSSAK [0204] 396-406: SPVVSGDTS.PR [0205]
386-405: TDHGAEIVYKS.PVVSGDTS.PR [0206] 407-428:
HLSNVSSTGSIDMVDS.PQLATL [0207] 260-266: IGS.TEML
[0208] FIG. 11: Binding of htau34 to microtubules, before (circles)
and after phosphorylation for 90 min (stage 1, triangles). The
reduction in binding capacity is very similar to that after 24
hours phosphorylation (compare FIG. 9b).
[0209] FIG. 12: SDS-PAGE and immunoblots of tau protein from
Alzheimer and normal human brain with antibodies SMI33, SMI31, and
SMI34. [0210] (a) Lane 1, SDS-PAGE of tau protein from a normal
human control brain, showing 5-6 bands between M.sub.r55 and 65 kD
(somewhat lower than the PHF tau of lane 3). Lane 2, normal human
tau after phosphorylation with kinase activity, resulting in an
upward shift of all bands. Lanes 3, 4, immunoblot of PHF tau with
antibody SE2 which recognizes all tau isoforms independently of
phosphorylation (Kosik et al., Neuron 1 (1988), 817-825). Lane 3,
PHF tau as isolated from an Alzheimer brain; lane 4, after
dephosphorylation with alkaline phosphatase. Note that the bands of
the dephosphorylated protein are shifted down on the gel. [0211]
(b) Immunoblot of (a) with SMI33. The anti-body recognizes normal
human tau (lane 1), and PHF tau after dephosphorylation (lane 4).
[0212] (c) Immunoblot of (a) with SMI31. Note that the antibody
recognizes normal human tau after phosphorylation, and PHF tau in
its natural state of phosphorylation (lanes 2, 3). [0213] (d)
Immunoblot of (a) with SMI34. This antibody recognizes normal human
tau only after phosphorylation (lane 2), and PHF tau (lane 3).
[0214] FIG. 13: Time course of phosphorylation of bacterially
expressed human isoform htau23 (similar to previous figure) and
immunoblots with antibodies SMI33, SMI31, SMI34, TAU1, and AT8.
[0215] (a) SDS-PAGE, phosphorylation times 0-24 hours, showing the
successive M.sub.r shifts. [0216] (b-f) Immunoblots with SMI31,
SMI34, SMI33, TAU1, and AT8. Antibodies SMI33 and TAU1 recognize
htau23 fully up to the end of stage 1 (2 hours), but the epitope
becomes blocked during the second stage. Antibodies SMI31, SMI34,
and AT8 are complementary in that they recognize the protein only
in the second and third stage of phosphorylation. [0217] (g-h)
Immunoblot of htau34 with SMI33 and SMI310 which recognize the
protein from the stage 2 phosphorylation onwards, similar to
SMI31.
[0218] FIG. 14: SDS-PAGE of tau and several constructs, and
immunoblots with the antibodies SMI33, SMI31, and SMI34. [0219] (a)
SDS-PAGE. Lanes 1 and 2: Construct K10 before and after
phosphorylation with the kinase for 24 hours. Lanes 3 and 4:
Construct K17 before and after phosphorylation.
[0220] Lanes 5 and 6: Construct K19 before and after
phosphorylation. All constructs except K19 show a shift upon
phosphorylation. With K10 one observes three shifted bands, with
K17 there is only one shifted band. [0221] (b) Immunoblot of (a)
with SMI33: The anti-body recognizes only K17 in the
unphosphorylated form (lane 3), suggesting that the epitope lies
before the repeats. [0222] (c) Immunoblot of (a) with SMI34. The
anti-body recognizes K10 and K17 in the phosphorylated form (only
top bands, lanes 2, 4). The antibody does not recognize K19 (the
repeat region), but requires sequences on both the N-terminal and
C-terminal side of the repeats. The epitope is therefore
non-contiguous (conformation-dependent). [0223] (d) Immunoblot of
(a) with SMI31. The anti-body recognizes only the top band of the
phosphorylated K10 (lane 2), suggesting that the epitope lies
behind the repeat region.
[0224] FIG. 15: Diagram of point mutants of htau40 and htau23.
[0225] FIG. 16: SDS gel of htau40 and the point mutants of FIG. 15,
and immunoblots with antibodies SMI33, SMI31, and SMI34. [0226] (a)
Lanes 1-8, SDS gel of htau40 and its mutants KAP235, KAP396, and
KAP235/396 in the unphosphorylated and phosphorylated form (+). In
each case phosphorylation leads to an upward shift in the SDS gel.
[0227] (b) Blot of (a) with SMI33. The antibody response is
strongly reduced when S235 is mutated, both in the dephosphorylated
and phosphorylated state (lanes 3+4, 7+8). This indicates that the
(dephosphorylated) first KSP motif is part of the epitope of SMI33.
When S396 is mutated to A the behavior is similar to the parent
molecule, i.e. strong antibody response in the dephosphorylated
state, no reaction in the phosphorylated state, so that S396 does
not contribute to the epitope of SMI33. [0228] (c) Blot of (a) with
SMI31. The antibody recognizes htau40 and all mutants in the
phosphorylated form (lanes 2, 4, 6, 8). This shows that
phosphorylation of the two KSP motifs is not the main determinant
of the epitope. [0229] (d) Blot of (a) with SMI34. The reaction is
similar to SMI31 but more pronounced, again indicating that the two
KSP motifs are not essential.
[0230] FIG. 17: Deletion mutants of tau and their antibody
response. (a) SDS gel of constructs containing only two repeats
(K5-K7) or one repeat (K13-K15), before and after phosphorylation.
(b) Immunoblot of (a) with SMI34. Note that the antibody recognizes
all phosphorylated proteins (K7 only weakly). (c) Immunoblot of (a)
with SMI31. Note that the antibody recognizes the phosphorylated
two-repeat molecules (K5-K5), but not the one-repeat molecules
(K13-K15). Lanes 7 and 8 show htau40 as a control. (d) SDS gel of
constructs K2, K3M, and K4, before and after phosphorylation. (e)
Blot of (d) with SMI34, recognizing only K4 phosphorylated. (f)
Blot of (d) with SMI31, recognizing only K2 phosphorylated.
[0231] FIG. 18: Diagram of htau40 and various mutants used in this
study.
[0232] FIG. 19: Diagram of tau isoforms and constructs used in
studies on tau dimerization and oligomerization [0233] (a)
T8R-1,553 residues, MW 57743, derived from htau40 (see below). It
has two inserts near the N-terminus (29 residues each, hatched), a
repeat domain of four repeats (numbered 1-4) which is duplicated
with a small spacer in between. [0234] (b) T8R-2, 511 residues, MW
53459; it lacks the N-terminal inserts, but has the four repeats
duplicated. [0235] (c) T7R-2,480 residues, MW 50212; similar to
T8R-2, but without the second repeat sequence in the first repeat
domain. [0236] (d) Htau40,441 residues, MW 45850, the largest of
the six human tau isoforms (Goedert et al.), with two N-terminal
inserts and a repeat domain containing four repeats. [0237] (e)
Htau23,352 residues, MW 36760, the smallest of the human tau
isoforms, without the N-terminal repeats and only three repeats.
[0238] (f) K11,152 residues, MW 16326, a repeat domain with four
repeats plus a short tail. [0239] (g) K12,121 residues, MW 13079, a
repeat domain with three repeats plus a short tail.
[0240] FIG. 20: SDS PAGE (4-20%) and gel chromatography of tau
constructs and cross-linked products. Gels a and c were run in
reducing conditions (3 .mu.M DTT in sample buffer), gel b in
non-reducing conditions (except lane 1 with 3 mM DTT in sample
buffer). [0241] (a) Constructs T8R-1, Htau23 and K12. Molecular
weight markers are given on the left. [0242] (b) Construct K12 and
cross-linked products. Cross-linking occurs spontaneously in the
absence of DTT; it can be prevented by DTT, or induced by addition
of PDM or MBS. Aggregation products are labeled on the right
(monomers, dimers, trimers, tetramers etc.). [0243] (c) Silver
stained SDS gel of a Superose 12 gel filtration run of K12
cross-linked by PDM. The dimers (top band) elute before the
monomers. Fractions 16 and 17 were used for electron microscopy.
[0244] (d) Elution profile of Superose 12 gel filtration of
construct K12 monomers and dimers cross-linked with PDM. The
elution positions of calibration proteins are plotted against their
effective hydrated Stokes radii on a logarithmic scale (right
axis). [0245] (e) CD spectrum of construct K12 (8 mg/ml in 40 mM
HEPES pH 7.2, path length 0.01 mm). There is no significant
.alpha.-helical or .beta.-sheet structure. Similar spectra are
obtained with other constructs as well as with full length tau.
[0246] FIG. 21: Synthetic paired helical filaments from construct
K12. [0247] (a) A tangle of synthetic PHFs from K12 (crossover
period of .apprxeq.70-75 nm indicated by arrowheads). The construct
was expressed and purified by the methods described previously
(Steiner et al.). It was dialysed against 0.5 M Tris-HCl, with pH
values between 5.0 and 5.5. The solution was negatively stained
with 2% uranyl acetate. [0248] (b) and (c) Single fibers of
synthetic paired helical filaments made from construct K12. Note
the crossover repeats (arrowheads) and the rod-like particles of
lengths around 100 nm (c, middle). Bar=100 nm.
[0249] FIG. 22: Synthetic paired helical filaments from K12 dimers
cross-linked with PDM and negatively stained with 1%
phosphotungstic acid (micrographs provided by M. Kniel). Bar=100
nm.
[0250] FIG. 23: Paired helical filaments from Alzheimer brain
(micrographs provided by Dr. Lichtenberg-Kraag). [0251] (a) PHFs
from neurofibrillary tangles prepared after Wischik et al., stained
with 1% phosphotungstic acid. This preparation contains homogeneous
long filaments which still retain their pronase sensitive "fuzzy
coat." The crossover repeat is 75-80 nm, the width varies between a
minimum of about 10 nm and a maximum of 22 nm. [0252] (b) PHFs
prepared after Greenberg & Davies. This preparation results in
soluble filaments of shorter length than in (a) and is more
heterogeneous. (1) is a paired helical filament with a 72 nm repeat
and a width varying between 8 and 18 nm; (2) is a straight filament
of 8 nm width; (3) is a twisted filament with a particularly wide
diameter (up to 25 nm); (4) is a straight filament with a wide
diameter (18 nm); (5) is a twisted rod-like particle about 80 nm
long, equivalent to about one crossover period. In many cases the
particles appear to have broken apart across the filament, e.g. the
two rods labeled (4), the twisted filament of (3) and the short
stub to the right of it, or the two straight rods above particle
(3). Bar=100 nm.
[0253] FIG. 24: Electron micrographs of tau isoform htau23 and
construct T8R-1 prepared by glycerol spraying and metal shadowing
[0254] (a) monomers of htau23, [0255] (b) dimers of htau23, [0256]
(c) monomers of T8R-1, [0257] (d) folded forms of T8R-1 (hair-pin
folds showing intramolecular antiparallel association), [0258] (e)
dimers of T8R-1. For lengths see Table 1 and FIG. 7. Interpretative
diagrams are shown on the right. Bar=50 nm.
[0259] FIG. 25: Length histograms of tau constructs and dimers.
[0260] FIG. 26: Electron micrographs of constructs K11 and K12.
[0261] (a) Monomers of K11, [0262] (b) dimers of K11 [0263] (c)
tetramers of K11 formed by longitudinal association of two dimers.
[0264] (c) Monomers of K12, [0265] (d) dimers of K12, [0266] (e)
tetramers of K12. Bar=50 nm.
[0267] FIG. 27: (a) K12 dimers cross-linked by PDM (i.e. Cys322 to
Cys322); [0268] (B) K12 dimers cross-linked by MBS (i.e. Cys322 to
nearby Lys). Bar=50 nm.
[0269] FIG. 28: Antibody labeling of htau23, K12 and cross-linked
products thereof. [0270] (a) htau23 dimers with an antibody at one
end (left) and with an antibody at each end (right) demonstrating
the antiparallel dimerization of htau23; [0271] (b) K12 dimers with
an antibody at one end (left), with antibodies at both ends
(middle) and presumable tetramers with antibodies at the free ends
(right) indicating that this type of association blocks the
epitope; [0272] (c) K12 dimers cross-linked with PDM, with an
antibody at one end (left), with antibodies at each end (middle)
and a tetramer with antibodies at the free ends (right); [0273] (d)
K12 dimers cross-linked by MBS with an antibody at one end (left),
with antibodies at each end (middle) and a tetramer with antibodies
at the free ends (right). Bar=50 nm.
[0274] FIG. 29: Time course of phosphorylation of htau40 by GSK3
and immune response. (1) SDS-PAGE of htau40 after incubation with
the kinase between 0 and 20 hours at 37.degree. C. The minor lower
band in lane 1 is a fragment. Note the progressive shift to higher
Mr values, similar to the effects of brain extract and MAP kinase.
(2) Autoradiography. (3) Immunoblot with the antibody TAU1 whose
reactivity is lost after .apprxeq.2 h (following the
phosphorylation of S199 and S202). (5) Immunoblot with antibody
SMI34 (conformation sensitive and against phosphorylated Ser). (6)
Blot with SMI31 (epitope includes phosphorylated S396 and S404).
(7) Blot with antibody SMI33 which requires a dephosphorylated
S235. There are some differences with respect to phosphorylation by
MAP kinase or the brain extract. The SMI33 staining persists for a
long period, suggesting that Ser235 is only slowly phosphorylated
by GSK3. The staining of SMI31 appears very quickly, before that of
AT8 or SMI34, showing that S396 and S404 are among the earliest
targets of GSK3.
[0275] FIG. 30: Mobility shift of htau23 versus mutant htau23/A404
upon phosphorylation with GSK3. Top, SDS gel, bottom,
autoradiogram. Lanes 1-3, htau23 unphosphorylated and
phosphorylated for 2 or 20 hours. Note the pronounced shift and the
clear incorporation of phosphate. Lanes 4-6, mutant Ser404-Ala,
unphosphorylated and phosphorylated for 2 and 20 hours. The shift
after 2 hours is much smaller and the degree of phosphorylation
much lower. This shows that the first strong shift and
phosphorylation is at Ser404, similar as with MAP kinase and the
brain extract kinase activity.
[0276] FIG. 31: Diagrams of tau constructs. Top, AP17, a derivative
of htau23 with all Ser-Pro or Thr-Pro motifs altered into Ala-Pro.
Middle, AP11, only Ser-Pro motifs changed into Ala-Pro. Bottom,
K18, only 4 repeats of tau (derived from htau40).
[0277] FIG. 32: Copolymerization of MAP kinase and GSK3 with
porcine brain microtubules. (a) SDS gel of microtubule purification
stages. Ex brain extract, supernatant after first cold spin.
S=supernatant of first hot spin=tubulin and MAPs not assembled into
microtubules after warming to 37.degree. C.; P=pellet of
redissolved microtubules. The other lanes (S, P) show two further
cycles of assembly and disassembly by temperature shifts (last
pellet of microtubule protein was concentrated). (b) Blot with
anti-MAP kinase, showing mainly the p42 isoform and some of the p44
isoform. (c) Blot with anti-GSK3.beta.; note that this antibody
shows some cross-reactivity with GSK3.alpha.. (d) Blot with
anti-GSK3.alpha.. The blots show that both kinases and their
isoforms co-purify with the cycles of microtubule assembly.
[0278] FIG. 33: (a) Identification of GSK3.alpha. and .beta. in
normal and Alzheimer brain extracts. M=markers, lane 1, SDS gel of
normal brain extract, lane 2, immunoblot with anti-GSK3.alpha.;
lane 3, immunoblot with anti-GSK33 (with some crossreactivity to
.alpha.). Lanes 4 and 5, same blots with Alzheimer brain
extracts.
[0279] FIG. 34: Binding curves of htau23 to microtubules (made from
10 .mu.M tubulin in the presence of 20 .mu.M taxol). Top curve
(squares), htau 23 unphosphorylated. Middle (circles), htau23
phosphorylated with GSK3, showing a comparable stoichiometry as the
unmodified tau protein (saturating 0.6 per tubulin dimer). Bottom
curve (triangles), control of htau23 phosphorylated with the brain
kinase activity, showing a pronounced decrease in stoichiometry.
The solid lines show the best fits assuming independent binding
sites.
[0280] FIG. 35: (a) Diagram of htau23 and point mutants used in
this invention. (b) Binding curves of htau23 and its point mutants
to microtubules, unphosphorylated and phosphorylated with brain
extract. The top and bottom curves show unphosphorylated and
phosphorylated wild type htau23, the other curves are after
phosphorylation. Mutants are (from top to bottom): Ser262-Ala,
Ser235-Asp/Ser396-Asp, Ser404-Ala, Ser202-Ala. The mutation at
Ser262 nearly eliminates the sensitivity of the tau-microtubule
interaction to phosphorylation. These curves were derived from
quantitating SDS gels by densitometry (see Example 6). Polymerized
tubulin is 30 .mu.M. The fitted stoichiometries n (=tau/tubulin
dimer) and binding constants K.sub.d(.mu.M) are: htau23 wt
non-phos. (n=0.49, K.sub.d=2.5); A262 phos. (n=0.45, K.sub.d=5.3);
D235/D396 phos. (n=0.32, K.sub.d=7.4); A404 phos. (n=0.32,
K.sub.d=9.3); A202 phos. (n=0.31, K.sub.d=9.4); htau23 wt phos.
(n=0.16, K.sub.d=4.9).
[0281] FIG. 36: Binding curves of htau40 to microtubules. Top,
unphosphorylated htau40 (triangles); middle, htau40 phosphorylated
with MAP kinase (circles); bottom, htau40 phosphorylated with brain
extract (squares). Fitted dissociation constants K.sub.d and
stoichiometries are as indicated.
[0282] FIG. 37: (a) Diagram of total mutant AP18. All Ser-Pro
Thr-Pro are replaced by Ala-Pro. In addition, Ser262 and 356 are
mutated into Ala. In the mutant AP17 Ser262 and Ser356 remain
unchanged. (b) Binding curves of htau 23 and the "total" mutants
AP17 and AP18 to microtubules without or with phosphorylation by
brain extract. Top, unphosphorylated htau23 (filled triangles);
middle, phosphorylated AP18 (circles), the two bottom curves are
phosphorylated AP17 (open squares) and htau23 (open triangles). The
difference in behavior between AP17 and AP18 is due to the
phosphorylation of Ser262 in AP17. Fitted stoichiometries and
binding constants are: htau23 wt non-phos. (n=0.49, K.sub.d=2.5);
AP18 phos (n=0.48, K.sub.d=6.1); AP17 phos (n=0.18, K.sub.d=6.6);
htau23 wt phos. (n=0.16, K.sub.d=4.9).
[0283] FIG. 38: Preparation of the kinase from porcine brain by
chromatographic steps. (a) Mono Q HR 10/10 FPLC. The
phosphorylation of recombinant human tau 34 and construct AP17 is
shown on the y-axis as moles P.sub.i transferred per mole of tau.
Fractions which decrease the binding of tau to MT elute around
fraction 12, 20 and 30, the peaks around fractions 20 and 30 being
the most effective. (b) Fractions 28-32 from Mono Q were gel
filtrated on a Superdex G-75 HiLoad 16/60 column. The column was
calibrated with standard proteins as shown by the filled symbols:
Ribonuclease, 14 kDal; chymotrypsinogen A, 25 kDal; ovalbumin, 43
kDal; bovine serum albumin, 67 kDal. Molecular weight is indicate
on the right y-axis on a logarithmic scale. The phosphorylation of
htau34 and construct K18 is shown on the left y-axis. The highest
activity elutes at a Mr of approx. 35 kDal. (c) Fractions 17-23
from the gel filtration column were pooled and rechromatographed on
a Mono Q HR 5/5 column. Fraction 10 was used for binding studies.
(d) SDS-gel showing the main purification stages. M: Marker
proteins; lane 1, whole brain extract, lane 2, Mono Q HR 10/10
FPLC, fraction 30; lane 3, Superdex gel filtration, fraction 22;
lanes 4-5, Mono Q HR 5/5 FPLC, fractions 10 and 9. Lane 5 shows the
purified 35 kDal band and a trace at 41 kDal.
[0284] FIG. 39: SDS gel and in-gel assay of kinase activity (for
details see Example 11). (a) 7-15% silver stained SDS gel of
fractions 9-11 (lanes 1-3) of second Mono Q run (see FIG. 38c). (b)
Autoradiogram of an in-gel experiment, with tau construct K9 (=four
repeats plus C-terminal tail of tau) in the gel and 5 .mu.l each of
fractions 9-11 (lanes 1-3). (c) Autoradiogram of control gel
containing no tau protein and showing no autophosphorylation of the
Mono Q fractions. Note that specific kinase activities are
difficult to quantify from these gels since the renatured protein
tends to diffuse out of the gels; this is especially true of the 35
kDal band.
[0285] FIG. 40: Effect of phosphorylation of tau by 35 kDal kinase
on gel shift and microtubule binding. (a) SDS gel of htau23 and
constructs phosphorylated by several kinases. M, marker proteins.
Lanes 1 and 2, htau23 without and with phosphorylation by 35 kDal
kinase. Lanes 3 and 4, same experiment with point mutant
htau23(Ser409-Ala) (no shift); lanes 5 and 6, point mutant
htau23(Ser416-Ala) (only part of the protein phosphorylated, but
otherwise same shift as in lane 2); lanes 7 and 8, point mutant
htau23(Ser404-Ala) (same shift as lanes 2 and 6). The mutants show
that the 35 kDal kinase induces a shift by phosphorylating Ser409.
Note that Ser404 is the target of MAP kinase, Ser416 of CaM kinase
(Steiner et al., 1990, ibid.), and Ser409 and Ser416 of PKA, each
of which induces a shift. Lanes 9-11 show a comparison of the
shifts induced in htau23 by the different kinases (CaM kinase, PKA
and MAP kinase). The shifts induced by PKA (lane 10) is the same as
that of the 35 kDal kinase, and that MAP kinase produces by far the
largest shift, typical of the Alzheimer-like state of tau. The bars
on the right indicate the shift level; from bottom to top,
unphosphorylated htau23 (control), CaM kinase shift level, PKA
shift level, MAP kinase shift level. All shift sites are near the
C-terminus. (b) Binding curves of htau23 and the mutants Ser262-Ala
to microtubules without or with phosphorylation by the 35 kDal
kinase (Mono Q fraction 10, 20 hours). Top, unphosphorylated htau23
(open circles, n=0.49, K.sub.d=2.5 .mu.M); middle, phosphorylated
mutant (squares, n=0.44, K.sub.d=11.6 .mu.M); bottom,
phosphorylated htau23 (filled circles, n=0.21, K.sub.d=8.8 .mu.M).
In the absence of Ser262 the reduction in stoichiometry is 0.05;
with phosphorylated Ser262 it is 0.28.
[0286] FIG. 41: Diagram of htau40, highlighting the first
microtubule-binding repeat and the Ser262 that is important for
microtubule binding.
[0287] FIG. 42: 1. Dephosphorylation ("dephos.") of p32-marked
htau40 ("ht40.sup.32P") with different PPases. Autodiagraphs of
7-15% SDS gradient gels. [0288] FIG. 1: Autoradiographs of 7-15%
SDS gradient gels. [0289] A. Dephos. with PP2a H-isoform (10
.mu.g/ml) [0290] Lane 1: ht40P before dephos. [0291] Lane 2: 10 min
dephos. [0292] Lane 3: 30 min. dephos. [0293] Lane 4: 120 min
dephos. [0294] B. Dephos. with PP2a M-isoform (10 .mu.g/ml), [0295]
Lanes 1-4: see A. [0296] C: Dephos. with PP2a L-isoform (10
.mu.g/ml), [0297] Lanes 1-4: see A. [0298] D: Dephos. with
catalytic subunit of PP1 (500 U/ml), [0299] Lanes 1-4: see A.
[0300] FIG. 43: 2. Dephosphorylation with PP2a-H: disappearing of
phosphorylation dependent antibody epitopes [0301] A. SDS-PAGE
(7-15%). [0302] Lane 1: ht40P before dephos. [0303] Lane 2: 10 min
dephos. [0304] Lane 3: 30 min. dephos. [0305] Lane 4: 120 min
dephos. [0306] Lane 5: 5 h dephos. [0307] Lane 6: 16 h dephos.
[0308] B. Autoradiographs [0309] C. Immunoblot AT-8 [0310] D.
Immunoblot Tau-1A [0311] E. Immunoblot SMI-33
[0312] FIG. 44: Kinetics of dephos. with PP2a-H [0313] a. time
course of dephos. of ht40P with different concentrations of PP2a
[0314] b. variation in the ht40P-concentration:
Michaelis-Menten-Diagramm.
[0315] FIG. 45: Preparation of the 70 kDal kinase which
phosphorylates the two IGS motifs and the two CGS motifs of tau
protein (Serines 262, 293, 324, 409). The kinase strongly reduces
the affinity of tau for microtubules. [0316] (a) Chromatography on
S-Sepharose. Kinase activity elutes at 250 mM NaCl. [0317] (b)
Chromatography on heparin agarose. Kinase activity elutes at 250 mM
NaCl. [0318] (c) Gel filtration on Superdex G-75. Kinase activity
elutes at 70 kDal.
[0319] FIG. 46: Time course of phosphorylation of htau40 with
cdk2/cyclin A. Lanes 1-9 correspond to time points 0, 10, 30, 90
min, 3, 6, 10, 24 hours, and 0 min (the 0 min lanes are the
control). [0320] (a) SDS polyacrylamide gel electrophoresis,
showing the shift of the protein upon phosphorylation. [0321] (b)
Autoradiogram showing increasing incorporation of phosphate. [0322]
(c) Immunoblot with TAU-1 antibody which recognizes only
unphosphorylated Ser199 and Ser202. [0323] (d) Immunoblot with AT-8
antibody which recognizes these two serines in a phosphorylated
state, as well as Alzheimer tau.
EXAMPLE 1
Preparation of Tau Protein
[0324] Preparation of Tau from Normal Brains: the Procedures of tau
preparation from human, bovine, or porcine brain,
dephosphorylation, and rephosphorylation were essentially as
described by Hagestedt et al., J. Cell. Biol. 109 (1989),
1643-1651.
[0325] Preparation of Tau from Alzheimer Brains: Human Brain
tissues from neuropathologically confirmed cases of Alzheimer's
disease were obtained from various sources. The autopsies were
performed between 1 and 25 hours post mortem. The brain tissue was
kept frozen at -70.degree. C. Tau from paired helical filaments
(PHF) was prepared according to Greenberg & Davies, Proc. Natl.
Acad. Sci. USA 87 (1990), 5827-5831.
EXAMPLE 2
Characterization and Partial Purification of the Tau
Phosphorylating Activity (Protein Kinase) of Porc Brain Extract
[0326] Porc brain extract supernatant was fractionated by ammonium
sulphate precipitation. The main kinase activity precipitated at
40% saturation. This fraction was desalted by gel filtration,
diluted fivefold and incubated in activation buffer (25 mM Tris, 2
mM EGTA, 2 mM DTT, 40 mM p-nitrophenylphosphate, 10 .mu.M okadaic
acid, 2 mM MgATP, protease inhibitors) for 2 hours at 37.degree. C.
During this incubation a phosphorylation of a 44 kD protein at
tyrosine residue(s) occurs as shown by Western blotting with
anti-phosphotyrosine mAb. The 44 kD protein could be identified as
MAP2 kinase by a second mAb.
[0327] The crude enzyme activity was further purified by ion
exchange chromatography (Mono Q FPLC, Pharmacia). Fractions
containing the activated MAP-Kinase, as shown by Western blotting,
exerted the most prominent tau phosphorylating activity (Peak I). A
second tau phosphorylating activity (Peak II) did not induce
comparable SDS-gel shifts and Alzheimer-specific antibody
reactivity in tau.
EXAMPLE 3
Construction of Plasmids Carrying Genes Encoding Recombinant Tau
Polypeptides for the Determination of Alzheimer Tau Protein
Specific Epitopes
[0328] Cloning and expression of tau constructs: Plasmid
preparations and cloning procedures were performed according to
Sambrook et al. (Molecular Cloning Laboratory Handbook, 2nd
edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989).
Amplifications by the polymerase chain reaction (PCR, Saiki et al.,
Science 239 (1988), 487-491) were carried out using Taq polymerase
as specified by the manufacturer (Perkin Elmer Cetus). The tau
genes and their constructs were expressed in the expression vector
pNG2, a derivative of pET-3b (Rosenberg et al., Gene 56 (1987),
125-135), modified by removal of PstI, HindIII, NheI and EcoRV
restriction sites for convenient engineering of the tau gene. For
the expression the BL21 (DE3) E. coli system (Studier et al., Meth.
Enzym. 185 (1990), 60-89) was used. Most constructs were derived
from the human isoform htau23 which contains 352 residues and three
internal repeats in the C-terminal microtubule binding region
(Goedert et al., Proc. Natl., Acad. Sci. USA 85 (1988), 4051-4055).
The numbering of residues used here refers to the sequence of
htau40, the largest of the human isoforms (441 residues, Goedert et
al., ibid.). For the isolation of the constructs use was made of
the heat stability of the protein; they were separated by FPLC Mono
S (Pharmacia) chromatography according to the procedure described
by Hagestedt et al., J. Cell. Biol. 109 (1989), 1643-1651.
[0329] K10: This represents the carboxy part of the htau23 isoform
consisting of 168 residues (Q244-L441 plus start methionine, but
without the second repeat V275-S305). The K10 tau cassette was
generated in the pNG2/htau23 vector by deletion of the NdeI-PstI
fragment and replacing it with a chemically synthesized hexamer
5'TATGCA3'. After religation the NdeI endonuclease site was
restored and PstI site was damaged.
[0330] Constructs K11 and K12 were made by a combination of
fragments derived from the htau23 and htau24 genes. K11 is a tau
derivative containing 4 repeats and consists of 152 amino acids
(Q244-Y394 plus start methionine). K12 is a tau derivative
containing 3 repeats and consists of 121 amino acids (Q244-Y394
plus start nethionine, but without the second repeat V275-S305,
htau40 numbering).
[0331] Htau23 and htau40 are human tau isoforms consisting of 352
and 441 amino acids, respectively (8).
[0332] K17: The K17 tau cassette (145 residues) is a shorter
derivative of K16. It was made in two steps: First K16 was
constructed using PCR to engineer the htau24 gene. The 5' "add on"
of restriction sites on both ends of the amplified fragment was
applied to facilitate the insertion of the PCR products into the
cloning vector. The start primer (JB50) had the sequence GGCG ("G/C
clamp"), the CATATG recognition site for the NdeI nuclease
(containing the universal ATG start codon), followed by coding
information for amino acids S198-T205. The stop primer (JB51) had a
"G/C clamp" and the GGATCC recognition sequence for BamHI followed
by a stop anticodon and anticoding sequence for the C terminal
amino acids P364-E372. The K16 tau cassette consists of 176
residues, 175 from htau40 (S198-E372) plus a start methionine. This
fragment represents part of the assembly domain consisting of 46
residues between S198 and the beginning of the first repeat
following by the sequence of four repeats finished at E372. In the
second step, a BstXI-BstXI fragment from the newly constructed tau
K16 cassette was exchanged against the similar BstXI-BstXI fragment
from the htau23 gene containing only three repeats and causing the
generation of the tau cassette K17. Thus K17 represents the
analogous part of the projection domain like K16 but missing the
second tau repeat.
[0333] K3M (355 residues) is a chimera consisting of 145 residues
from the amino terminus of bovine Tau4 (from the plasmid
pETNde43-12, Himmler et al., Mol. Cell. Biol. 9 (1989), 1381-1388)
and 190 residues from carboxy part of human htau23 (from the
plasmid pUC18/htau23, Goedert et al., 1988 ibid.). It is a molecule
with three repeats and two amino terminal inserts, consisting of 29
residues each. K3M was constructed by excision of XmaI-BclI
fragment from pETNde42-12 and replacing it with analogous XmaI-BclI
fragment originated from the htau23 gene. This manipulation removed
64 residues (XmaI-XmaI segment from bTau4) and replaced the 4
repeats carboxy terminus against three repeats carboxy
terminus.
[0334] K19 represents the three repeats of htau23 and consists of
99 residues (Q244-E372, plus start methionine, without repeat 2).
The K19 molecule was constructed from K17 by replacing the 144 nt
long NdeI-PstI fragment with the synthetic hexamer 5'TATGCA3'. This
modification retains the intact NdeI restriction site in the
beginning of the molecule and removes the PstI site.
[0335] Construction of the D-mutant of htau23: In order to replace
S199 and S202 by D in htau23, a double stranded DNA cassette
encoding the amino acids G164-P219 was designed. This DNA segment
was assembled from 8 oligonucleotides (30 to 60 nucleotides in
length) and contained SfiI and XmaI sticky ends. The insertion of
the assembled cassette into linearized pNG2/htau23 vector with
removed native SfiI-XmaI fragment created the required gene.
[0336] Construction of htau23/A404: htau23/A404 is a mutated htau23
molecule where Ser404 was replaced by the Ala in order to remove
this phosphorylation site. For convenient manipulation of the
htau23 gene, an artificial NcoI restriction site in the position
1161 (htau40 numbering) was introduced. This mutation was done
using PCR-SOE (splicing by overlap extension, Higuchi et al., Nucl.
Acids. Res. 16, (1988), 7351-7367). The new NcoI does not influence
the amino acid sequence of tau protein. For the introduction of the
Ala residue in the position 404 a synthetic DNA cassette was used,
representing the 120 bp DNA fragment between NcoI and NheI
restriction sites and encoding the amino acids His388-Thr427. This
DNA segment was assembled from 4 oligonucleotides (54 to 66
nucleotides in length) and contained NcoI and NheI sticky ends. The
insertion of the assembled cassette into the linearized
pNG2/htau23/NcoI vector with removed native NcoI-NheI fragment
created the htau23/A404 gene. The mutation of Ser396 to Ala was
created in similar way like that in the position 404.
[0337] K2 (204 residues) is a chimera consisting of 36 residues
from the amino terminus of bovine Tau4 and 168 residues from the
carboxy part of htau23; it contains three repeats. K4-K7 are
deletion mutants of htau23 containing only two repeats: K4 has
repeats No. 1 and 3 (270 residues, D345-A426 excised); K5 has
repeats No. 1 and 3 (310 residues, D345-T386 excised); K6 has
repeats No. 3 and 4 (322 residues, T245-K274 excised); K7 has
repeats No. 1 and 4 (321 residues, V306-Q336 excised); note that
repeat No. 2 is always absent in htau23. K13-K15 are deletion
mutants of htau23 containing only one repeat: K13 has repeat No. 4
(291 residues, T245-Q336 excised); K14 has repeat No. 3 (279
residues, T245-S305 and D345-D387 excised); K15 has repeat No. 1
(278 residues, D345-D387 excised).
EXAMPLE 4
Determination of Alzheimer Specific Epitope in The Tau Protein
[0338] A panel of antibodies against PHFs from Alzheimer brain was
closely examined for their reactivity and one (AT8) was found that
was specific for PHF tau. FIG. 1 shows the reactivity of the
antibody AT8 against different tau species. In the case of tau from
Alzheimer paired helical filaments (PHF) the antibody recognizes
all isoforms (FIG. 1b, lane 1). When the mixture of tau isoforms
from normal bovine or human brain was tested (known to be in a
mixed state of phosphorylation, FIG. 1a, lanes 2-5), reactivity
with the AT8 antibody (FIG. 1b) was detected. The same is true for
the six individual human isoforms expressed in E. coli
(unphosphorylated, FIGS. 1a and 1b, lanes 6-11). It is concluded
that AT8 is indeed specific for Alzheimer tau; in particular, it
reacts with a phosphorylated epitope that occurs only in PHFs, but
not in normal tau. Moreover, there is a correlation between the AT8
reactivity, phosphorylation, and electrophoretic mobility; it
appears as if there was an Alzheimer-like phosphorylation that
caused an upward shift in the SDS gel.
[0339] In order to identify the kinase(s) that were responsible for
this behavior, and the corresponding phosphorylation sites, a
kinase activity from porcine brain extract was prepared as
described in Example 2. The six human isoforms expressed in E. coli
were phosphorylated according to standard procedures with this
activity in the presence of okadaic acid, a phosphatase inhibitor.
FIG. 2a shows that each isoform changes considerably its
electrophoretic mobility in the gel (upward shift) and shows a
strong immunoreactivity with the AT8 antibody (FIG. 2b). These
results show that the phosphorylation of tau by this kinase
activity is analogous to that of the Alzheimer state. Moreover,
since all isoforms are affected in a similar way the
phosphorylation site(s) must be in a region common to all of
them.
[0340] The strategy to identify said common region was to use first
the engineered mutants prepared as described in Example 3 in order
to narrow down the site, and then to determine it by direct
sequencing. FIG. 3 describes some of the mutants used, K19, K10,
K17, and K3M (see also Example 3). Except for K19, all of these
mutants are phosphorylated by the kinase activity and show an
upward M shift in the SDS gel (FIG. 4a). K19 is a construct that
comprises just three repeats of 31 or 32 residues. It does not
become phosphorylated by the kinase activity and therefore does not
show an M.sub.r shift in the SDS gel (FIG. 4c).
[0341] This means that the phosphorylation site(s) are outside the
region of the repeats. Phosphorylation can take place on either
side of the repeats and induces an upward shift in the gel; the
shift is larger for phosphorylation after the repeats. The antibody
AT8 recognizes none of the unphosphorylated forms (as expected);
after phosphorylation it reacts only with the construct K17 (FIG.
4b, lane 6), not with K10 or K3M (FIG. 4b, lanes 4 and 8). In other
words, K17 retains the epitope, while K10 and K3M have lost it. By
reference to FIG. 3 it is concluded that the epitope is not in the
region of the pseudo-repeats nor in C-terminal tail where we found
a CaM kinase site previously (since K10 and K19 are non-reactive),
but rather it has to be between S198 and T220 (FIG. 3, peptide P),
i.e. in the region following the major chymotryptic cleavage site
(behind Y197) in the "assembly" domain of tau.
[0342] Next a total tryptic digest of radioactively labeled htau34,
an isoform with 4 internal repeats (Goedert et al., 1989, ibid.)
was carried out. The peptides were isolated by HPLC and sequenced.
One of them was in the area of interest, S195-R209 (FIG. 5). This
peptide contained two phosphates at S199 and S202. Both are
followed by a proline, suggesting that the enzyme active in the
extract was a proline-directed kinase.
[0343] These results suggested that the phosphorylation sensitive
AT8 epitope might be in the vicinity of residue 200. This was
tested by engineering a mutant of htau22 (3 repeats, no N-terminal
insert) where S199 and S202 were both changed to D. This choice was
made in order to rule out the phosphorylation of these residues by
a kinase, but also to mimic in part the "phosphorylated" state in
terms of negative charges. On SDS gels this mutant showed a small
upward shift to higher M (FIG. 6, lane 4). The immunoblots show
that only the parent protein htau23 reacted with the anti-body
after phosphorylation (FIG. 6, lane 6), but not the
unphosphorylated htau23 (as expected) nor the mutant, whether
phosphorylated or not (lanes 7, 8).
[0344] It is concluded that the epitope of AT8 is in the region
5199-5202 and depends on the phosphorylation of these two serines.
They can be phosphorylated by a proline-directed kinase present in
brain extract which turns the protein into an Alzheimer-like state.
The region is perfectly conserved in all tau variants known so far
and explains why all of them respond to phosphorylation and to the
antibody in the same way.
EXAMPLE 5
Characterization of the Protein Kinase Activity
[0345] Phosphorylation of tau proteins was carried out in the
following way: Tau protein (0.5 mg/ml) was incubated for various
times (up to 24 hours) at 36.degree. C. with the brain extract in
40 mM HEPES containing 2 mM MgCl.sub.2, 1 mM DTT, 5 mM EGTA, 1.5 mM
PMSF, 2 mM ATP, 20 .mu.g/ml protease inhibitor mix (pepstatin,
leupeptin, alpha-macroglobulin, aprotinin), with or without 1 mM
okadaic acid. After that 500 mM DTT were added, the solution was
boiled for 10 min and centrifuged for 15 min at 15000 g at
4.degree. C. The supernatant was dialyzed against reassembly buffer
(RB, 100 mM Na-PIPES pH 6.9, 1 mM EGTA, 1 mM GTP, 1 mM MgSO.sub.4'
1 mM DTT) and used for binding studies.
[0346] Radioactive labeling was done with gamma-[.sup.32P]ATP (NEN
Du Pont) at 10 mCi/ml, 3000 Ci/mmol, diluted to 15-30 Ci/mol ATP
for autoradiography on SDS gels. The phosphate incorporated into
the protein was quantified as follows: 1 .mu.g of phosphorylated
protein was applied to SDS gels, the bands were cut out and counted
in the scintillation counter in Cerenkov mode. The counter was
calibrated with known samples of .sup.32P (detection efficiency
about 50% in Cerenkov mode). The corrected counts were translated
into moles of P.sub.i per mole of tau on the basis of the known
specific activity of radioactive ATP used during
phosphorylation.
[0347] A remarkable feature found for this kinase is that it shifts
the M.sub.r of all tau isoforms in three distinct stages (see FIGS.
7a and 8a for the case of htau23). During the first two hours of
phosphorylation the protein is converted from a M.sub.r0=48 kD
protein to a slower species, with an M.sub.r1 of about 52 kD. Upon
completion of this first stage, a second one sets in which is
finished around 6.10 hours (M.sub.r2=54 kD), The third stage takes
about 24 hours (M.sub.r3=56 kD), after that no more shift is
observed.
[0348] During the initial stage each band of the tau doublet
incorporates phosphate (e.g. at a level of about 0.5 P.sub.i per
molecule in the presence of OA at 30 min, see FIG. 7b, lane 4).
This means that there must be at least two distinct phosphorylation
sites, one that is responsible for the shift (the "shift site",
upper band), and one that has no effect on the M.sub.r (lower
band). The lower band gradually disappears, and at two hours each
tau molecule contains about 2 P.sub.i. In other words, the upper
band contains tau molecules in which the "shift site" is
phosphorylated, irrespective of the other site(s); whereas the
lower band contains only molecules where the shift site is not
phosphorylated. The effect of OA is seen mainly in the lower band,
indicating that the phosphatase operates mainly on the non-shift
site(s). These considerations apply to the first stage of
phosphorylation; during the second and third stages there are
further shifts, but a detailed analysis of shift sites and
non-shift sites is not possible because of the overlap of bands.
Overall about two additional phosphates can be incorporated in
every stage, giving a maximum of 6 for htau23 and 7 for htau34.
These values refers to the presence of OA; without it we usually
find .apprxeq.1-2 P.sub.i less. When the purified kinase is used,
one finds 12-14 P.sub.i.
[0349] Since the major shift occurs during the first stage, and
since a large shift is considered a hallmark of Alzheimer tau, it
was suspected that the first stage phosphorylation might induce an
Alzheimer-like state. This was checked by immunoblotting according
to standard procedures with Alzheimer-specific antibodies. FIG. 8a
shows a similar phosphorylation experiment as above (with 10 .mu.M
OA throughout), FIG. 8b is the immunoblot with the monoclonal
antibody SMI34 which reacts with a phosphorylated epitope in
Alzheimer tangles (Sternberger et al., ibid.). The antibody
recognizes the bacterially expressed tau phosphorylated by the
kinase, but only from stage 2 onwards. A similar behavior is found
with other Alzheimer-specific antibodies tested. The result from
these studies is that the major phosphorylation-dependent M.sub.r
shift (stage 1) is distinct from the ones that generate the
Alzheimer-like antibody response (stages 2, 3).
EXAMPLE 6
Tau Protein in Microtubule Binding Studies
[0350] Another point of interest with respect to the correlation
between abnormal phosphorylation of tau proteins and Alzheimer's
disease was whether the phosphorylation had an influence on tau's
affinity for microtubules. This was tested using a microtubule
binding assay. Accordingly, PC tubulin was incubated at 37.degree.
C. in the presence of 1 mM GTP and 20 .mu.M taxol. After 10 min tau
protein was added in different concentrations and incubated for
another 10 min. The suspensions were centrifuged for 35 min at
43000 g at 37.degree. C. The resulting pellets were resuspended in
CB buffer (50 mM PIPES PH 6.9, 1 mM EGTA, 0.2 mM MgCl.sub.2, 5 mM
DTT, 500 mM NaCl). In the case of htau 23 and htau 34 the pellets
and supernatants were boiled for 10 min and recentrifuged for 10
min at 43000 g at 4.degree. C. (this step served to remove the
tubulin component which otherwise would overlap with these tau
isoforms on SDS gels). Pellets and supernatants (containing the
bound and the free tau, respectively) were subjected to SDS PAGE
(gradient 7-15% acrylamide) and stained with Coomassie brilliant
blue R250. The gels were scanned at 400 dpi on an Epson GT 6000
scanner and evaluated on a PC 368AT using the program GelScan (G.
Spieker, Aachen). The protein concentration on the gel was always
within the linear range (up to 1.5 optical density). The
intensities were transformed to concentrations using calibration
curves and used in the binding equation.
[0351] Tau.sub.bound=n[Mt][Tau.sub.free]/{Kd+[Tau.sub.free]}, from
which the dissociation constant K.sub.d and the number n of binding
sites per dimer were obtained by fitting. [Mt] is the concentration
of tubulin dimers polymerized in microtubules (usually 30
.mu.M).
[0352] With fully phosphorylated protein (stage 3, 24 hours) a
dramatic decrease in binding capacity of htau23 was observed (FIG.
9b), from about one tau per two tubulin dimers to one tau per six
tubulin dimers. In other words, it appears that unphosphorylated
tau packs tightly onto a microtubule surface, whereas fully
phosphorylated tau covers the microtubule surface less densely, as
if it occupied more space. FIG. 9c shows the same experiment with
htau34. The results are similar, i.e. there is a threefold
reduction in binding capacity. Tau isoforms with four repeats, such
as htau34, bind to microtubules particularly tightly in the
unphosphorylated state (K.sub.d.sup.-1-2 .mu.M).
[0353] Since the major M.sub.r shift (see Example 5) occurs during
the initial two hours it was of interest to find out which residues
become phosphorylated during the first stage, and how they affected
microtubule binding. As mentioned above, there are about two
phospates incorporated during this period, one of which causes the
shift from M.sub.r0 to M.sub.r1. FIG. 10 illustrates the binding of
htau34 to microtubules after 90 min of phosphorylation. The
striking result is that the limited phosphorylation decreases the
affinity as efficiently as the full phosphorylation. This means
that the reduction in microtubule affinity precedes the
Alzheimer-like immunoreactivity (FIG. 8).
[0354] The analysis of tryptic peptides after 90 min showed four
major peaks of radioactivity, with phosphates on serines 202, 235,
404, and 262. Three of these are SP sites that are not in the
repeat region, but rather flank that region in nearly symmetric
positions (FIG. 11); the fourth (S262) is a non-SP site in the
first repeat. It is in particular note-worthy that S396 was not
among the phosphorylated residues. This was unexpected since Lee et
al. (1991, ibid.) had shown that S396 (the center of a KSP motif)
was phosphorylated in tau from paired helical filaments. Thus S396
must become phosphorylated during the second or third stages of
phosphorylation, concomitant with the immunoreactivity (FIG.
8b).
[0355] Several point mutants were generated according to standard
procedures to find out which site(s) were responsible for the
initial M.sub.r shift. When ser404 was turned into ala the M.sub.r
shift during the first stage disappeared, whereas it remained
visible when ser199, 202, 235, or 396 were mutated. This means that
the phosphorylation of ser404 accounts for the one P.sub.i present
in the upper band of FIG. 7a or 8a. The additional
.apprxeq.1P.sub.i present after 2 hours is distributed among
serines 202, 235, and 404.
[0356] Whereas the results on the "shift site" S404 of tau are
clear cut, the factors responsible for the reduction of microtubule
binding are more complex. The S404-A mutant binds to microtubules
similarly as the parent htau34; after 90 min of phosphorylation the
stoichiometry decreases about 2-fold, i.e. less than the factor of
3 observed with the parent molecule. If S404 were the only residue
whose phosphorylation was responsible for the loss of microtubule
binding we would not expect any decrease in the mutant. The fact
that a decrease is observed means that other factors play a role as
well; these factors are presumably related to the incorporation of
more than one P.sub.i at one or more of the other sites before or
at the beginning of the repeat region (e.g. 202, 235, 262).
However, these residues cannot by themselves be responsible for the
full decrease of affinity either. In fact, point mutations at
positions 202 or 235 show a similar effect as that of 404, i.e.
only a partial reduction of binding. One possible explanation is
that different phosphorylation sites interact in a cooperative
manner and generate a new confirmation.
EXAMPLE 7
Time Course of Phosphorylation as Determined by Stage Specific
Antibodies
[0357] Neurofilament specific antibodies SMI31, SMI34, SMI35 and
SMI310 against a phosphorylated epitope and SMI33 against a
non-phosphorylated epitope [(Sternberger et al., Proc. Natl. Acad.
Sci. USA 82 (1985), 4274-4276)] were used to detect stage specific
phosphorylation of tau protein. SMI33 recognizes normal human brain
tau (FIG. 12, lane 1) but does not recognize PHF tau, except when
it is dephosphorylated (lane 4). This suggests that the epitope of
SMI33 is specifically blocked by some phosphorylation in the
Alzheimer state which does not occur in normal brain tau. SMI31 and
SMI34 both react in a complementary fashion to SMI33: Only PHF tau
is recognized (FIGS. 12c and 12d, lane 3), but not when it is
dephosphorylated (lane 4), nor the normal tau control (lane 1).
[0358] The testing of the various antibodies during the time course
of phosphorylation shows that SMI33 loses reactivity during the
second stage of phosphorylation (see FIG. 7).
[0359] For antibody SMI31 no reactivity is observed with the
unphosphorylated protein (time 0) or during the first stage, but
the reactivity appears gradually during the second stage and
remains throughout the third. A similar time course is found with
antibody SMI34 (FIG. 13c and compare FIG. 12d, lane 3), SMI35, and
SMI310 (FIG. 13g,h). For comparison the blots with AT8 (FIG. 13f),
a phosphorylation sensitive Alzheimer tangle antibody (Binder et
al., J. Cell. Biol. 101 (1985), 1371-1379 are included) and TAU1,
an antibody against dephosphorylated tau. AT8 reacts similarly to
SMI31, SMI34, SMI35, and SMI310, while TAU1 is similar to SMI33.
The striking feature of the blots is that in each case it is the
stage 2 phosphorylation that determines the antibody response.
[0360] These experiments could be interpreted by assuming that the
antibodies react with the same region of tau in a dephosphorylated
or phosphorylated form; but this assumption is too simple, as shown
later. Two other features should be pointed out, however: One is
that the largest gel shift (stage 1) is not the one that causes the
Alzheimer-like immunoreactivity (appearing in stage 2). Thus not
every gel shift of tau is diagnostic of the Alzheimer state,
although conversely the Alzheimer state always shows a gel shift.
Secondly, there is a surprisingly precise relationship between gel
shift, phosphorylation, and immunoreactivity with several different
antibodies.
[0361] The major phosphorylated motifs of neurofilaments are
repeated sequences of the type KSPV where S is the phosphate
acceptor; see e.g. Geisler et al., FEBS Lett. 221 (1987), 403-407.
Tau has one such motif, centered at S396, and another KSP motif is
centered at S235. The two KSP sites lie on either side of the
repeat region and are conserved in all tau isoforms. By analogy one
may suspect that these sites are involved in the reaction with the
SMI antibodies that were raised against neurofilaments. We tested
this in three ways, by mutating one or two of the serines, by
making smaller tau constructs, and by direct sequencing of tryptic
peptides.
[0362] Constructs K10, K17, and K19 were examined before or after
phosphorylation with the kinase (FIG. 14a). K10 and K17 show an
M.sub.r shift, but not K19. Note also that K10 and K17 are only
partly converted to the higher M.sub.r form in this experiment,
indicating that their phosphorylation is less efficient. K10 shows
three shifted bands, indicating that there are three
phosphorylation sites in the C-terminal region. K17 shows only one
shifted band so that there is only one shift-inducing site in the
region before the repeats. FIG. 14b-d show the immunoblots with
SMI33, SMI31, and SMI34; the data on SMI35 and SMI310 are similar
to SMI31 (not shown). Antibody SMI33 reacts only with K17 in the
dephosphorylated state, but not with K10 and K19 (FIG. 14, lane 3).
This suggests that the epitope is in a region before the repeats,
between S198 and Q244, outside the sequences covered by the other
constructs. This would be consistent with an epitope at the first
KSP site. Antibody SMI31 reacts with K10 in its phosphorylated
form, but not K17 or K19 (FIG. 14). Using similar arguments as
before, the epitope is in the region T373-L441, consistent with the
second KSP site. Finally, antibody SMI34 labels htau23, K10 and
K17, but not K19 (FIG. 14c). The latter property would argue
against the repeat region as an epitope, but the remaining reaction
with K10 and K17 would seem mutually exclusive. Our interpretations
is that SMI34 has a conformational epitope that depends on tails on
either side of the repeats and becomes fully stabilized only when
at least one tail is present. However, the phosphorylation
dependence is in each case the same as that of the intact
molecule.
[0363] Since it was suspected that the two KSP motifs were
phosphorylated by the kinase, it was tried to prove this directly.
Radioactively labeled tryptic peptides of htau34 were identified by
HPLC and protein sequencing, and phosphorylated residues were
determined. There are two major phosphorylated tryptic peptides in
these regions; peptide 1 (T231-K240, TPPKS.sub.pPPSSAK) contains
the first KSP motif, phosphorylated at S235, peptide 2 (T386-R406,
TDHGAEIVYKS.sub.pPVVSGDTS.sub.pPR) contains the second KSP site,
phosphorylated at S396 and S404. S416, the single phosphorylation
site of CaM kinase described earlier (Steiner et al. EMBO J. 9
(1990), 3539-3544, S405 in the numbering of htau 23 used earlier)
is not phosphorylated by the kinase used here.
[0364] Next point mutants of the phosphorylated residues 235 and
396 (FIG. 15) were made and analysed in terms of gel shift and
antibody reactivity (FIG. 16). The parent protein htau40 and its
KAP mutants have nearly identical M.sub.r values, and they all
shift by the same amount after phosphorylation (FIG. 16, lanes
1-8). The reactivity of SMI33 is strongly reduced when S235 is
mutated to A (FIG. 16, lanes 3, 7) and obliterated after
phosphorylation (FIG. 16, lanes 2, 4, 6, 8). This means that the
epitope of SMI33 is around the first KSP site, but phosphorylation
at other sites have an influence as well (perhaps via a
conformation). The mutation at S396 (second KSP site) has no
noticeable influence on the SMI33 staining (FIG. 16b, lanes 5,
6).
[0365] As mentioned above, the epitope of SMI31 depends on the
phosphorylation of sites behind the repeat region. When S396 is
mutated to Ala the antibody still reacts in phosphorylation
dependent manner so that this serine is not responsible for the
epitope by itself (FIG. 16c, lane 6). Mutation S404 to Ala yields
the same result. However, if both serines are mutated, the antibody
no longer reacts upon phosphorylation (not shown). This means that
the epitope includes the two phosphorylated serines. The binding of
this antibody also has a conformational component: constructs with
only one repeat (K13-K15) are not recognized (FIG. 17, lanes 10,
12, 14).
[0366] SMI34 shows the most complex behavior because its reactivity
depends on phosphorylation sites before and after the repeat
region. This antibody recognizes all KAP mutats, so that S235 and
S396 cannot play a major role. However, the fact that SMI34
recognizes phosphorylated K17, K10, but not K19 (FIG. 17) suggests
that the regions before and/or behind the repeats must cooperate
with the repeats to generate the epitope. One possibility would be
that the epitope is noncontiguous, another one is that it may
depend on the number and conformation of the repeats. In order to
check these possibilities constructs with different combinations of
two repeats (K5, K6, K7, FIG. 18), and constructs with one repeat
only (K13, K14, K15) were done. All of these showed a shift upon
phosphorylation, and all of them were recognized by SMI34 (the
reaction is less pronounced when the third repeat is absent,
indicating that this repeat is particularly important for the
conformation, FIG. 17, lane 6). This means that the epitope of
SMI34 does not depend on the number of repeats. However, the nature
of the region just before the repeats seems to be important and in
particular sensitive to charges. This can be deduced from
constructs such as K2 or K3M where charged sequences have been
brought close to the repeat region, resulting in a loss of SMI34
reactivity. In other words, it seems as if the charged sequences
are capable of masking the epitope, independently of the
phosphorylation itself (FIG. 17, lanes 2, 4). The interactions
between the constructs and the antibodies are summarized in Table
1.
EXAMPLE 8
[0367] Cloning and expression of tau constructs: Plasmid
preparations and cloning procedures were performed according to
Sambrook et al. PCR amplifications were carried out using Taq
polymerase as specified by the manufacturer (Perkin Elmer Cetus)
and a DNA TRIO-Thermoblock (Biometra).
[0368] Tau cDNA clones and their constructs were subcloned into the
expression vector pNG2 (a derivative of pET-3b, Studier et al.,
modified in the laboratory by removal of PstI, HindIII, NheI and
EcoRV restriction sites for convenient engineering of the tau
clones), or in expression vector pET-3a. For the expression, the
BL21 (DE3) E. coli system was used (Studier et al.). All residue
numbers refer to the sequence of htau40, the largest of the human
isoforms (441 residues, Goedert et al.). For the isolation of the
constructs the heat stability of the protein was used; they were
separated by FPLC Mono S (Pharmacia) chromatography (for details
see Hagestedt et al.).
[0369] Construction of T8R-1: This is a tau derivative containing 8
repeats. It was constructed on the basis of the bovine tau4 isoform
(Himmler et al.). Two plasmids, pETNde43-12 (containing the btau4
gene) and pET-K0 (containing K0 which consists mainly of the four
repeats plus leading and trailing sequences from the vector,
Steiner et al.) were used for the construction of T8R-1. The
NdeI-RsaI DNA fragment from btau4 was connected with "filled in"
XmaI-BamHI fragment of K0 leading to chimeric molecule consisting
of 553 amino acids. The T8R-1 gene encodes Met1-Bal393 connected
through the artificially introduced Ser residue with the
Gly248-Tyr394 tau fragment, followed by a 23 residue tail from the
bacteriophage T7 sequence (htau40 numbering).
[0370] Construction of T7R-2 and T8R-2: T7R-2 is a tau derivative
containing 7 repeats, T8R-2 contains 8 repeats. Both molecules were
constructed on the basis of the human htau23 and htau24 isoforms
(Goedert et al.). For the engineering of the T7R-2 and T8R-2
molecules, PCR repeat cassettes A1 (encoding 4 repeats), A2
(encoding the whole carboxy part of the tau24 molecule including
the four repeat sequence and the tau sequence behind them) and A3
(encoding 3 repeats) were prepared. The T8R-2 molecule was
generated by combination of A1 and A2 with NdeI-PstI DNA fragment
isolated from htau23. This tau derivative consists of 511 amino
acids, the first 311 N-terminal residues of htau24 (Met1-Lys369,
containing 4 repeats), followed by Gly-Thr link, then by 198
residues of the C-terminus of htau24 (Gln244-Leu441, four more
repeats). The T7R-2 gene was generated similarly to T8R-2 but the
A3 cassette was used instead of A1. The T7R-2 protein consists of
480 amino acids, the first 280 N-terminal residues of htau23
(Met1-Lys369, including 3 repeats), followed by Gly-Thr link, then
by 198 residues of the carboxy terminal part of htau 24
(gln244-Leu441, containing 4 repeats, htau40 numbering).
EXAMPLE 9
Conformation of Tau Protein and Higher Order Structures Thereof
(a) Conformation and Dimerization of Tau Constructs
[0371] FIG. 19 illustrates the types of constructs used in this
example. Three types of molecules were used: (i) tau isoforms
occurring in brain ranging from htau23 (the smallest isoform, 352
residues) to htau40 (the largest 441 residue, see Goedert et al.).
They differ mainly by the number of internal repeats in the
C-terminal domain (3 or 4) and the number of inserts near the
N-terminus (0, 1, or 2). The internal repeats are involved in
microtubule binding and in the formation of paired helical
filaments; attention was then focused on the those constructs which
would presumably yield information on the structure of the repeat
region; (ii) engineered constructs with an increased number of
repeats, e.g. seven or eight (T7, T8); (iii) constructs containing
essentially the repeats only (e.g. K11, K12).
[0372] The SDS gels of FIG. 20 illustrate some of these proteins.
Most tau constructs have M.sub.r values larger than expected from
their actual mass (FIG. 20a). A notable feature is the tendency to
form dimers and oligomers. This is particularly pronounced with
some constructs, for example K12 (FIG. 20b). The formation of
dimers can already be observed by letting the protein stand for
some time (FIG. 20b, lane 2), presumably because the dimers become
fixed by a disulfide bridge; this can be prevented by DTT (lane 1).
To test this, the cross-linker PDM which predominantly links
cysteines was used. This generates essentially the same products as
in the absence of DTT (lane 3). Chemical cross-linking for
construct K12 (2-5 mg/ml) was carried out by incubation in 40 mM
HEPES pH 7.5 with 0.5 mM DTT for 30 min at 37.degree. C. and
followed by reaction for 30 min at room temperature with 0.7 mM PDM
(Sigma) or 1.5 mM MBS (Pierce) added from freshly prepared stock
solutions in DMSO. The reactions were quenched by addition with 5
mM DTT or 5 mM DTT and 5 mM ethanolamine, respectively. Finally,
dimers and higher oligomers can also be generated by MBS, which
links cysteines and lysines (lane 4). The cross-linked species can
be separated by chromatography on a Superose 12 column (FIG. 20c),
allowing the study of a homogeneous population of dimers. For this
purpose, the covalently cross-linked dimers were separated from the
monomers by gel filtration on a Pharmacia Superose 12 FPLC column
equilibrated and eluted with 50 mM Tris-HCL pH 7.6 containing 0.5 M
NaSCN, 0.5 M LiCl and 2 mM DTT operated at a flow rate of 0.3
ml/min. Column fractions were analysed by SDS-PAGE, pooled and
concentrated by centrifugation through centricon 3
microconcentrators (Amicon). The column was calibrated with the
proteins from the Pharmacia low molecular weight gel filtration
calibration kit. Effective hydrated Stokes radii (r) of the
calibration proteins were taken from the kit's instruction manual
and partition coefficients (a) were determined from the elution
volumes and fitted to an equation of the form .sigma.=-A log r+B,
yielding the Stokes radii for the tau construct monomers and
dimers. The axial ratios were calculated following Perrin (for
further details, see Cantor & Schimmel, Biophysical chemistry,
Part II: Techniques for the study of biological structure and
function. Freeman & Co, San Francisco, 1980) The elution
profile (FIG. 20d) yields Stokes radii of 2.5 nm for the monomer of
K12, and 3.0 nm for the dimer. Given the molecular weights of 13
and 26 kDal this yields axial ratios of 10 and 8, consistent with
the rod-like shape observed by electron microscopy (the equivalent
lengths of prolate ellipsoids would be 6.8 and 8.5 nm which
underestimates the actual lengths; see below).
[0373] Other tau species show similar cross-linking results, but
they are somewhat more complex for the following reason: Tau has
cysteines only in repeats 2 and 3 (residues Cys291 and Cys322).
Repeat 2 is absent from some isoforms, for example htau 23 or
construct K12, leaving only the lone Cys322. With Cys-Cys
cross-linkers such as PDM, these molecules can only form dimers,
but no higher aggregates (FIG. 20b, lane 3). In contrast, bivalent
molecules with two cysteines (such as htau40, K11) can form
intramolecular cross-links, dimers and higher oligomers. This
diversity is similar to what is found after cross-linking K12 with
MBS (FIG. 20b, lane 4) because tau contains many lysines.
[0374] The conformation of several tau constructs in solution was
probed by analytical ultracentrifugation and CD spectroscopy
according to standard procedures. For example, htau40 had a
sedimentation constant of 2.6 S on the mixture of tau from brain.
For a globular particle of the mass of htau40 (45.8 kDal) one would
expect .apprxeq.4.2 S; the lower observed value indicates an
elongated structure with a hydrodynamic axial ratio of .apprxeq.15.
The CD spectra of htau40 and construct K12 (FIG. 20e) were
indistinguishable; they showed very little secondary structure.
This means that both the N-terminal and C-terminal domains of tau
lack internal regularity such a .alpha.-helix or .beta.-sheet.
(b) Synthetic Paired Helical Filaments.
[0375] Tau isolated from brain tissue can self-assemble into
fibrous structures (see e.g. Montejo de Garcini & Avila, J.
Biochem. 102 (1987), 1415-1421; Lichtenberg-Kraag & Mandelkow,
J. Struct. Biol. 105 (1990), 46-53). This property became
particularly interesting in view of the fact that tau is one of the
main components of the neurofibrillary tangles of Alzheimer's
disease. In the earlier studies the relationship of the filaments
formed in vitro to the Alzheimer PHrs remained ambiguous,
especially since the protein was heterogeneous. It was therefore
desirable to check if recombinant tau constructs were capable of
self-assembly. This was tested in a variety of conditions of pH,
salt buffer type, etc. Typically, solutions of tau constructs or
chemically cross-linked dimers were dialyzed against various
buffers (e.g. .apprxeq.50-500 mM MES, Tris-HCl, Tris-maleate, pH
values 5-9, 5-30 mM MgCl.sub.2, CaCl.sub.2, AlCl.sub.3) for 12-24
hours at 4.degree. C. The solution was briefly centrifuged (Heraeus
Biofuge A, 1 min, 10,000 g) and the pellet was stored for several
days at 4.degree. C. and then processed for negative stain electron
microscopy (2% uranyl acetate or 1% phosphotungstic acid).
Alternatively the solution was used for grid dialysis on gold grids
following Van Bruggen et al., J. Microsc. 141 (1986), 11-20. Of the
constructs tested only K11 and K12 yielded filaments resembling
PHFs. The optimal conditions were 0.3-0.5 M Tris-HCl and pH
5.0-5.5, and without any additional salts. The results obtained
with construct K12 are illustrated in FIG. 21. In the pH range of
5.0-5.5 there was extensive formation of filaments. Their length
was variable, but typically in the range of 200-1000 nm. Most
appeared rather smooth, others showed a regular variation of width,
with axial periodicities around 70-75 nm (arrowheads). The minimum
diameter was about 8 nm and the maximum around 15 nm. Short
rod-like particles, about 80-150 nm in length were also observed,
which appeared to represent just one or two crossover periods of
the filaments (FIG. 21, middle). It was not possible to discern
reliably any axial fine structure that might indicate an
arrangement of protein subunits. This was therefore either below
the resolution limit of negative stain, and/or due to lack of
contrast. In general the filaments tended to be bundled up in
clusters, as if they had a high affinity for one another (FIG.
21a). Similar PHF-like filaments were also obtained with K12 dimers
cross-linked with PDM (FIG. 22). This suggests that the dimer might
be an intermediate stage in filament assembly.
[0376] Many of these features are similar to those of paired
helical filaments isolated from Alzheimer's disease brains, shown
for comparison in FIG. 23. Their appearance depends somewhat on the
isolation procedure. FIG. 5a shows "insoluble" filaments prepared
from neurofibrillary tangles after Wischik et al., J. Cell Biol.
100 (1985), 1905-1912. These filaments are long, straight, and have
a homogeneous ultrastructure characterized by the distinct
.apprxeq.75 nm repeat. By contrast, when the filaments are
"solubilized" by sarkosyl following Greenberg & Davies, Proc.
Natl. Acad. Sci. USA 83 (1990), 5827-5831, they are shorter and
less homogeneous (FIG. 23b). In particular, this preparation
includes very short particles (equivalent to about 1-2 crossover
periods), and smooth filaments that do not have the twisted
appearance (reminiscent of straight filaments). There is a striking
similarity between the synthetic PHFs based on the repeat domain
(e.g. K11, K12, K12 dimers, FIG. 21, 22) with the soluble PHFs from
Alzheimer brains (FIG. 23b), judged by three different criteria:
(i) The filaments are shorter than the insoluble PHFs of FIG. 23a;
(ii) they are less homogeneous in their periodicity, and some lack
the twisted appearance altogether (straight filaments), (iii) they
include very short rod-like particles, down to the length of one
crossover period.
[0377] Thus far, synthetic PHF-like fibers have only been observed
with constructs such as K12 and K11 containing essentially the
repeat domain (3 or 4 repeats, FIG. 19), but not with larger tau
isoforms. These data are all consistent with the assumption that
the repeat domain is the basic unit that is capable of
self-assembling into PHFs very similar to those of Alzheimer
neurofibrillary tangles. This also agrees with experiments in
several laboratories showing that the pronase-resistant core of
Alzheimer PHFs contains the repeat region (e.g. Goedert et al.,
ibid., Jakes et al., EMBO J. 10 (1991), 2725-2729). It was also
noted that the filament-forming constructs were not phosphorylated
so that this does not, in contrast to the genuine Alzheimer PHF,
play a role in self-assembly here.
(c) Electron Microscopy of Tau Monomers and Dimers.
[0378] The results on the synthetic PHFs suggested that the repeat
region had a special role in the interaction between tau molecules.
It was therefore desirable to define their structure in more detail
by comparing different constructs in the electron microscope. The
method of choice was metal shadowing at a very shallow angle,
combined with glycerol spraying; this helps to make the particles
visible which otherwise would not be seen because of their low
contrast. Spraying was done following Tyler & Branton, J.
Ultrastruct. Res. 71 (1980), 95-102. The samples were diluted 1:10
in spraying buffer (50 mM ammonium acetate pH 8.0, 150 mM NaCl, 1
mM MgCl.sub.2, 0.1 mM EGTA), made up to 70% glycerol and sprayed
onto freshly cleaved mica. The sprayed samples were vacuum dried
for 2 hours, shadowed with platinum/carbon (thickness about 1.5 nm,
shadowing angle 4.degree.) using a BAE 080T shadowing unit (Balzers
Union), followed by 20-30 nm carbon. Finally the replicas were
floated off on doubly distilled water and picked up with 600 mesh
copper grids.
[0379] Molecules of htau23 (352 residues, FIG. 24a) are rod-like
and have a mean length of 35.+-.7 nm (lengths summarized in Table 2
and FIG. 25). This value is less than that reported by Hirokawa et
al., J. Cell Biol. 107 (1988), 1449-1459, but this might be due to
differences in the experimental approach (freezing vs. glycerol
spraying; a mixture of all isoforms vs. the smallest isoform). The
apparent width of the metal shadowed htau23 molecules is about 3-5
nm, and the contrast is low--much less than that of control samples
(single and double stranded DNA, .alpha.-helical proteins). Careful
inspection of the micrographs reveals a population of particles
with enhanced contrast, somewhat larger diameter (5-7 nm),
sometimes split into two parts, and lengths similar or slightly
more than the monomer (around 40 nm). These particles are
interpreted as (nearly) juxtaposed monomers forming dimers (FIG.
24b), consistent with the results on cross-linked dimers and
antibody decoration shown later.
[0380] Clearly longer particles are obtained with the construct
T8R-1 which average 58.+-.15 nm, 23 nm more than htau23 (FIG. 24c,
25b). This construct contains eight repeats (a duplication of the
four basic ones, FIG. 19), that is five repeats more than htau23,
plus the two 29-mer inserts near the N-terminus. T8R-2 has a
similar length (61.+-.17 nm), even though it lacks the N-terminal
inserts. Construct T7R-2 also has a similar length of 60.+-.16 nm,
even though it has only seven repeats (3+4) and no N-terminal
inserts. At first sight these results appear puzzling: On the one
hand, larger constructs become longer, but on the other hand
certain parts of the sequence do not affect the length.
Anticipating the results below, the contradiction can be explained
by a unifying hypothesis: The length of the tau constructs is
determined mainly by the repeat region; by comparison, the
N-terminal domain and the C-terminal tail are only of minor
influence. The repeat region itself must be considered a unit,
roughly 20-25 nm long, whose length is approximately independent of
the second repeat. The hypothesis implies that the N-terminal
inserts have only a minor influence on the length. It predicts that
constructs with 3 or 4 repeats have roughly the same length (e.g.
T7R vs. T8R), and that the addition of one repeat domain adds about
20-25 nm in length (as in htau23 vs. T7R or T8R).
[0381] T8R and other constructs also form particles folded into a
hairpin (FIG. 24d), as if the two "units" (of four repeats each in
this case) could interact; this is suggestive of an antiparallel
arrangement, supporting the antibody data described infra. T8R
particles were also observed whose width and contrast indicate
dimers similar to htau23 (FIG. 24e).
[0382] As in the previous cases, the repeat domain constructs that
form the PHF-like fibers formed by K11 and K12 described above
(FIG. 26) are rod-like. Using the criteria of thickness or contrast
and the comparison with the dimers, K11 displays a population of
low contrast monomers with a mean length of 26.+-.5 nm (FIG. 26a),
and a population of more contrasty dimers, about 32.+-.6 nm (FIG.
26b). This means that the two molecules must be juxtaposed for most
of their length. For K12, monomers of length 25.+-.4 nm, (FIG.
26d), and dimers of about 30.+-.4 nm (FIG. 26e) are found. The
monomers have about 70-75% of the length of htau23, although they
contain only a third of the residues (FIG. 25c, e). With both
constructs, longer particles are found which are interpreted as
dimers associated into tetramers (FIG. 26c, f).
[0383] Thus far the classification into monomers and dimers was
judged by relating the width and contrast of the particles to model
structures. However, it is possible to isolate the covalently
cross-linked dimers by gel chromatography and study them directly
by electron microscopy and other methods. As an example, dimers of
K12 cross-linked by PDM via the single Cys322 (FIG. 27a) are shown.
In the electron microscope, their contrast is similar to the dimers
described above; but more importantly, they are only slightly
longer than the monomers (29.+-.6 nm FIG. 27a, FIG. 25e, g). This
means that the PDM dimers are formed by two molecules lying next to
one another and nearly in register. The dimers of K12 induced by
MBS (34.+-.6 nm) are also similar, except that they tend to be
somewhat longer (by .apprxeq.5 nm) than those obtained with PDM,
probably because a greater variety of Cys-Lys bonds are possible
(FIG. 27b, 25h).
[0384] Taken together, the results obtained with K11 and K12 (and
other constructs containing essentially the repeat domain) are
consistent with the hypothesis that the repeat domain forms a
folding unit of rather uniform length, independently of whether it
contains 3 or 4 repeats.
[0385] For all constructs tested, the glycerol spray experiments
show a certain tendency to form fibrous structures. In most cases,
they are rather uniform in diameter, they show no obvious
relationship to paired helical filaments and may result from a
distinct pathway of self-assembly.
(d) Antiparallel Alignment of Dimers
[0386] It is clear from the above data that tau and its constructs
tend to align laterally into dimers. This raised the question of
polarity: Are the particles parallel or antiparallel? First
indications came from the hairpin fold observed with the 8-repeat
constructs (e.g. FIG. 24d), suggesting antiparallel orientations of
the two halves. Direct evidence for this was obtained by labeling
with the monoclonal antibody 2-4 whose epitope is on the last
repeat and therefore close to the C-terminus in terms of the
sequence (Dingus et al., J. Biol. Chem. 266 (1991), 18854-18860).
FIG. 28a (left) shows particles of htau23 with one antibody
molecule bound. The antibodies bind at or near one end, showing
that one of the physical ends of the rod coincides roughly with the
C-terminus. The lengths of the rod portions shown are similar to
those of unlabeled htau23; in terms of apparent width, they could
be monomers or dimers. In the same fields, one also finds doubly
labeled particles (FIG. 28a, right). The antibodies bind at
opposite ends, proving that the two subunits of a dimer have
opposite polarities.
[0387] The same features are found with construct K12; rodlike
stubs with an antibody at one end (FIG. 28b, left); dumbbells, i.e.
antiparallel dimers (FIG. 28b, middle). Finally, there are
particles with two antibodies and two stubs, with a kink in the
middle (pairs of "cherries," FIG. 28b, right). Each of the arms has
roughly the length of a unit stub so that the particles appear
equivalent to the tetramers of FIGS. 26c and f. The interaction
between the dimers at the center appears to prevent the binding of
an antibody which could otherwise be expected there.
[0388] PDM dimers of construct K12 (formed by Cys322-Cys322
crosslinks) are shown in FIG. 28c. Particles with one anti-body
label are on the left, doubly labeled ones in the middle, showing
that the chemically crosslinked dimer consists of antiparallel
monomer. A presumptive tetramer is on the right. Essentially the
same data are obtained with MBS crosslinked dimers (Cys322 to
nearby Lys, FIG. 28d).
[0389] Based on the knowledge described in this Example, in vitro
methods for testing drugs effective in dissolving Alzheimer paired
helical filaments as for testing drugs effective in the reduction
or prevention of the formation of Alzheimer paired helical
filaments may be developed, as is described above.
EXAMPLE 10
Effect of Glycogen Synthase Kinase-3 (GSK-3) and cdk2-Cyclin A on
Phosphorylation of the Tau Protein
[0390] Experiments described in Examples 4 and 5 were repeated
using GSK3 (also referred to as phosphatase activating factor
F.sub.A, Vandenheede et al., J. Biol. Chem. 255 (1980),
11768-11774) as the phosphorylating enzyme.
[0391] GSK3 (.alpha. and .beta. isoforms) were purified from bovine
brain as described in Vandenheede et al., ibid., with an additional
Mono S chromatography step which separates the two isoforms. Most
experiments described here were done with immunoprecipitates of
GSK-.alpha. on TSK beads (following Van Lint et al., Analyt.
Biochem. 1993, in press), but control experiments with the .beta.
subunits showed the same behavior.
[0392] Polyclonal anti-peptide antibodies to the .alpha. and .beta.
isoforms of GSK3 were raised in rabbits and affinity purified on
peptide columns. Immunoprecipitates of GSK3 were prepared from
PC-12 cytosols in 20 mM Tris-HCl, 1% NP-40, 1 mM PMSF, 2 .mu.g/ml
aprotinin, 1 .mu.g/ml leupeptin and 0.2 .mu.g/ml pepstatin. 100
.mu.l of cytosols were incubated with 1 .mu.l of .alpha.- or
.beta.-GSK antibodies (1 mg/ml) or control rabbit antibodies and
incubated for 4 h at 4.degree. C., 5 .mu.l of TSK-protein A beads
were added and incubated for another hour, and finally the beads
were washed with 10 mg/ml BSA in 20 mM Tris-HCl, 0.5 M LiCl in Tris
buffer, and 20 mM Hepes pH 7.2 with 10 mM MgCl.sub.2 and 1 mM DTT.
In phosphorylation assays, 2 .mu.l of pellets were incubated with 8
.mu.l of substrate (3 .mu.M) in 40 mM Hepes pH 7.2, 10 mM
MgCl.sub.2, 2 mM ATP, 2 mM EGTA, 0.5 mM DTT and 1 mM PMSF.
(a) Time Course of Phosphorylation and Antibody Response Induced by
GSK3
[0393] FIG. 29 shows a time course of phosphorylation of htau40
with GSK3, and the corresponding autoradiogram and immunoblots. In
most respects the behavior is similar to that obtained with the
brain kinase activity or with purified MAP kinase. Phosphorylation
induces a gel shift in three main stages; it incorporates
.apprxeq.4 P.sub.i; it induces the reactivity of antibodies AT8,
SMI34, and SMI31, but reduces the reactivity of TAU1 and SMI33.
(b) Phosphorylation Sites of GSK3 on Tau
[0394] The main phosphorylation sites can be determined from
anti-body epitopes and point mutants (FIG. 29). TAU1 requires that
both Ser199 and Ser202 are unphosphorylated, AT8 requires them both
phosphorylated. Thus when only one of the two serines is
phosphorylated these antibodies do not react. This means that
Ser199 and Ser202 both become phosphorylated during stage 2 (FIG.
29, panels 3, 4). Similarly, antibody SMI31 requires the
phosphorylation of both Ser396 and Ser404, which means that both
serines become phosphorylated rapidly during stage 1 (FIG. 29,
panel 6). SMI33 reacts only when Ser235 is unphosphorylated so that
the gradual loss of reactivity means that this residue becomes
phosphorylated only slowly (panel 7). Together these residues would
account for 5 Pi, but only .apprxeq.4 Pi were observed by
autoradiography, indicating that not all of these serines are
phosphorylated at 100%. There are some subtle differences in the
time course of immune response, compared to MAP kinase. For
example, the SMI31 reactivity sets in early and precedes that of
AT8 and SMI34, while the reactivity of SMI33 persists for a longer
time, indicating that the mode of action of GSK3 is not identical
to that of MAP kinase.
[0395] Additional information can be obtained by point mutations.
As shown in Examples 5 and 6, the initial strong mobility shift
induced by the kinase activity from brain extracts and by MAP
kinase is due to the phosphorylation of Ser404. The same is true
for GSK3, as illustrated in FIG. 30 (lanes 1-3). When Ser404 is
mutated into Ala, the initial rapid shift disappears, and initial
phosphorylation is reduced to a low level (FIG. 30, compare lanes 2
and 5).
[0396] Another conclusion from the immunoblots is that GSK3
strongly prefers Ser-Pro motifs, in contrast to MAP kinase which
also affects Thr-Pro. This follows since the .apprxeq.4 Pi
incorporated are needed to account for the phosphorylated epitopes.
To test this construct AP11 was prepared, a derivative of htau23
where all 6 Ser-Pro are replaced with Ala-Pro (FIG. 31, middle).
AP11 is phosphorylated only to a minimal extent, <0.1 Pi per
molecule, confirming that the Thr-Pro motifs remain largely
unphosphorylated. The same result is obtained with construct AP17
(all 6 Ser-Pro and 8 Thr-Pro replaced by Ala-Pro, FIG. 31, top).
Another construct, K18 containing only the four repeats (FIG. 31,
bottom), is also not phosphorylated, indicating that no major sites
are within the microtubule binding region. Thus, GSK3 and MAP
kinase are similar in that they are both proline directed, but MAP
kinase is also active with respect to Thr-Pro motifs.
(c) GSK3 and MAP Kinase are Associated with Microtubules and with
PHFs
[0397] Considering that tau is a microtubule-associated protein one
might expect that kinases that phosphorylate tau might be localized
in the vicinity. It was therefore tested whether MAP kinase or GSK3
were microtube-associated proteins according to the usual criterium
of co-purification through repeated circles of assembly and
disassembly. This was indeed the case. FIG. 32b shows that both the
p42 and p44 isoforms of MAP kinase co-purified with porcine brain
microtubules, FIG. 32c,d demonstrates the same for the case of GSK3
.alpha. and .beta.. Interestingly, the microtubule-associated MAP
kinase was not in an activated state since it was not
phosphorylated on Tyr (as judged by immunoblotting, not shown).
[0398] Considering this result, it was of interest to investigate
whether the kinases were also associated with Alzheimer PHFs. The
immunoblots of FIG. 33a demonstrate that GSK3 is present in normal
and in Alzheimer brain in roughly equivalent amounts and thus
resembles MAP kinase in this respect. Moreover, the kinases
co-purify directly with PHFs isolated by two different procedures,
following Wischik et al., J. Cell. Biol. 100 (1985), 1905-1912
(FIG. 33b, lane 1) and Wolozin et al., Science 232 (1986), 648-650
(lane 2).
[0399] The fact that GSK3 is associated with microtubules and PHFs
and phosphorylates tau would suggest that the kinase might be able
to affect the interaction between tau and microtubules. This would
be in agreement with a common notion about the pathological effects
of tau phosphorylation. Surprisingly, however, there was no
influence on the binding. FIG. 34 shows the binding of htau23 to
microtubules without phosphorylation, with phosphorylation by GSK3,
and by the kinase activity of the brain extract. In the latter
case, there is a strong reduction in affinity, but the effect of
GSK3 itself is minimal.
(d) Phosphorylation of Tau by Cdk2-Cyclin A
[0400] The protein kinase cdk2-cyclin A (a proline-directed ser/thr
kinase; see Hunter, ibid.) induces the Alzheimer-like state, as
judged by phosphorylation, gel shift and antibody response. The
kinase cdk2 incorporated 3.5 Pi into htau40 and generated a similar
shift in the gel as MAP kinase and GSK-3. The antibodies AT-8,
SMI31, SMI34 recognize the phosphorylated tau, TAU-1 and SMI33 do
not, again similar to MAP kinase and GSK-3. All ser-pro motifs (Ser
199, 202, 235, 396, 405, 422) can be phosphorylated to some extent;
see FIG. 46.
[0401] The preparation was as follows: Cells overproducing the
cdk2/cyclin A complex were obtained by Dr. Piwnica Worms,
Boston.
[0402] Cyclin A was fused to glutathione-S-transferase. Thus, the
complex is easily purified using glutathione agarose beads as
outlined below:
Kinase Assays on Glutathione Beads:
[0403] 3.times.10.sup.6 cells were infected with viruses encoding
human p33.sup.cdk2 and human cyclin A (fused to
glutathione-S-transferase) each at an m.o.i. of 10. At 40 hours
post infection, cells were rinsed (2.times.) in PBS. Cells were
frozen on plate at -70.degree. C. (Cells are kept frozen until
experiments are carried out.)
Preparation of Cell Lysates:
[0404] Lyse cells in 1 ml of the following buffer: [0405] 50 mM
Tris pH 7.4 [0406] 250 mM NaCl [0407] 50 mM NaF [0408] 10 mM NaPPi
[0409] 0.1% NP40 [0410] 10% glycerol [0411] protease inhibitors
(0.15 units/ml aprotinin, 2 nM PMSF, 20 .mu.M leupeptin)
[0412] Plates were rocked for 15 min at 4.degree. C., lysates were
collected, placed in Eppendorf tube and spun at 10K for 10 min at
4.degree. C. Clarified lysates were placed in fresh Eppendorf
tube.
Glutathione Precipitation
[0413] 100 .mu.l (50% slurry of agarose in PBS) of glutathione
agarose (from Sigma) were added to the clarified lysate, rocked
.apprxeq.1 hour at 4.degree. C. and were spun briefly to pellet
beads.
[0414] Beads were washed two times in 1 ml of above lysis buffer
and washed two times with incomplete kinase buffer (50 mM Tris pH
7.4, 10 mM MgCl.sub.2). As much buffer as possible was removed from
the beads after the final wash.
For Kinase Assays:
[0415] Exogenous substrate was added and then complete kinase
buffer was added: [0416] 50 mM Tris, pH 7.4 [0417] 10 mM MgCl.sub.2
[0418] 1 mM DTT [0419] 10 .mu.M unlabeled ATP [0420] 2 .mu.l of
gamma .sup.32P-labelled ATP (NEN: 3000 Ci/mM) and incubated at
30.degree. C. for the desired amount of time.
EXAMPLE 11
Phosphorylation of Ser 262 of Tau Protein by a Novel Kinase and
Effect Thereof on Binding to Microtubules by Tau Proteins
[0421] So far it has been shown that the Alzheimer-like state of
tau protein includes phosphorylation of Ser-Pro and Thr-Pro motifs,
and that this state can be mimicked by a brain extract-kinase
activity and by MAP kinase, as judged by the response with
Alzheimer-specific antibodies. As will be demonstrated in the
following, a crucial regulation of tau's binding to microtubules
occurs at Ser262, a residue phosphorylated by the brain extract
activity but not by MAP kinase. A novel kinase from mammalian brain
which phosphorylates this residue and thereby strongly reduces the
interaction between microtubules and tau protein has furthermore
been purified.
[0422] Binding studies between tau and taxol-stabilized
microtubules were done as described in Example 6. This provides a
direct measure of the attachment of tau to pre-formed microtubules
and yields dissociation constants and binding stoichiometries
(n=tau.sub.bound/tubulin dimer); the reduction in stoichiometry is
the most conspicuous and reproducible parameter. The drop in
stoichiometry in a wild type tau isoform upon phosphorylation,
D.sub.n,wt=(n.sub.unphos-n.sub.phos).sub.wt, is taken as 100% and
can be compared to the effect of phosphorylation on a mutant.
D.sub.n,mut.
[0423] Preparation of the kinase from brain: An extract from 250 g
of porcine brain tissue was prepared and submitted to ammonium
sulfate precipitation as described in Example 2. The precipitate
obtained between 30 and 45% saturation was homogenized in buffer 1
(25 mM Tris-HCl pH 7.4 containing 25 mM NaCl, 2 mM EGTA, 2 mM DTT,
1 mM PMSF) and dialyzed against 1 liter of this buffer with two
changes overnight. Total protein concentration was determined using
the Pierce BCA assay kit. After clarification of the dialysate by
ultracentrifugation, portions of up to 250 mg of protein were
loaded on a Mono QHR 10/10 column (Pharmacia) equilibrated with
buffer 1. Elution was performed with a linear gradient of 25-500 mM
NaCl in 120 ml of buffer 1 with a flow rate of 2 ml/min. Fractions
were screened by phosphorylation of bacterially expressed tau and
tau constructs as described below. Active peaks were pooled and
concentrated 10 to 40-fold by centrifugation through Centriprep 10
microconcentrators (Amicon) and chromatographed on a Superdex 75
HiLoad 16/60 size exclusion column (Pharmacia) equilibrated and
eluted with buffer 1 containing 50 mM NaCl. Active fractions were
pooled and rechromatographed on a Mono Q HR 5/5 column with a
gradient of 0-600 mM NaCl in 30 ml of buffer 1 with a flow rate of
0.5 ml/min. Active fractions were dialyzed against buffer 1 and
stored at 0.degree. C. The gel filtration column was calibrated
with the Pharmacia low weight marker set. Phosphorylation assays
were performed as described (Steiner et al., 1990, ibid.).
[0424] In-gel assays of tau phosphorylation were done following
Geahlen et al., Anal. Biochem. 153 (1986), 151-158. MonoQ-fractions
with kinase activity were subjected to 11% SDS PAGE (0.5 mm thick
slab gels). Tau protein was added to the separation gel solution
just prior to polymerisation (final concentration 0.1 mg/ml). The
following steps were then performed: (1) To remove SDS, the gels
were washed with two changes of 20% propanol in 50 mM Tris-HCl pH
8.0 for 30 min at room temperature, then 50 mM Tris-HCl pH 8.0
containing 5 mM .beta.-mercaptoethanol (=buffer A) for another 30
min at RT. (2) The enzyme was denatured by two changes of 6 M
guanidine-HCL for 1 hour at room temperature (RT). (3) The enzyme
was renatured by five changes of buffer A containing 0.04% Tween 40
for .apprxeq.15 hours at 4.degree. C. (4) Pre-incubation with
phosphorylation buffer without ATP for 30 min at RT (40 mM Hepes pH
7.5, 5 mM EGTA, 3 mM MgCl, 0.1 mM PMSF, 2 mM DTT). (5)
Phosphorylation with added 0.1 mM ATP and 130 Ci/Mol (gamma-32) ATP
was performed by incubation of the gel in a plastic bag at
37.degree. C. for 20 hours on a rotating wheel. (6) Removal of
excess (gamma-32) ATP: The gel was washed by incubation in five
changes of 300-500 ml of 5% TCA containing 1% sodium pyrophosphate
until unbound radioactivity was negligible. (7) Staining and
autoradiography were done according to conventional methods.
(a) Phosphorylation of Ser262 Strongly Reduces the Binding of Tau
to Microtubules
[0425] As shown in Example 6, when tau protein is phosphorylated by
the brain extract kinase activity, the stoichiometry typically
dropped from .apprxeq.0.5 tau per tubulin dimer down to
.apprxeq.0.1-0.15, i.e. about 3-4-fold; this effect on the wild
type protein will be taken as 100% in this Example. The parameters
affected by phosphorylation have distinct time courses. A major
part of the gel shift occurs early (stage 1 phosphorylation, up to
2 hours) and can be ascribed to a single site, Ser 404 (numbering
of htau40). Most of the Alzheimer-like antibody response, as well
as an additional gel shift, sets in during stage 2 (up to
.apprxeq.6 hours); a further shift combined with more incorporation
of phosphate occurs during stage three (up to 24 hours). However,
the effect on microtubule binding was already fully visible after
stage 1. At this point, the protein bound about two moles of
P.sub.i (out of a maximum of .apprxeq.5-6). About one of these was
at Ser404, identifiable by the first gel shift. The other phosphate
was distributed among Ser202, 235, and 262, but exact
quantification by autoradiography and phosphopeptide sequencing was
difficult.
[0426] It was therefore decided to approach the problem by
site-directed mutagenesis. The Ser residues in question were
replaced by Ala (making them non-phosphorylatable) or Asp
(mimicking the negative charge of the phosphorylated state; see
FIG. 35a). These mutants were then assayed with respect to gel
shift, phosphate incorporation, and microtubule binding (FIG. 35b).
The mutant Ser404-Ala loses its shift during stage 1
phosphorylation, but the phosphorylation of this protein still has
a sizable effect in reducing the microtubule binding capacity
(difference in stoichiometry D.sub.n=0.17, i.e. 52% of the
unmutated control with D.sub.n=0.33). This suggests that one or
more of the remaining Ser202, 235, and 262 are responsible for a
major fraction of the phosphorylation effect on binding. Similar
results are obtained when Ser202, 235, and 396 are mutated into Ala
or Asp, indicating that neither of these residues accounts for the
low stoichiometry after phosphorylation observed with wild type
htau23. However, when Ser262 was altered, the binding to
microtubules was nearly unaffected by phosphorylation
(D.sub.n=0.04). In other words, it appears that mutating one
residue, Ser262 in the first repeat, nearly eliminates the
phosphorylation sensitivity of tau towards microtubule binding; or
conversely, phosphorylation of Ser262 reduces the binding of tau to
microtubules dramatically.
(b) MP Kinase Induces the Alzheimer-Like Immune Response of Tau But
does not Impair Microtubule Binding
[0427] The binding data in section (a) were obtained with a brain
extract, but most of the properties of extract phosphorylation
could be induced by purified MAP kinase from Xenopus oocytes or
porcine brain. Extract and MAP kinase induce a gel shift, they have
a similar time course of phosphorylation, and both induce a similar
pattern of antibody responses (including the onset of the
"Alzheimer-like" response in stage 2 phosphorylation). The majority
of sites found with the extract are in Ser-Pro motifs; all of them
are phosphorylated by MAP kinase as well, plus Thr-Pro motifs, i.e.
purified MAP kinase is more efficient as a Pro-directed Ser/Thr
kinase than the brain extract. Finally, MAP kinase is a major
phosphorylating component in the brain extracts.
[0428] However, when the effect of highly purified MAP kinase on
tau's microtubule binding was tested it turned out to be
surprisingly small (D.sub.n=0.09) compared with the brain extract
(D.sub.n=0.31 in FIG. 36). This was consistent with the above
experiments, suggesting that phosphorylation of Ser-Pro or Thr-Pro
motifs by itself was only of secondary importance with respect to
microtubule binding.
[0429] This was tested by employing two "total" mutants, AP17 and
AP18 derived from htau23 (FIG. 37a). AP18 is similar to AP17, but
in addition Ser262 and 356 (the two serines not followed by Pro
found earlier in extract phosphorylations) were changed into Ala.
While MAP kinase phosphorylates all Ser-Pro and Thr-Pro sites of
wild type htau23 (typically up to a maximum of 10-12 moles of
P.sub.i per htau23), AP17 incorporates at most 1.4 P.sub.i,
illustrating the high specificity of MAP kinase for Ser-Pro or
Thr-Pro motifs. AP17 binds tightly to microtubules, independently
of phosphorylation by MAP kinase, with similar parameters as
unphosphorylated wild type htau23. The same results are obtained
with AP18 and MAP kinase (<1 P.sub.i incorporated).
[0430] However, when AP17 and AP18 are phosphorylated with the
brain extract activity the two mutants are dramatically different
(FIG. 37b). AP18 incorporates about 0.5 P.sub.i and shows only a
minor reduction of the stoichiometry of tau bound to microtubules
upon phosphorylation (D.sub.n=0.01). AP17 incorporates .apprxeq.1.3
P.sub.i, and yet its reduction of the binding of tau to
microtubules upon phosphorylation is the same as that of wild type
htau23 (D.sub.n=0.31).
[0431] These results made it clear that the brain extract
apparently contains some phosphorylating component distinct from
MAP kinase which phosphorylates Ser262 in the first repeat of tau
protein, and that this single Ser, when phosphorylated, is capable
of dramatically altering the interaction of tau with microtubules.
By contrast, MAP kinase affects the other indicators of the
Alzheimer state of tau, the gel shift and the immune response.
(c) The 35 kDal and 70 kDal Kinases in Brain Reduces Microtubule
Binding by Phosphorylating Ser 262
[0432] The sequence around Ser 262 does not fit obvious consensus
motifs of known kinases so that it did not seem promising to test
them. Instead, the kinase was purified from the brain extract.
Active fractions were identified by the criteria of tau
phosphorylation and effect on microtubule binding.
[0433] The first step was ion exchange chromatography on Mono Q
(FIG. 38a), yielding 3 main peaks of kinase activity. The fractions
with the largest effect on microtubule binding were further
subjected to gel chromatography (FIG. 38b). The main active
fraction eluted at an Mr around 35 kDal. This was followed by
another ion exchange run. The protein did not bind to Mono S,
suggesting an acidic pI, but it eluted as one major peak on Mono Q
(FIG. 38c). Silver stained gels of fraction 9 showed a 35 kDal band
with >95% purity, and minor (<5%) bands around 41 kDal (FIG.
38d, lane 5). Other fractions and an additional band at .apprxeq.45
kDal, but this had no kinase activity (see below).
[0434] To determine directly which of the bands in the gel were
capable of phosphorylating tau an in-gel assay following the method
of Geahlen et al., Anal. Biochem. 153 (1986), 151-158 was
performed. Tau protein was polymerized into the gel matrix, the
Mono Q fractions were separated on the gel by SDS electrophoresis,
the bound proteins were renatured in situ, incubated with
radioactive ATP and assayed for activity by autoradiography. FIG.
39 shows that the 35 kDal and 41 kDal bands contained kinase
activity, but not the 45 kDal band.
[0435] Quantification of the amount of phosphate incorporated into
tau constructs by the kinase yielded the following results: 3.2
P.sub.i for htau34, 3.4 for htau40, 3.3 for htau23, but only 2.8
for the mutant htau23(Ser262.fwdarw.Ala). The total mutant AP17
incorporated 3.0 P.sub.i, indicating that Ser-Pro or Thr-Pro motifs
were not targets of the kinase, and the 3-repeat construct K18
contained 1.4 P.sub.i.
[0436] Tau phosphorylated by the kinase is shifted upward in the
SDS gel. FIG. 40a shows a comparison of different tau gel shifts
and kinases. The shift by the 35 kDal kinase is of medium magnitude
(lane 2), like that of PKA (lane 10), larger than that of CaM
kinase (lane 9) but distinctly smaller than that of MAP kinase
(lane 11) which induces the Alzheimer-like immune response. The
mutant Ser409-Ala (lanes 3, 4) is not shifted by phosphorylation,
but other mutants are (e.g. at Ser416, lanes 5, 6, or at Ser404,
lanes 7, 8), indicating that Ser409 is the residue whose
phosphorylation by the 35 kDal kinase generates the shift. This
same shift is found with PKA (lane 10) which also phosphorylates
Ser409. Since phosphorylation sites within the repeat region
generally do not produce a shift these data confirm that the shift
sites (mostly in the C-terminal tail) are distinct from the sites
controlling microtubule binding (e.g. Ser262).
[0437] The effect of the purified kinase of the binding of tau
(FIG. 40b) is similar to that of the brain extract (FIG. 37b). For
example, the stoichiometry of htau23 is reduced by D.sub.n=0.28
upon phosphorylation, but only by 0.05 in the point mutant
Ser262-Ala, again emphasizing the importance of Ser262.
[0438] A diagram of htau40, highlighting the first
microtubule-binding repeat and the Ser262 that is important for
microtubule-binding is depicted in FIG. 41.
[0439] A similar effect on the binding of tau to microtubules is
observed when tau is phosphorylated by the 70 kDal kinase (see FIG.
45). This kinase incorporates about 3-4 Pi into the repeat region
of tau, specifically at serines 262, 293, 324, 409. It is prepared
by the following steps: (a) Preparation of high spin supernatant of
brain extract. (b) Chromatography on Q-Sepharose. (c)
Chromatography of flowthrough on S-Sepharose. Kinase activity
elutes at 250 mM NaCl. (d) Chromatography on heparin agarose.
Kinase activity elutes at 250 mM NaCl. (e) Gel filtration. Kinase
activity elutes at 70 kDal. (f) Chromatography on Mono Q. Kinase
activity elutes at 150 mM NaCl.
EXAMPLE 12
Dephosphorylation of Tau Protein by Phosphatases PP2a and PP1
[0440] htau 40 was phosphorylated with porcine MAP kinase (p42) and
.sup.32P-ATP according to methods described throughout this
specification. Subsequently, htau40 was dephosphorylated with
several isoforms of PP2a (FIG. 42 A to C) as PP1 (FIG. 42D). The
results show that htau40 is dephosphorylated by all isoforms of
PP2a, and, although much slower, by PP1 FIG. 43 shows that upon
dephosphorylation the antibody-specific epitopes disappear as well.
In FIG. 44 the time course of dephosphorylation and the
Michaelis-Menten-kinetics are shown.
[0441] Thus, PP2a and PP1 serve as antagonist to MAP-kinases and
may therefore be used in pharmaceutical compositions for the
treatment of Alzheimer disease.
TABLE-US-00002 TABLE 1 Interactions of tau constructs with
antibodies in the phosphorylated or unphosphorylated state (+ or
-). The staining an immunoblots ranges from very weak (x), to very
strong xxx. phosph. construct +/- SMI33 SMI31 SMI34 htau40 - xxx +
xxx xxx htau23 - xxx + xxx xxx K3M - + (x) K2 - + xxx K17 - xxx +
xx K10 - + xxx xxx K19 - + htau40/A235 - (x) + xxx xxx htau40/A396
- xxx + xx xxx htau40/A235/A396 - (x) + xx xxx htau23/A404 - xxx +
xxx xxx htau23/A396/A404 - xxx + xxx K4 - xxx + xx K5 - xxx + xx
xxx K6 - xxx + xx xxx K7 - xxx + x xx K13 - xxx + xx K14 - xxx + xx
K15 - xxx + xx
TABLE-US-00003 TABLE 2 Summary of lengths of various tau
constructs. construct length (nm) s.d. (nm) number htau23 35 7 232
T8R-1 58 15 304 T8R-2 61 17 75 T7R-2 60 16 73 K11 26 5 32 K11 dimer
32 6 24 K12 25 4 27 K12 dimer 30 4 25 K12 PDM dimer 29 6 79 K12 MBS
dimer 34 6 85
Sequence CWU 1
1
* * * * *