NEW TAU SPECIES
20200031891 ยท 2020-01-30
Inventors
- Luc BUEE (Lille Cedex, FR)
- Malika HAMDANE (Lille Cedex, FR)
- David BLUM (Lille Cedex, FR)
- Maxime DERISBOURG (Lille Cedex, FR)
- Coline LEGHAY (Lille Cedex, FR)
- Giovanni CHIAPPETTA (Paris Cedex 5, FR)
- Joelle VINH (Paris Cedex 5, FR)
- Yann VERDIER (Paris Cedex 5, FR)
Cpc classification
International classification
Abstract
The invention relates to the identification of a new Tau species starting at residue Met11 (Met11-Tau) which is N-alpha-terminally acetylated form (N-alpha-acetyl-Met11-Tau species: Ac-Met11-Tau). Several monoclonal antibodies specific of this new Tau species have been developed. One of this antibody, 2H2/D11, was used in THY-Tau22 mouse model (that develops with age neurofibrillary degeneration (NFD) and memory deficits), and N-alpha-Ac-Met11-Tau species were clearly detected early in neurons displaying NFD on hippocampal brain sections while it is not reactive in hippocampus from elderly controls. Finally, by using ELISA sandwich specific of Ac-Met11-Tau species, Alzheimer Disease (AD) brain samples are clearly discriminated from human elderly control brains. Thus the invention relates to this new Tau species starting from the methionine residue at position 11 said methionine being N-alpha acetylated. The invention also relates to antibody that specifically binds this new tau species, a method of detection of this new Tau species and a method of diagnosis of Tauopathy disorder.
Claims
1. An isolated Tau polypeptide which comprises at least 9 consecutive amino acids starting from the methionine residue at position 11 in any one of SEQ ID NOS: 1-7 wherein said methionine residue at position 11 is N-alpha acetylated.
2. The isolated Tau polypeptide of claim 1 which comprises the amino acid sequence starting from the methionine residue at position 11 to the amino acid at position 776 (SEQ ID N0:7).
3. An isolated polypeptide selected from the group consisting of: (i) the amino acid sequence Tau N-Alpha Acetyl-Met11-352 (SEQ ID N0:1); (ii) the amino acid sequence Tau N-Alpha Acetyl-Met11-381 (SEQ ID N0:2); (iii) the amino acid sequence Tau N-Alpha Acetyl-Met11-383 (SEQ ID N0:3) (iv) the amino acid sequence Tau N-Alpha Acetyl-Met11-410 (SEQ ID N0:4) (v) the amino acid sequence Tau N-Alpha Acetyl-Met11-412 (SEQ ID N0:5); (vi) the amino acid sequence Tau N-Alpha Acetyl-Met11-441 (SEQ ID N0:6); (vii) the amino acid sequence Tau N-Alpha Acetyl-Met11-776 (SEQ ID N0:7); (viii) an amino acid sequence at least 80% identical to the sequence of any one of (i) to (vii); and (ix) a fragment of at least 9 consecutive amino acids starting from the N-Alpha Acetyl methionine residue at position 11 of the sequence of any one of (i) to (viii).
4. An isolated polypeptide which comprises the amino acid sequence (N--acetyl)MEDHAGTYGLG (SEQ ID N0:8).
5. The isolated polypeptide according to claim 4, wherein the isolated polypeptide is at most 766 amino acids in length.
6. (canceled)
7. An antibody that specifically binds to the isolated Tau polypeptide of claim 1.
8. The antibody according to claim 7 wherein the antibody does not bind to a non N-alpha-acetylated form of Methionine 11 Tau polypeptide (SEQ ID NO: 9 and/or a N-alpha-acetyl-Met1-Tau polypeptide (SEQ ID NO: 10).
9. The antibody according to claim 7, wherein the antibody is polyclonal or monoclonal.
10. (canceled)
11. A method for detecting and/or evaluating the amount of the Tau polypeptide of claim 1, in a biological sample, comprising contacting said sample with an antibody that specifically binds to the Tau polypeptide, and detecting formation of a complex between the Tau polypeptide and the antibody.
12-13. (canceled)
14. The method according to claim 15, wherein the tauopathy is Alzheimer disease.
15. An in vitro method for diagnosing a tauopathy in a subject in need thereof, comprising detecting, in a biological sample from the subject, the Tau polypeptide of claim 1, wherein detection of the presence of the Tau polypeptide indicates that the subject has a tauopathy.
Description
FIGURES
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EXAMPLE 1
[0121] Materials and Methods
[0122] Human tissue samples. Human brain autopsy samples were from the Lille NeuroBank collection (Centre de Ressources Biologiques du CHU de Lille). Informed consent was obtained from all subjects. The Lille NeuroBank has been declared to the French Research Ministry by the Lille University Hospital (CHU-Lille) on Aug. 14, 2008 under the reference DC-2000-642 and fulfills the criteria defined by French Law regarding biological resources, including informed consent, ethics review committee approval and data protection. The study was approved by the ethics review committee of Lille NeuroBank. The stages of Tau pathology were categorized on the basis of neuropathological characteristics according to Braak and Braak (2011).
[0123] Identification of Ac-Met11-Tau species. Ac-Met11-Tau was identified using the experimental approaches detailed in Derisbourg et al (2015). Briefly, Tau species were enriched by immunoprecipitation from human occipital cortex brain, labeled with biotin and analyzed by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS).
[0124] Generation of 2H2/D11 antibody. 2H2/D11 antibody was generated by immunization with an N-alpha-terminal acetyl Tau peptide (Ac-Met11-Tau peptide: {N-acetyl}MEDHAGTYGLG: SEQ ID N 8) corresponding to the Tau sequence from Methionine at position 11 to Glycine at position 21. This sequence is encoded by exon1; shared by all Tau isoforms. A cysteine residue has been added to the C-terminus of Ac-Met11-Tau peptide for KLH Conjugation. Balb/c mice were immunized subcutaneously with 3 boosts at days 14, 45 and 63. The lymphocytes from the spleen of the mouse displaying the highest titer were then fused with NS1 myeloma cells, according to the method described in (Pandey, 2010). The hybridoma supernatants were screened in indirect ELISA against the following different peptides:
[0125] Ac-Met 11-Tau peptide: {Na-acetyl}MEDHAGTYGLG; (SEQ ID N 8) the same sequence than Tau fragment used as antigen.
[0126] Met11-Tau peptide: MEDHAGTYGLG; (SEQ ID N 9)
[0127] Ac-Met1-Tau peptide: {Na-acetyl} MAEPRQEFEVMEDHAGTYGLG (SEQ ID N 10); the peptide starts at Methionine 1 of Tau harboring an N-alpha-terminal acetylation.
[0128] Indirect ELISA screening of hybridoma supernatants allowed selection of a set of clones that specifically detect the Ac-Met11-Tau species with a slight or without any cross-reactivity with the free non-N-alpha-terminally-acetylated Methionine 11, nor with the non-truncated Methionine 11, nor with any N-alpha-acetyl-Methionine when it is not in the same amino acid context than Methionine11. Isotype and the type of light chain have been determined for the selected hybridoma (Table 1, Bellow).
TABLE-US-00001 TABLE 1 Hybridoma designation Isotype light chain 1C10 IgG1/IgM Kappa/Lamda 2H2 IgG2a Kappa 3F2 IgG2A/IgM Kappa/Lamda 2C12 IgG1 Kappa 9H4 IgG2a Kappa
[0129] The hybridoma 2H2 was further subcloned and we selected the hybridoma clone that produces 2H2/D11 antibody. The specificity of 2H2/D11 antibody towards N-alpha-terminally acetylated methioninel 1 of Tau protein was reproducibly validated by indirect ELISA, western blotting and immunohistochemistry. The VH and VL sequences of 2H2D11 are provided.
[0130] An antibody against total Tau proteins was also generated following immunization with Met11-Tau peptide: MEDHAGTYGLG; (SEQ ID N 9). We selected the hybridoma clone 7C12/E12 (IgG1, Kappa. The epitope according to the longest human Tau isoform is: aa 11-20). The VH and VL sequences of 7C12/E7) are provided.
[0131] Indirect ELISA. Nunc 96-well microtiter plates (Maxisorp F8; Nunc, Inc.) were coated overnight at 4 C. with 10Ong/well of either Ac-Met11-Tau, Met11-Tau or Ac-Met1-Tau peptide (see above) in 50 mM NaHCO3, pH 9.6. After 3 washes with PBS containing 0.05% Tween (PBS-T), plates were blocked and hybridoma supernatants or purified antibody were tested by use of goat anti-mouse IgG horseradish peroxidase-conjugated antibody (A3673; Sigma) at 1:4000 dilution. Tetramethyl benzidine (T3405, Sigma) was the substrate. The reaction was stopped by addition of sulfuric acid, changing the color from blue to yellow. Plates were measured with a spectrophotometer (Multiskan Ascent Thermo Labsystem) at 450 nm.
[0132] Sandwich ELISA. Nunc 96-well plates (VWR) were coated with 100 l of 2H2/D11 antibody (for detection of Ac-Met 1 1-Tau species) or AT120 (INNOTEST hTau, FUJIREBIO) or 7C12/E7 antibody (for detection of total Tau proteins) at a concentration of 1 g/ml in Carbonate buffer (NaHCO3 0.1M, Na2CO3 0.1M, pH 9.6) overnight at 4 C. The plates were subsequently blocked with a WASH1X buffer (INNOTEST hTau Ag kit, FUJIREBIO) containing 0.1% casein at 37 C. for 1 hour and washed with WASH1X buffer 3 times. Protein samples were standardized at 1 g/l and diluted in SAMPL DIL buffer (INNOTEST hTau Ag kit, FUJIREBIO). Protein samples and biotinylated antibodies (HT7/BT2, INNOTEST hTau Ag kit, FUJIREBIO) were added and the plates were incubated at room temperature overnight. The wells were washed four times then incubated with Peroxidase-labeled streptavidin at room temperature for 30 min and washed four times. Detection was performed using Tetramethyl benzidine substrate for 30 min at room temperature; the assay was stopped with H2SO4 and absorbance was read with spetrophotometer (Multiskan Ascent, Thermo Labsystems) at 450 nm.
[0133] Tau Plasmid constructs. Expression vectors carrying cDNA for Full-length Tau (Tau-412) and Met11-Tau were generated using the In-Fusion cloning Kit (Clontech), and PCR primers were designed to clone inserts into the EcoRI site of pcDNA4/TO (Invitrogen). Each cDNA fragment was amplified by PCR (DyNAzyme EXT DNA polymerase, New England BioLabs) from pcDNA3.1-Tau4R (Mailliot et al., 2000). Forward primers were designed to contain the Kozak consensus sequence and are as follows:
TABLE-US-00002 ForTau-412, (SEQIDNo11) 5-CAGTGTGGTGGAATTCGCCACCATGGCTGAGCCCCGCCAGGAGTT- 3; ForMet11-Tau, (SEQIDNo12) 5-CAGTGTGGTGGAATTCGCCACCATGGAAGATCACGCTGGGACGT-3;
[0134] The reverse primer for the two amplifications was:
TABLE-US-00003 (SEQIDNo13) 5-GATATCTGCAGAATTCTCACAAACCCTGCTTGGCCAGGG AGGCA-3.
[0135] DNA sequencing was carried out for construct validation.
[0136] Tau cell lines. SH-SY5Y cells that constitutively express tetracycline repressor (detailed described in Hamdane et al., 2003) were transfected with Tau plasmid constructs using ExGen500 (Euromedex, France) according to manufacturer's instructions. Individual stable clones were generated following Zeocin selection (100 g/ml), and those that exhibited the weakest basal expression of Tau were selected. The cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal calf serum with pyruvate, 2 mM L-glutamine, 50 units/ml penicillin/streptomycin and 1 mM non essential AA. For induction of Tau-412 and Met11-Tau expression, cells were maintained in medium with Tetracycline at 1 g/ml (Invitrogen) in a 5% CO2 humidified incubator at 37 C.
[0137] Protein extractions. Cells were washed using PBS and harvested in ice-cold RIPA buffer: 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, completed with protease inhibitors. For humain Brain cortex samples, tissues were homogenized by sonication in a buffer containing 0.32 M sucrose, 100 mM NaCl, 110 mM KOAc, 0.5% Triton X-100, and 10 mM Tris-HCl, pH 7.4 with protease inhibitors (Complete w/o EDTA, Roche), sonicated and centrifuged at 2500g for 5 min. After sonication and homogenization, protein concentrations were determined using the BCA Assay Kit (Pierce).
[0138] Western Blotting. Protein extracts were standardized at 1 g/l with LDS 2X supplemented with a reducing agent (Invitrogen) and denatured at 100 C. for 10 min. Proteins were then separated with SDS-PAGE using precast 4-12% Bis-Tris NuPage Novex gels (Invitrogen). Proteins were transferred to 0.45 M nitrocellulose membranes (Amersham Hybond ECL), which were saturated with 5% dry non-fat milk in TNT buffer; 140 mM NaCl, 0.5% Tween20, 15 mM Tris, pH 7.4, or 5% bovine serum albumin (Sigma) in TNT buffer, depending of the primary antibody. Membranes were then incubated with the primary antibody (Table 2, bellow) overnight at 4 C., washed with TNT buffer three times for 10 min, incubated with the secondary antibody (Vector) and washed again before development. Immuno labeling was visualized using chemiluminescence kits (ECLTM, Amersham Bioscience) on an LAS-4000 acquisition system (Fujifilm).
[0139] Peptide blocking experiments. Before proceeding to Western Blot or IHC immunostaining, 2H2/D11 antibody was incubated with agitation overnight at 4 C., in blocking buffer without or with excess of the blocking peptide (molar ratio: 1/50): either Ac-Met11-Tau peptide ({N-acetyl}MEDHAGTYGLG) or Met11-Tau peptide (MEDHAGTYGLG). Thereafter, the antibody samples were used to perform staining protocol as described in Western Blotting and IHC sections.
[0140] Immunohistochemistry (IHC). Thy-Tau22 mice were anesthetized (chloral hydrate 8%) and transcardially perfused with cold PBS followed by 4% paraformaldehyde for 20 min. The brains were removed rapidly, post-fixed overnight in 4% paraformaldehyde, placed in 20% sucrose for 24 hours and finally kept frozen at 80 C. until use. Serial free-floating sagittal sections (40 m) were obtained using a cryostat (Leica Microsystems GmbH, Germany). Sections of interest were used for free floating immunohistochemistry. Concerning human brain sections, the hippocampus was dissected, fixed and sliced in the same conditions than mice.
[0141] Brain sections were washed with PBS-Triton (0.2%) and treated for 30 min with 0.3% H2O2, and nonspecific binding was blocked with MOM (Mouse IgG blocking reagent) or horse serum (1/100 in PBS; Vector Laboratories) for 1 hour. The sections were then incubated with the primary antibody (Table 2, bellow) in PBS-Triton 0.2% overnight at 4 C. After 3 washes (10 min), labeling was amplified using a biotinylated anti-mouse IgG or rabbit-IgG (1/400 in PBS-Triton 0.2%; Vector Laboratories) for 1 hour, followed by the ABC kit (1/400 in PBS; Vector Laboratories), and labeling was completed using 0.5 mg/ml DAB (Vector Laboratories) in 50 mmol/1 Tris-HCl, pH 7.6, containing 0.075% H202. Brain sections were mounted on SuperFrost slides, dehydrated through a graded series of alcohol and toluene, and then mounted with Vectamount (Vector Laboratories) for microscopic analysis using Zeiss AXIOSCAN Zlslide scanner and Zen software.
[0142] Immunofluorescence (co-labeling experiments). Mice brain sections were washed with PBS-Triton 0.2%, and nonspecific binding was blocked through incubation with MOM (1/100 in PBS-Triton 0.2%; Vector Laboratories) for 1 hour. The sections were then incubated with the first primary antibody in PBS-Triton 0.2% overnight at 4 C. After 3 washes (10 min), the sections were incubated with the second primary antibody in PBS-Triton 0.2% overnight at 4 C. Sections were then washed and incubated with the two secondary antibodies (Invitrogen) coupled to either Alexa 468 or Alexa 588 (1/1000 in PBS) for 1 hour at room temperature. After 3 washes (10 min), the sections were mounted with Vectashield containing DAPI to label the nuclei (Vector Laboratories). Confocal microscopy was performed on a Zeiss LSM 710 inverted confocal microscope (60 magnification). Images were collected in the z direction at 1-m or 0.8-m intervals.
TABLE-US-00004 TABLE 2 Table 2 - Summary of primary antibodies used for Western Blotting, Immunohistochemistry and ELISA Antibody Species Dilution Application(s) Supplier Tau-Cter (total rabbit 1/10000 WB Homemade Tau, 426-441) Tau P-Ser422 rabbit 1/1000 IHC Homemade Tau P-Ser199 rabbit 1/500 IHC Homemade 2H2/D11 mouse 1/5000-1/200 WB-ELISA-IHC Homemade (Ac-Met11- Tau) NSE rabbit 1/50000 WB GeneTex 7C12/E7 mouse 1/5000-1/200 WB-ELISA-IHC Homemade (total Tau, 11-21) hT7 mouse 1/500 ELISA Invitrogen Bt2 mouse 1/500 ELISA Invitrogen
[0143] Results
[0144] We recently used an optimized proteomics approach and succeeded in identifying new Tau species from the human brain (Derisbourg et al., 2015). Among these latter, the Tau species starting at residue Met11 (Met11-Tau) is also detected as N-alpha-terminally acetylated form (N-alpha-acetyl-Met11-Tau species: Ac-Met11-Tau); such modification has never been described for Tau protein (
[0145] Thereafter, 2H2/D11 specificity was validated by Western Blotting analysis using protein extracts from cell lines. For this, we generated inducible stable cell lines by transfecting human neuroblastoma cells with tetracycline inducible expression vectors containing the coding sequences of either Full length or Met11 Tau species (FL-Tau (Tau-412) and Met11-Tau, respectively). It should be noted that when we analyzed Met11-Tau cells by the LC-MS/MS proteomic approach, the truncated Met11-Tau protein was found as a mix of non-modified form as well as N-alpha-terminally-acetylated form in this cell line (mass spectra analyses showed a shift of 42 Da corresponding to the addition of an N-alpha-acetyl group to Methionine 11; data not shown).
[0146] The transgenes expression by Western Blot, using an antibody against the C-terminal part of Tau that recognizes all Tau species, is displayed in cells treated with tetracycline. Western Blot analysis using 2H2/D11 antibody only labels extracts from the cells expressing the Met11-Tau protein without any cross reactivity with FL-Tau (
[0147] After the validation of 2H2/D11 specificity, we used this antibody to establish whether there is an association between Ac-Met11-Tau species and Tau pathology, we used THY-Tau22 mouse model that develops with age neurofibrillary degeneration and memory deficits (For full description, please see Schindowski et al., 2006; Van der Jeugd et al., 2013; Burnouf et al., 2012; 2013). We examined sections of hippocampus from these mice with 2H2/D11 monoclonal antibody. Immunohistochemistry analysis interestingly showed that 2H2/D11 antibody displayed no immunoreactivity with Wt mice while in Thy-Tau22 mice the antibody labels Tau pathology as displayed by characteristic pathological inclusions in neurons. Moreover, this immunolabeling is detected early and seems to increase with age.
[0148] The specificity of this finding was further confirmed by experiments where peptide blocking has been performed before proceeding with the immunostaining. When the antibody is neutralized by the N-alpha-Acetylated-Met11-Tau peptide, the staining is absent, while the immunostaining with the antibody blocked with the Met11-Tau peptide is the same than that with the antibody alone.
[0149] Furthermore, we performed double labeling experiments using some well-characterized antibodies specific to phosphorylated Tau. We used Phospho-Serine 199 antibody that labels Tau in neurons independently of the presence of neurofibrillary degeneration and Phospho-Serine 422 antibody that only labels pathological Tau. Co-labeling experiments showed that 2H2/D11 antibody detected a subpopulation of neurons that is immunoreactive with the pathological phospho-serine-422 Tau. Overall our data suggest that in mice model, Ac-Met11-Tau protein is found mostly in neurons displaying Tau pathology, and that Ac-Met11 is likely a marker of early stages of pathological process.
[0150] We have next examined sections of human brain hippocampus. Immunohistochemistry analysis showed that 2H2D/11 antibody is not reactive in hippocampus from elderly Control. Interestingly, in AD hippocampus the antibody labels characteristic features of neurofibrillary tangles that are the signature of AD.
[0151] Giving that we attempt to examine whether Ac-Met11-Tau species is potential biomarker for diagnosis, we developed a sandwich ELISA by using protein extracts from stable cell lines (described in Mat & Meth section and
[0152] Conclusion
[0153] We have identified new Tau species starting at methionine 11 and bearing an N-alpha-terminally acetylation. We have developed hybridomas, including the hybridoma that produces 2H2/D11 antibody, allowing specific detection of N-alpha-terminally-acetylated-Met11 Tau species. Our data based on IHC and ELISA assays, from transgenic mice model and human brains, have shown Ac-Met11-Tau specie to be a pathological modification.
[0154] Our ongoing studies aim to determine the role of this new Tau specie in AD pathogenesis and to establish whether Ac-Met11-Tau species and its related Met11 species is a signature feature of AD and its related Tauopathies as well as if it is indicative of AD stage. Hence, we are performing detailed immunohistochemistry characterization to further examine this species in AD and other Tauopathies. We will use quantitative and qualitative approaches based on our newly developed reagents in biochemical and histological assays (immunohistochemistry, western Blotting and ELISA) using brains from control subjects, well-characterized AD patients with different staging, and patients with other Tauopathies;. This work will have immediate application for diagnosis.
TABLE-US-00005 TABLE3 Usefulaminoacidsequencesforpracticingtheinvention SEQIDNO Nucleotideoraminoacidsequence 1:TauN-AlphaAcetyl- N-AlphaAcetyl-MEDHAGTYGLGDRKDQGGYT Met11-352 MHQDQEGDTDAGLKAEEAGIGDTPSLEDEA AGHVTQARMVSKSKDGTGSD DKKAKGADGKTKIATPRGAAPPGQKGQANA TRIPAKTPPAPKTPPSSGEPPKSGDRSGYS SPGSPGTPGSRSRTPSLPTPPTREPKKVAV VRTPPKSPSSAKSRLQTAPVPMPDLKNVKS KIGSTENLKHQPGGGKVQIVYKPVDLSKVT SKCGSLGNIHHKPGGGQVEVKSEKLDFKDR VQSKIGSLDNITHVPGGGNKKIETHKLTFR ENAKAKTDHGAEIVYKSPVVSGDTSPRHLS NVSSTGSIDMVDSPQLATLADEVSASLAKQ GL 2:TauN-AlphaAcetyl- N-AlphaAcetylMEDHAGTYGLGDRKDQGGYT Met11-381 MHQDQEGDTDAGLKESPLQTPTEDGSEEPG SETSDAKSTPTAEAEEAGIGDTPSLEDEAA GHVTQARMVSKSKDGTGSDDKKAKGADGKT KIATPRGAAPPGQKGQANATRIPAKTPPAP KTPPSSGEPPKSGDRSGYSSPGSPGTPGSR SRTPSLPTPPTREPKKVAVVRTPPKSPSSA KSRLQTAPVPMPDLKNVKSKIGSTENLKHQ PGGGKVQIVYKPVDLSKVTSKCGSLGNIHH KPGGGQVEVKSEKLDFKDRVQSKIGSLDNI THVPGGGNKKIETHKLTFRENAKAKTDHGA EIVYKSPVVSGDTSPRHLSNVSSTGSIDMV DSPQLATLADEVSASLAKQGL 3:TauN-AlphaAcetyl- N-AlphaAcetylMEDHAGTYGLGDRKDQGGYT Met11-383 MHQDQEGDTDAGLKAEEAGIGDTPSLEDEA AGHVTQARMVSKSKDGTGSD DKKAKGADGKTKIATPRGAAPPGQKGQANA TRIPAKTPPAPKTPPSSGEPPKSGDRSGYS SPGSPGTPGSRSRTPSLPTPPTREPKKVAV VRTPPKSPSSAKSRLQTAPVPMPDLKNVKS KIGSTENLKHQPGGGKVQIINKKLDLSNVQ SKCGSKDNIKHVPGGGSVQIVYKPVDLSKV TSKCGSLGNIHHKPGGGQVEVKSEKLDFKD RVQSKIGSLDNITHVPGGGNKKIETHKLTF RENAKAKTDHGAEIVYKSPVVSGDTSPRHL SNVSSTGSIDMVDSPQLATLADEVSASLAK QGL 4.TauN-AlphaAcetyl- N-AlphaAcetylMEDHAGTYGLGDRKDQGGYT Met11-410 MHQDQEGDTDAGLKESPLQTPTEDGSEEPG SETSDAKSTPTAEDVTAPLVDEGAPGKQAA AQPHTEIPEGTTAEEAGIGDTPSLEDEAAG HVTQARMVSKSKDGTGSDDKKAKGADGKTK IATPRGAAPPGQKGQANATRIPAKTPPAPK TPPSSGEPPKSGDRSGYSSPGSPGTPGSRS RTPSLPTPPTREPKKVAVVRTPPKSPSSAK SRLQTAPVPMPDLKNVKSKIGSTENLKHQP GGGKVQIVYKPVDLSKVTSKCGSLGNIHHK PGGGQVEVKSEKLDFKDRVQSKIGSLDNIT HVPGGGNKKIETHKLTFRENAKAKTDHGAE IVYKSPVVSGDTSPRHLSNVSSTGSIDMVD SPQLATLADEVSASLAKQGL 5.TauN-AlphaAcetyl- N-AlphaAcetyl-MEDHAGTYGLGDRKDQGGYT Met11-412 MHQDQEGDTDAGLKESPLQTPTEDGSEEPG SETSDAKSTPTAEAEEAGIGDTPSLEDEAA GHVTQARMVSKSKDGTGSDDKKAKGADGKT KIATPRGAAPPGQKGQANATRIPAKTPPAP KTPPSSGEPPKSGDRSGYSSPGSPGTPGSR SRTPSLPTPPTREPKKVAVVRTPPKSPSSA KSRLQTAPVPMPDLKNVKSKIGSTENLKHQ PGGGKVQIINKKLDLSNVQSKCGSKDNIKH VPGGGSVQIVYKPVDLSKVTSKCGSLGNIH HKPGGGQVEVKSEKLDFKDRVQSKIGSLDN ITHVPGGGNKKIETHKLTFRENAKAKTDHG AEIVYKSPVVSGDTSPRHLSNVSSTGSIDM VDSPQLATLADEVSASLAKQGL 6:TauN-AlphaAcetyl- N-AlphaAcetyl-MEDHAGTYGLGDRKDQGGYT Met11-441(Isoform2) MHQDQEGDTDAGLKESPLQTPTEDGSEEPG SETSDAKSTPTAEDVTAPLVDEGAPGKQAA AQPHTEIPEGTTAEEAGIGDTPSLEDEAAG HVTQARMVSKSKDGTGSDDKKAKGADGKTK IATPRGAAPPGQKGQANATRIPAKTPPAPK TPPSSGEPPKSGDRSGYSSPGSPGTPGSRS RTPSLPTPPTREPKKVAVVRTPPKSPSSAK SRLQTAPVPMPDLKNVKSKIGSTENLKHQP GGGKVQIINKKLDLSNVQSKCGSKDNIKHV PGGGSVQIVYKPVDLSKVTSKCGSLGNIHH KPGGGQVEVKSEKLDFKDRVQSKIGSLDNI THVPGGGNKKIETHKLTFRENAKAKTDHGA EIVYKSPVVSGDTSPRHLSNVSSTGSIDMV DSPQLATLADEVSASLAKQGL 7.TauN-AlphaAcetyl- N-AlphaAcetyl-MEDHAGTYGLGDRKDQGGYT Met11-776 MHQDQEGDTDAGLKESPLQTPTEDGSEEPG SETSDAKSTPTAEDVTAPLVDEGAPGKQAA AQPHTEIPEGTTAEEAGIGDTPSLEDEAAG HVTQEPESGKVVQEGFLREPGPPGLSHQLM SGMPGAPLLPEGPREATRQPSGTGPEDTEG GRHAPELLKHQLLGDLHQEGPPLKGAGGKE RPGSKEEVDEDRDVDESSPQDSPPSKASPA QDGRPPQTAAREATSIPGFPAEGAIPLPVD FLSKVSTEIPASEPDGPSVGRAKGQDAPLE FTFHVEITPNVQKEQAHSEEHLGRAAFPGA PGEGPEARGPSLGEDTKEADLPEPSEKQPA AAPRGKPVSRVPQLKARMVSKSKDGTGSDD KKAKTSTRSSAKTLKNRPCLSPKHPTPGSS DPLIQPSSPAVCPEPPSSPKYVSSVTSRTG SSGAKEMKLKGADGKTKIATPRGAAPPGQK GQANATRIPAKTPPAPKTPPSSATKQVQRR PPPAGPRSERGEPPKSGDRSGYSSPGSPGT PGSRSRTPSLPTPPTREPKKVAVVRTPPKS PSSAKSRLQTAPVPMPDLKNVKSKIGSTEN LKHQPGGGKVQIINKKLDLSNVQSKCGSKD NIKHVPGGGSVQIVYKPVDLSKVTSKCGSL GNIHHKPGGGQVEVKSEKLDFKDRVQSKIG SLDNITHVPGGGNKKIETHKLTFRENAKAK TDHGAEIVYKSPVVSGDTSPRHLSNVSSTG SIDMVDSPQLATLADEVSASLAKQGL 8.TauN-AlphaAcetyl- N-AlphaAcetyl-MEDHAGTYGLG Met11-21(antigen) 9.TauMet11-21 MEDHAGTYGLG 10.TauN-AlphaAcetyl- N-AlphaAcetyl-MAEPRQEFEVMEDHAGTYGLG Met1-21 11.Forwardprimersfor cagtgtggtggaattcgccaccatggctgagccccgccaggagtt Tau-412 12.Forwardprimersfor cagtgtggtggaattcgccaccatggaagatcacgctgggacgt Met11Tau 13.Reverseprimersfor gatatctgcagaattctcacaaaccctgcttggccaggg both
EXAMPLE 2
[0155] Materials and Methods
[0156] Indirect ELISA. Nunc 96-well microtiter plates (Maxisorp F8; Nunc, Inc.) were coated overnight at 4 C. with 100 ng/well of Tau 1-peptide (SEQ ID N 10), Met11-Tau peptide (SEQ ID N 9), or Ac-Met11-Tau peptide (SEQ ID N 8) in 50 mM NaHCO3, pH 9.6. After 3 washes with PBS containing 0.05% Tween (PBS-T), plates were blocked with 0.1% casein solution (PBS) at 37 C. for 1 h, followed by incubation with 2H2/D11 antibody, or 7C12/E7 antibody for 1 h at 37 C. Immunodetection was performed by using a goat anti-mouse IgG horseradish peroxidase-conjugated antibody (A3673; Sigma) at 1:4000 dilution. Tetramethyl benzidine (T3405, Sigma) was the substrate. The reaction was stopped by addition of sulfuric acid, changing the color from blue to yellow. Plates were measured with a spectrophotometer (Multiskan Ascent Thermo Labsystem) at 450 nm.
[0157] Protein extractions. Human brain autopsy samples (hippocampus) were from the Lille NeuroBank collection (Centre de Ressources Biologiques du CHRU de Lille). Tissues were homogenized by sonication in a buffer containing150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, completed with protease inhibitors (Complete w/o EDTA, Roche); protein concentrations were determined using the BCA Assay Kit (Pierce).
[0158] Sandwich ELISA. Nunc 96-well plates (VWR) were coated with 100 l of 2H2/D11 antibody (for detection of Ac-Met11-Tau species) or AT120 (INNOTEST hTau, FUJIREBIO) (for detection of total Tau proteins) at a concentration of 1 g/ml in Carbonate buffer (NaHCO3 0.1M, Na2CO3 0.1M, pH 9.6) overnight at 4 C. The plates were subsequently blocked with a WASH1X buffer (INNOTEST hTau Ag kit, FUJIREBIO) containing 0.1% casein at 37 C. for 1 hour and washed with WASH1X buffer 3 times. Protein samples were standardized at 1 g/l and diluted in SAMPL DIL buffer (INNOTEST hTau Ag kit, FUJIREBIO). Protein samples and biotinylated antibodies (HT7/BT2, INNOTEST hTau Ag kit, FUJIREBIO) were added and the plates were incubated at room temperature overnight. The wells were washed four times then incubated with Peroxidase-labeled streptavidin at room temperature for 30 min and washed four times. Detection was performed using Tetramethyl benzidine substrate for 30 min at room temperature; the assay was stopped with H2SO4 and absorbance was read with spetrophotometer (Multiskan Ascent, Thermo Labsystems) at 450nm.
[0159] Results
[0160] Indirect ELISA reproducibly validated the specificity of 2H2/D11 antibody towards N-alpha-terminally acetylated methioninell of Tau protein. Indeed, 2H2/D11 displays no reactivity with the free non-N-alpha-terminally-acetylated Methionine 11, or with the non-truncated Methionine 11, or with an N-alpha-acetyl-Methionine when it is not in the same amino acid context than Methioninell. Regarding 7C12/E7 antibody against total Tau proteins, it displays the similar immunoreactivity towards the 3 peptides (
[0161] Protein extracts from hippocampus of elderly controls (n=6) and AD cases (n=10, from Braak IV-VI) were used in 2H2D/11-based sandwich ELISA. Data interestingly showed that our antibody specifically reacted in AD hippocampus samples (p=0.0002; compared with Mann-Whitney test) (
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