A method for determining hyperphosphorylated Tau in human cerebrospinal fluid by LC-MS

Abstract

The present invention relates to a method for measuring pS396Tau in a sample comprising steps i) treating a CSF sample from a subject suffering from or suspected of suffering from Tau pathology with trypsin, ii) exposing the sample from step i) to a dephosphorylating agent, subsequently reserving a portion of the sample for step iv) and then proceeding to step iii) with the remaining sample, iii) exposing the dephosphorylated sample from step ii) to a second trypsin treatment, and iv) measuring the amount of Tau peptide corresponding to Tau residues 396-406 (SPVVSGDTSPR) in the samples from steps ii) and iii) using LC-MS.

Description

[Technical field]

[0001] The present invention relates to a method for determining hyperphosphorylated Tau, in particular Tau phosphorylated at serine residue 396 (S396) of Tau, in human cerebrospinal fluid (CSF) using trypsin digestion and liquid chromatography / mass spectrometry workflow (LC-MS). [Background technology]

[0002] A method for tracking the progression of Tau pathology in Tau pathologies such as Alzheimer's disease (AD) or Down's syndrome may be highly suitable for diagnosing patients or monitoring their stage. Currently, there is a strong clinical need for diagnostic and predictive biomarkers, as well as biomarkers for classification purposes, to identify patients at an early stage of Tau pathology, such as those found in patients with mild cognitive impairment (MCI), which may later progress to AD.

[0003] Promising cerebrospinal fluid (CSF) biomarker candidates are total Tau protein and phosphorylated Tau protein (pTau) that may prove to have sufficient diagnostic accuracy and predictive power. Using Tau protein phosphorylated at serine 396 (pS396), the inventors have developed an assay that can detect hyperphosphorylated Tau and have shown that this assay / biomarker can indicate, for example, the efficacy of antibody therapy in transgenic mice expressing high levels of human Tau and Tau pathology.

[0004] The inventors of the present invention have found a reliable way to measure pS396-Tau using a method that exploits the fact that trypsin does not cleave after amino acid lysine 395 when Tau is phosphorylated at serine 396 (pS396), a hallmark of hyperphosphorylated Tau pathology. Thus, only Tau proteins that are not phosphorylated at this site are cleaved by trypsin, resulting in the production of peptides. [ka] (SEQ ID NO: 1) is generated, where [ka] is non-phosphorylated S396 serine.

[0005] The Tau protein is a highly complex structure consisting of (at least) six proteoforms (ranging from 352 to 441 amino acids) and containing more than 80 possible sites for post-translational modifications (PTMs). The heterogeneous structure represents a challenge for mass spectrometry analysis of the protein, as the mass of the signature peptide of interest can vary depending on the possible PTMs.

[0006] In the present invention, the signature peptide of interest contains serine 396. The cleavage points of trypsin are on the carbonyl (C) side of the amino acids Lys (K) and Arg (R), except when the latter amino acid is phosphorylated. Thus, in the case of analysis of pS396-Tau, trypsin cleavage produces the signature peptide [ka] (SEQ ID NO:2). However, this peptide may be phosphorylated (or not) at amino acids T386, Y394, S400, T403 and S404. The various possible combinations of these phosphorylations result in hundreds of different peptides, each of which has a specific chromatographic retention time and / or molecular mass for LC-MS analysis. If direct quantitative analysis of signature peptides containing pS396 is performed using LC-MS, quantification of each individual variant of the phosphorylated peptide may be required. This approach requires extensive analytical work and the more significant analytical signal of pS396 may be "diluted" in these various peptides. Since the expected concentration of pS396 in human CSF is in the low pM range, the demands on the sensitivity of the mass spectrometer may be a limitation when applying such direct analysis.

[0007] Thus, an embodiment of the present invention consists in trypsin digestion of samples obtained from CSF, in which an indirect analytical approach is applied, which allows to focus the analysis on the single human Tau-derived peptide SPVVSGDTSPR[aa396-406] (SEQ ID NO: 1).

[0008] This method exploits the fact that phosphorylation at S396 prevents trypsin from cleaving the protein chain at this position, and thus tryptic digestion of S396-phosphorylated Tau protein results in the peptide TDHGAEIVYK[p]SPVVSGDTSPR (SEQ ID NO: 2). Thus, only Tau protein that is not phosphorylated at S396 is cleaved by trypsin in the first workflow, resulting in the peptide SPVVSGDTSPR (SEQ ID NO: 1) (after removing all possible phosphorylations by phosphatase treatment). Thus, workflow 1 analysis of SPVVSGDTSPR corresponds to the amount of non-phosphorylated S396.

[0009] In workflow 2, a second trypsin digestion is applied. The peptide SPVVSGDTSPR (SEQ ID NO: 1) is not affected by this treatment, whereas TDHGAEIVYKSPVVSGDTSPR will now be cleaved between amino acids lysine 395 and serine 396, since the peptide is no longer phosphorylated at serine 396 (removed by phosphatase treatment). The second trypsin digestion therefore generates an additional amount of SPVVSGDTSPR, and workflow 2 analysis corresponds to the sum of non-phosphorylated and phosphorylated S396 (equal to the total amount of Tau).

[0010] Therefore, the difference in the amount of SPVVSGDTSPR[396-406] (SEQ ID NO: 1) measured by workflow 1 and workflow 2 is equal to the amount of Tau protein that was initially phosphorylated at S396 (pS396-Tau). Summary of the Invention

[0011] The present invention provides a method for measuring pS396Tau in a CSF sample, comprising the steps of: i) treating an (in vitro) CSF sample from a subject suffering from or suspected of suffering from Tau pathology with trypsin; ii) exposing the sample of step i) to a dephosphorylating agent, followed by reserving a portion of the sample for step iv) and then proceeding to step iii) with the remaining sample; iii) subjecting the dephosphorylated sample from step ii) to a second trypsinization; iv) measuring the amount of Tau peptide (SPVVSGDTSPR (SEQ ID NO: 1)) corresponding to Tau residues 396-406 in the samples from step ii) and step iii) using LC-MS; The present invention relates to a method comprising the steps of:

[0012] The method may be used to diagnose or monitor disease in patients with Tau pathology, or it is believed that the method may be used to monitor the effectiveness of treatment in patients with Tau pathology. [Brief description of the drawings]

[0013] [Figure 1] Schematic diagram of full-length Tau protein and peptides selected for LC-MS analysis, Tau(260-267) and Tau(396-406). The diagram also shows the location of phosphorylation on S396 (pS396) and peptide TDHGAEIVYK[p]SPVVSGDTSPR(Tau(386-406)), which is the result of miscleavage at lysine 395. pS396-Tau is indirectly measured as the difference in the concentration of Tau peptide (amino acids 396-406) before and after dephosphorylation. [Diagram 2]Theoretical LC-MS / MS chromatograms showing the signature peptides obtained at the individual steps of the sample workflow for the analysis of S396 phosphorylated Tau exemplified by 30% phosphorylation at S396. Although SPVVSGDTSPR (SEQ ID NO: 1) is measured in the method, TDHGAEIVYKSPVVSGDTSPR (SEQ ID NO: 2) is included in the figure for reference only. The upper section (A+B) shows the analytical peaks after the first trypsin digestion and dephosphorylation. A: The analytical peak of TDHGAEIVYKSPVVSGDTSPR corresponds to Tau phosphorylated at S396 (missed cleavage), whereas B: The analytical peak of SPVVSGDTSPR corresponds to Tau not phosphorylated at S396. The lower section (C+D) shows the analytical peaks obtained after the second trypsin digestion. C: TDHGAEIVYKSPVVSGDTSPR is now cleaved at lysine 395 (S396 is no longer phosphorylated) and is therefore undetectable. D: The analytical peak of SPVVSGDTSPR increases with an area corresponding to the peak of section A. The additional area corresponds to the amount of Tau phosphorylated at S396 (pS396). [Figure 3A] Figure 3A-B. LC-MS / MS chromatograms for the analysis of endogenous levels of Tau(396-406)SPVVSGDTSPR peptide in human CSF. Panel A shows the chromatogram obtained after workflow 1 (i.e., after the first trypsin digestion and dephosphorylation) and Panel B shows the chromatogram after workflow 2 (i.e., after the second trypsin digestion). Note that the analytical peak area increases from 30489 counts in WF1 to 52585 counts in WF2, which is equivalent to a difference of 22096 counts or 42%. This difference corresponds to the amount of phosphorylation at S396 (pS396). [Figure 3B] Same as above. [Figure 4-1]Figure 4A-D. LC-MS / MS chromatograms for the analysis of Tau(396-406)SPVVSGDTSPR peptide in calibration standards prepared in artificial CSF. Panel 1 shows a blank sample, panel 2 shows the calibration standard at 2 pM, which corresponds to the lower limit of quantification (LLOQ), panel 3 shows the calibration standard at 20 pM (normal level in human CSF), and panel 4 shows the calibration standard at 100 pM, which corresponds to the upper limit of quantification (ULOQ). In each panel, panel a is the unlabeled peptide and panel b is the internal standard (15N-SPVVSGDTSPR). [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. [Figure 4-6] Same as above. [Diagram 4-7] Same as above. [Figure 4-8] Same as above. [Diagram 5] Data obtained from analysis of CSF samples from transgenic mice (rtg4510) after 6 weeks of treatment (once a week) with either a control hIgG antibody (B12, not expected to bind Tau) or a monoclonal hIgG anti-Tau antibody developed to target the pS396-Tau epitope. The young mice were 3.5 months old when CSF was collected and known to have low levels of Tau pathology. The old mice were 9 months old when CSF was collected and known to have severe levels of Tau pathology at that age. The top panel shows the results of the analysis of Tau(396-406) and the bottom panel shows the results of S396 phosphorylated Tau calculated as the difference between Tau(396-406) before and after the second trypsin digestion. It can be observed that in the control antibody treated mice, the measured concentrations of Tau(396-406) and pS396 are higher in the old mice compared to the young mice. Furthermore, in aged mice (with Tau pathology), a dose-dependent decrease in tau(396-406) and pS396 is observed after treatment with anti-Tau hIgG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Sequences Incorporated by Reference SEQ ID NO: 1 SPVVSGDTSPR (Tau396-406) SEQ ID NO: 2 TDHGAEIVYKSPVVSGDTSPR (Tau386-406) SEQ ID NO: 3 IGSTENLK (Tau260-267).

[0015] Description of the invention The present invention relates to an assay for measuring the amount of Tau phosphorylated at serine residue S396 (pS396) in a human cerebrospinal fluid (CSF) sample. The aim is to obtain a measure of hyperphosphorylated Tau in the CSF of a subject by measuring pS396, in particular in subjects for whom a diagnosis is desired or in whom the effect of a treatment is to be determined or monitored. These subjects suffer or are suspected of suffering from Tau pathologies such as Alzheimer's disease (AD) and Down's syndrome.

[0016] The inventors of the present invention used the analysis of pS396Tau as an indirect measure of hyperphosphorylated Tau in CSF. By using a trypsin cleavage step, the inventors of the present invention took advantage of the inability of trypsin to cleave Tau just before the serine residue 396 (S396) of Tau when it is phosphorylated.

[0017] Thus, an aspect of the invention is trypsin digestion of a sample obtained from CSF. If Tau is not phosphorylated at S396, trypsin can cleave just before residue S396 to generate the peptide SPVVSGDTSPR [amino acids 396-406]. This fragment is prevalent, for example, in healthy individuals without Tau pathology. In disease stages of Tau pathology (e.g., AD), Tau becomes hyperphosphorylated and residue S396 also becomes phosphorylated. Phosphorylation at S396 to generate pS396 interferes with the cleavage ability of trypsin, and therefore trypsin digestion will not generate the signature peptide SPVVSGDTSPR. Instead, the larger peptide TDHGAEIVYKSPVVSGDTSPR is generated.

[0018] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: i) treating a CSF sample from a subject (e.g., a human) with trypsin; ii) exposing the remaining sample from step i) to a dephosphorylating agent (such as a phosphatase) followed by setting aside a portion of the sample for step iv) before proceeding to step iii); iii) exposing the dephosphorylated sample from step ii) to trypsinization; iv) measuring the amount of peptide SPVVSGDTSPR in the samples from step ii) and step iii) (e.g., by LC-MS); v) optionally comparing the amount of Tau peptide (SPVVSGDTSPR) from step ii) and step iii) measured in step iv); vi) optionally comparing the amount of Tau peptide (SPVVSGDTSPR) from steps ii) and iii) measured in step iv) with a control, such as a trypsin-digested Tau peptide fragment not affected by phosphorylation (e.g., a Tau peptide comprising residues 260-267 (IGSTENLK) (SEQ ID NO: 3); The present invention relates to a method comprising the steps of:

[0019] Since trypsin can cleave between the amino acids lysine 395 and serine 396 only if serine 396 is not phosphorylated, the SPVVSGDTSPR measured in step ii) above measures the portion of SPVVSGDTSPR that corresponds to the unphosphorylated S396 in the CSF sample.

[0020] In step iii) a second trypsin digest is applied after the dephosphorylation step of step ii). The part of phosphorylated and uncleaved Tau peptide TDHGAEIVYKSPVVSGDTSPR from step i) will now be cleaved between amino acids lysine 395 and serine 396, since Tau is no longer phosphorylated at serine 396. The second trypsin digestion therefore results in an additional amount of SPVVSGDTSPR corresponding to the sum of unphosphorylated S396 and phosphorylated S396 (total amount of Tau).

[0021] Furthermore, the difference between the amount of SPVVSGDTSPR (non-phosphorylated S396) measured directly after step i) and the amount of SPVVSGDTSPR (total Tau) measured after the second digestion in step iii) is the amount of Tau protein phosphorylated at S396. Since IGSTENLK[260-267] does not contain any phosphorylation sites, this peptide serves as a reference peptide and the same concentration should be measured before and after the second trypsin digestion step. It serves as a confirmation peptide to measure the total concentration of Tau protein.

[0022] In order to remove trypsin residues before the dephosphorylation step, the trypsin digested peptides in step i) are subjected to solid phase extraction (SPE) before the dephosphorylation treatment (to avoid cleavage at S396 when phosphorylation is removed). Before dephosphorylation, the pH is adjusted to 5.5. At this pH, the enzymatic activity of trypsin is minimized, but the activity of lambda protein phosphatase (λPP) is maintained. In step v), a suitable method for the analysis of Tau protein in the above assay can be LC-MS analysis of peptides Tau(396-406) and Tau(260-267).

[0023] The methods according to the invention can be used to assess or monitor Tau pathology in a subject, such as a mammal (mouse or monkey), particularly a human.

[0024] Tau pathologies include Alzheimer's disease, Down's syndrome, argyrophilic grain disease (AGD), psychiatric disorders, particularly psychiatric disorders due to AD or in AD patients, apathy due to AD or in AD patients, psychiatric symptoms in patients with dementia with Lewy bodies, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART), neurofibrillary tangle-predominant senile dementia, and boxing. Tau-related neurodegeneration can be seen in many diseases such as dementia, chronic traumatic encephalopathy, stroke, stroke recovery, neurodegeneration associated with Parkinson's disease, chromosomally linked parkinsonism, Lytico-Bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease and lipofuscinosis. It is expected that the above disclosed method can be used to diagnose patients with Tau pathology such as Alzheimer's disease or Down's syndrome. Furthermore, it is believed that the method is also useful for monitoring the disease in subjects with Tau pathology.

[0025] Furthermore, it is expected that the methods disclosed above can be used to monitor the efficacy of treatment in subjects with Tau pathology, such as Alzheimer's disease or Down's syndrome. In particular, the methods are believed to be useful for monitoring any treatment that may target Tau pathology, such as anti-Tau antibody treatment. EXAMPLES

[0026] Example 1: Materials and Reagents Lambda protein phosphatase (λPP, 50 μL of 400000 units / mL) and 10 mM manganese(II) chloride (1 mL of 10 mM MnCl 2 ) were obtained from Bioke.

[0027] Sep-Pak® tC18 100mg 96-well SPE plates were obtained from Waters, US.

[0028] Phosphatase inhibitor cocktail III was obtained from Sigma Aldrich.

[0029] Trypsin (from porcine pancreas, T0303) was obtained from Sigma Aldrich.

[0030] Trypsin inhibitor was obtained from Sigma Aldrich.

[0031] Biological samples Human CSF was obtained from BioIVT or PrecisionMed.

[0032] Mouse CSF was obtained from our in-house laboratory. CSF was obtained from rtg4510 mice expressing human 0N4R tau harboring the disease-associated mutation P301L.

[0033] [Table 1]

[0034] The following reagents are freshly prepared during sample preparation. Preparation details are described in Sample Preparation: Digestion Mixture 1: 100 μg / mL trypsin in 250 mM ABC in water Trypsin Stop Solution 1.00 mg / mL trypsin inhibitor in water Manganese(II) chloride solution 3.125 mM MnCl2 in water Lambda protein phosphatase (λPP) solution 36364 units / mL of λPP in water Digestion Mixture 2 300 μg / mL trypsin in 250 mM ABC in water.

[0035] Stock solution Stock solution 15 N-Tau protein (1.00 mg / mL) (=21.6 μM, MW=46353.2 g / mol) The equivalent of 100 μg is dissolved in 100 μL of 0.1% Tween 20 in PBS.

[0036] Stock solution pS396Tau protein (approximately 1.20 mg / mL) (= 26.1 μM, MW = 45902.2 g / mol) This solution is used to demonstrate the suitability of the method and to determine the lowest percentage of S396-phosphorylation that can be reliably measured.

[0037] Example 2: Procedure for sample preparation - Workflow 1 First trypsin digestion Frozen CSF samples were thawed at room temperature and homogenized by gentle inversion (at least 5 times) and vortexing for 30 seconds. Samples were centrifuged for 1 minute at approximately 500xg and 20°C, and 200 μL of sample was pipetted into a 1 mL plate. 10.0 μL of 0.1% Tween 20 in PBS was added to the (double) blank and 10.0 μL of internal standard working solution (2.7 nM 15 N-Tau protein) is added to all other wells.

[0038] Freshly prepare digestion mixture 1 (100 μg / mL trypsin) as described in Materials and Reagents. Mix 100 μL of 5 mg / mL trypsin in 1 mM HCl solution with 4900 μL of digestion solvent (250 mM ABC in water) in a tube. Add 40.0 μL of freshly prepared digestion mixture to each well and vortex the plate at 1200 rpm (thermomixer) and 37 °C for 60 min.

[0039] Freshly prepare digestion stop solution (1.00 mg / mL): Weigh out an amount of approximately 1.5 mg of trypsin inhibitor and dissolve in the amount of Milli-Q required to achieve a concentration of 1.00 mg / mL.

[0040] Add 12.0 μL of freshly prepared Digestion Stop Solution to each well and vortex the plate at 1200 rpm (Thermomixer or Mixmate) and 37° C. for 2 minutes.

[0041] Add 60.0 μL of digestion solvent (250 mM ABC in water) and 300 μL of 0.1% Tween 20 in PBS to each well and vortex mix.

[0042] solid phase extraction A solid phase extraction step is applied to remove trypsin residues (to avoid cleavage at S396 when phosphorylation is removed) and to concentrate the sample before the dephosphorylation step.

[0043] The SPE column (Sep-Pak® tC18 100 mg) was conditioned with 500 μL of methanol followed by 1000 μL of Milli-Q water. The complete sample was loaded onto the SPE column. The SPE column was washed twice with 1000 μL of SPE wash solution (100 mM ammonium acetate, pH=5.5).

[0044] Place the SPE plate over the (clean) 500μL plate and centrifuge the SPE plate over the 500μL plate at 400*g for 1 minute to collect any remaining SPE wash (waste). Elute the samples with 450μL methanol into a new clean 500μL LoBind plate deep well plate.

[0045] Evaporate the solvent to dryness at 65 °C for approximately 45 min under a gentle stream of nitrogen and reconstitute the samples in 20.0 µL of dephosphorylation solvent (100 mM ammonium acetate, pH = 5.5), at which pH the enzymatic activity of trypsin is minimized but lambda protein phosphatase (λPP) activity is maintained.

[0046] Dephosphorylation step Prepare a 3.125 mM solution of MnCl2 by mixing 1000 μL of 10 mM MnCl2 with 2200 μL of Milli-Q in a tube. Add 20.0 μL of the (freshly) prepared 3.125 mM MnCl2 solution to the sample. Prepare a λPP solution freshly by mixing 50.0 μL of λPP solution (containing 20000 units) with 500 μL of Milli-Q in the original cup. Add 10.0 μL of the (freshly) prepared λPP solution to the sample and vortex the plate at 1200 rpm (thermomixer) and 37 °C for 120 min. Add 50.0 μL of dephosphorylation solvent (100 mM ammonium acetate, pH = 5.5) to the plate and vortex the plate at 1200 rpm (thermomixer) and 37 °C for 1 min.

[0047] Finish workflow 1 and prepare for workflow 2 Transfer 50.0 µL of the sample to a new 500 µL protein plate. This sample will be used in Workflow 2 and will undergo a second trypsin digestion (Workflow 2 plate).

[0048] To the original plate, add 50.0 μL of 1% formic acid in water and vortex the plate for 1 minute at 1200 rpm and room temperature. The plate is ready for LC-MS / MS analysis.

[0049] Second trypsin digestion Freshly prepare digestion mixture 2 by mixing 300 μL of 5 mg / mL trypsin in 1 mM HCl solution with 4700 μL of 250 mM ABC solution in a tube (300 μg / mL trypsin). Add 30.0 μL of freshly prepared digestion mixture 2 to each well of the Workflow 2 plate and vortex at 1200 rpm (thermomixer) and 37 °C for 60 min.

[0050] The digestion is stopped by adding 20.0 μL of 1% formic acid in water to the Workflow 2 plate and vortexing the plate for 1 minute at 1200 rpm and 37° C. The plate is now ready for LC-MS / MS analysis.

[0051] Example 3: LC-MS / MS analysis Separation from endogenous interfering compounds is achieved by LC-MS / MS using an Acquity HSS T3 (100x2.1mm, 1.8μm) analytical column set at 30°C with 0.1% formic acid and 0.5% DMSO in water as mobile phase A and methanol as mobile phase B. An 11 min gradient is applied at a flow rate of 0.500mL / min, running at 5% mobile phase B for the first minute, increasing linearly to 14% B after 9 minutes, then stepping to 90% B, maintaining this for 1 minute, and decreasing again to 5% B. The injection volume is 40μL.

[0052] The expected retention time for SPVVSGDTSPR[396-406] is 7.2 min and for IGSTENLK[260-267] is 6.5 min.

[0053] A SCIEX triple quad 6500 mass spectrometer equipped with a turbo ion spray source is used for detection in positive ion mode. The turbo ion spray source is operated in positive ion mode at an ion spray voltage of 5500 V and a temperature of 500° C. The curtain gas is set at 30.

[0054] Quantification is based on multiple reaction monitoring (MRM) using the transitions specified in the table below. A linear (analyst) calibration curve with a 1 / x2 weighting factor is used, ranging from 2 to 100 pM of total Tau protein in CSF.

[0055] [Table 2]

Claims

1. A method for measuring pS396Tau in a sample, comprising steps i to iv: i) treating a CSF sample from a subject suffering from or suspected of suffering from a Tau pathology with trypsin; ii) exposing the sample of step i) to a dephosphorylating agent, followed by reserving a portion of the sample for step iv) and then proceeding to step iii) with the remaining sample; iii) subjecting the dephosphorylated sample from step ii) to a second trypsinization; iv) measuring the amount of Tau peptide corresponding to Tau residues 396-406 (SPVVSGDTSPR) in the samples from step ii) and step iii) using LC-MS; A method comprising:

2. 2. The method of claim 1, further comprising the step of comparing the amount of Tau peptide (SPVVSGDTSPR) from step ii) and step iii) measured in step iv).

3. 2. The method of claim 1, further comprising the step of comparing the results obtained in step iv) with a control containing Tau residues 260-267 (IGSTENLK).

4. The method of claim 1 , wherein the subject is a human.

5. The Tau pathologies include Alzheimer's disease, Down's syndrome, argyrophilic grain disease (AGD), psychosis, in particular psychosis due to AD or in AD patients, apathy due to AD or in AD patients, psychiatric symptoms in patients with dementia with Lewy bodies, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART), and neurofibrillary tangles.

2. The method of claim 1, wherein the disease is selected from dominant senile dementia, dementia pugilistica, chronic traumatic encephalopathy, stroke, stroke recovery, neurodegeneration associated with Parkinson's disease, chromosomally linked parkinsonism, Lytico-Bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis complex, Hallervorden-Spatz disease, and lipofuscinosis.

6. 2. The method of claim 1, wherein the Tau pathology is Alzheimer's disease or Down's syndrome.

7. Use of the method according to any one of claims 1 to 6 for diagnosing patients with Tau pathology.

8. Use of the method according to any one of claims 1 to 6 for disease monitoring in patients with Tau pathology.

9. Use of the method according to any one of claims 1 to 6 for monitoring the effect of a treatment in a subject suffering from a Tau pathology.

10. 10. The use of claim 9, wherein the treatment is an anti-Tau antibody treatment.

11. The Tau pathologies include Alzheimer's disease, Down's syndrome, argyrophilic grain disease (AGD), psychosis, in particular psychosis due to AD or in AD patients, apathy due to AD or in AD patients, psychiatric symptoms in patients with dementia with Lewy bodies, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD or its variants), TBI (traumatic brain injury, acute or chronic), corticobasal degeneration (CBD), Pick's disease, primary age-related tauopathy (PART), and neurofibrillary tangles.

8. The use according to claim 7, wherein the disease is selected from the group consisting of dominant senile dementia, dementia pugilistica, chronic traumatic encephalopathy, stroke, stroke recovery, neurodegeneration associated with Parkinson's disease, chromosomally linked parkinsonism, Lytico-Bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, Huntington's disease, lead encephalopathy, tuberous sclerosis complex, Hallervorden-Spatz disease and lipofuscinosis.

12. 8. The use according to claim 7, wherein the Tau pathology is Alzheimer's disease or Down's syndrome.

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