JP2021523357A - Diagnostic and therapeutic methods based on site-specific taurine oxidation - Google Patents

Abstract

The present disclosure predicts the time to onset of mild cognitive impairment due to Alzheimer's disease by quantifying taurine oxidation at specific amino acid residues, grades Alzheimer's disease, guides treatment decisions, and is the subject of clinical trials. Provided are methods for making selections and assessing the clinical efficacy of certain therapeutic interventions.
[Selection diagram] Fig. 1

Description

Cross-reference to related applications This application is the priority of US Provisional Application No. 62 / 666,504 filed May 3, 2018 and US Provisional Application No. 62 / 666,509 filed May 3, 2018. Allegedly, these disclosures are incorporated herein by reference.

Government Rights The invention was made with government support under NS065667 and NS095773 conferred by the National Institutes of Health. The government has certain rights to the present invention.

Sequence Listing Reference This application includes a sequence listing, which is submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy was made on May 3, 2019, and the file name is 623217_ST25. It is txt and the file size is 24KB bytes.

Microtubule-associated protein tau (MAPT or tau) plays an important role in neuronal morphology and physiology. Tau has six different isoforms in its full-length protein and is capable of undergoing multiple post-translational modifications, including acetylation, glycosylation, and phosphorylation. Phosphorylation is important in regulating the normal function of tau in axonal stabilization and can occur at more than 80 different residues. However, excessive phosphorylation of tau appears to increase the likelihood that tau will aggregate into intracellular insoluble anti-spiral fibrils (PHF) and neurofibrillary tangles (NFT). PHF and NFT consist primarily of hyperphosphorylated tau.

Intracellular neurofibrillary tangles in the cerebral cortex are the pathological features that determine Alzheimer's disease (AD) and correlate with the onset of clinical manifestations well after the appearance of extracellular amyloid β (Aβ) plaques. There is. Extracellular amyloid β plaques begin to develop up to 20 years before the onset of symptoms. In AD, soluble p-tau and non-phosphorylated tau are doubled in cerebrospinal fluid (CSF). It has been proposed that these changes reflect the effects of neuronal death (neurodegeneration), which passively releases tau and NFT into CSF. However, in other tauopathy with marked NFT pathology and neurodegeneration (eg, progressive supranuclear palsy, frontotemporal lobar degeneration-tau), soluble p-tau and total tau CSF levels do not increase. These findings suggest that Aβ may trigger the process of inducing AD-specific tauopathy, and this idea is supported by cellular and animal models. This concept is further supported by increased active production of soluble tau in the presence of amyloid plaques in humans.

Although tau constitutes a hallmark of AD pathology and can be measured in aggregated or lysed form, how post-translational modifications of this important neuroprotein lead to the development of NFTs and neurodegeneration in humans. There remains a significant gap in our understanding. For example, the association between tau and amyloid β plaque is unknown. Similarly, it is unclear what pathophysiological changes, if any, occur in tau before the onset of AD and during the clinical phase. Therefore, it is also unclear to what extent tau, if possible, can be used to stage subjects and guide treatment decisions before the onset of AD-related symptoms.

Therefore, improved methods for quantifying taurine oxidation are still needed in the art.

In one embodiment, the disclosure includes a method of diagnosing a subject at high risk of conversion to mild cognitive impairment (MCI) due to Alzheimer's disease (AD). In this method, (a) an isolated tau sample obtained from a subject is prepared, and tau phosphorylation at one or more amino acid residues selected from T181, T205, and T217 in the isolated tau sample is performed. Measuring and optionally measuring total tau; and (b) measured phosphorylation levels (s) measured by PET imaging and / or brain by Aβ42 / 40 measurements in CSF. Includes diagnosing a subject as having a high risk of conversion to MCI by AD if it deviates significantly from the mean in the control population without amyloid plaques. Using tau phosphorylation measurements at T181, T205, and / or T217, and optionally in combination with tau total measurement, or in addition to this, from the measured phosphorylation levels (s) The calculated ratio, or the ratio calculated from the measured phosphorylation level (s) and total tau, may be used. The ratio calculated from the measured phosphorylation level (s) may be the ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217. .. The ratio calculated from the measured phosphorylation level (s) and total tau may be the ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. .. Mathematical operations other than ratios may also be used.

In another aspect, the disclosure includes a method of grading subjects prior to the onset of mild cognitive impairment (MCI) due to Alzheimer's disease (AD). In this method, (a) an isolated tau sample obtained from a subject is prepared, and taurin oxidation at one or more amino acid residues selected from T181, T205, and T217 in the isolated tau sample is performed. Measuring and optionally measuring total tau; and (b) measured phosphorylation levels (s) measured by PET imaging and / or brain by Aβ42 / 40 measurements in CSF. Includes diagnosing a subject as having a certain number of years before the onset of MCI due to AD if it deviates significantly from the mean in the control population without amyloid plaques. Tau phosphorylation measurements at T181, T205, and / or T217 were used and optionally combined with or in addition to a total tau measurement, calculated from the measured phosphorylation levels (s). Ratios, or ratios calculated from measured phosphorylation levels (s) and total tau, may be used. The ratio calculated from the measured phosphorylation level (s) may be the ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217. .. The ratio calculated from the measured phosphorylation level (s) and total tau may be the ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. .. Mathematical operations other than ratios may also be used.

In another aspect, the disclosure includes a method of grading a subject after the onset of Alzheimer's disease (AD) symptoms. In this method, (a) an isolated tau sample obtained from a subject is prepared, and taurin oxidation at one or more amino acid residues selected from T181, T205, and T217 in the isolated tau sample is performed. Measuring and optionally measuring total tau; and (b) measured phosphorylation levels (s) measured by PET imaging and / or brain by Aβ42 / 40 measurements in CSF. Includes diagnosing a subject as having passed a certain number of years since the onset of MCI due to AD if it deviates significantly from the average in the control population that was free of amyloid plaques. Tau phosphorylation measurements at T181, T205, and / or T217 were used and optionally combined with or in addition to a total tau measurement, calculated from the measured phosphorylation levels (s). Ratios, or ratios calculated from measured phosphorylation levels (s) and total tau, may be used. The ratio calculated from the measured phosphorylation level (s) may be the ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217. .. The ratio calculated from the measured phosphorylation level (s) and total tau may be the ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. .. Mathematical operations other than ratios may also be used.

In another aspect, the present disclosure includes methods for treating a subject in need of treatment. In this method, (a) an isolated tau sample obtained from a subject is prepared, and tau phosphorylation at one or more amino acid residues selected from T181, T205, and T217 in the isolated tau sample is performed. Measuring and optionally measuring total tau; and (b) measured phosphorylation levels (s) measured by PET imaging and / or brain by Aβ42 / 40 measurement in CSF. Includes administration of a pharmaceutical composition to a subject when it deviates significantly from the mean in the control population that was free of amyloid plaque. Tau phosphorylation measurements at T181, T205, and / or T217 were used and optionally combined with or in addition to a total tau measurement, calculated from the measured phosphorylation levels (s). Ratios, or ratios calculated from measured phosphorylation levels (s) and total tau, may be used. The ratio calculated from the measured phosphorylation level (s) may be the ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217. .. The ratio calculated from the measured phosphorylation level (s) and total tau may be the ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. .. Mathematical operations other than ratios may also be used.

In another aspect, the disclosure includes a method for enrolling a subject in a clinical trial. In this method, (a) an isolated tau sample obtained from a subject is prepared, and tau phosphorylation at one or more amino acid residues selected from T181, T205, and T217 in the isolated tau sample is performed. Measuring and optionally measuring total tau; and (b) measured phosphorylation levels (s) measured by PET imaging and / or brain by Aβ42 / 40 measurement in CSF. Includes enrolling a subject in a clinical trial if it deviates significantly from the mean in a control population that was free of amyloid plaques. Tau phosphorylation measurements at T181, T205, and / or T217 were used and optionally combined with or in addition to a total tau measurement, calculated from the measured phosphorylation levels (s). Ratios, or ratios calculated from measured phosphorylation levels (s) and total tau, may be used. The ratio calculated from the measured phosphorylation level (s) may be the ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217. .. The ratio calculated from the measured phosphorylation level (s) and total tau may be the ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. .. Mathematical operations other than ratios may also be used.

Other aspects and iterations of the invention are described in more detail below.

The file of this application contains at least one color photograph. A copy of the Patent Application Gazette, including color photographs, will be provided by the JPO upon request and upon payment of the required fees.

It is a figure of the longest human tau isoform (2N4R) and the epitope of the tau antibody. The N-terminus, mid-domain, MTBR, and C-terminus have been identified for this isoform and are predictably altered for other tau isoforms (eg, 2N3R, 1N4R, 1N3R, 0N4R, and 0N3R). do. It is a figure which shows the principle of a parallel reaction monitoring experiment. The data obtained from the PRM screening of the monophosphorylated tau sequence of 103-126 (0N isoform) is shown. A unique LC-MS / MS pattern containing a series of fragments that elute near unmodified peptides 103-126 and are expected to be phosphorylated at T111 (a), S113 (b), or T123 (c). Identified. The hypothesized y-ion fragments from each p-tau peptide are underlined in the sequence. The possibility of simultaneous elution of three putative monophosphorylated peptides was analyzed. The phosphate group-free ion fragment y15 is specific for the peptide phosphorylated at residue T111 (y15 (a)), and the phosphate group y8 fragment is the p-tau peptide at residue T123 (y15 (a)). It is specific to y8 (c)). Corresponding extracted ion chromatograms (XICs) were detected in small amounts, above the detection limit, which supports the identification of two corresponding monophosphorylated tau peptides. Conversely, the y15 fragment with a phosphate group, which is common to pT111 and pS113 (y15 (a + b)), and the y8 fragment, which does not have a phosphate group, is common to pS113 and pT123 (y8 (b + c)). ), These are much larger in quantity. These signal differences support the presence of taupeptide (b) monophosphorylated at residue S113 as the major species of pattern. The data obtained from the PRM screening of the monophosphorylated tau sequence 68-126 (1N isoform) having 6 phosphorylated sites is shown. The three phosphorylation sites are common to peptides with residues 103-126, as described in FIG. 3 (df). Six LC-MS patterns were identified. The Y28 fragment with a phosphate group is common to pS68 (a) or pT69 (b), which was found in the two LC-MS patterns 4 and 5. This demonstrates the presence of two phosphorylated peptides, but the corresponding LC-MS pattern is strictly attributed to the detection of the ion fragment y29 and the indistinguishability of pS68 and pT69. Can't. Specific fragments corresponding to pT71 (c) and pT111 (d) are found in LC-MS patterns 6 and 1, respectively. Specific fragments of pS113 (e) and pT123 (f) (y15 with a phosphate group) are found in LC-MS pattern 2. This pattern contains both the y10 fragment with and without the phosphate group, suggesting that these two phosphorylated peptides co-elut. Since y10 having no phosphate group has a larger signal than y10 having a phosphate group, the degree of pT123 seems to be lower than that of pS113. LC-MS pattern 3 is due to a small amount of conformational isomers or LC artifacts derived from the phosphorylated peptide of pattern 4, as seen for non-phosphorylated peptides. The data obtained from the PRM screening of the monophosphorylated tau sequence 45-67 (1N and 2N isoforms) is shown. A strong signal from the conformational isomer was identified prior to the non-phosphorylated peptide LC-MS pattern. Therefore, it was expected that the corresponding LC-MS pattern of the phosphorylated peptide would also have a conformation isomer separable by LC. In fact, as a result of PRM scan interpretation, pT50 (b) was detected as the major phosphorylation site in this sequence (Pattern 2). Pattern 1 has a similar fragmentation fingerprint, which was assigned to the conformational isomer of pT50. pS46 (a) can also distinguish the signal from pT50 (b), but because it was not detected, this phosphorylation is absent, or in small amounts, other phosphorus that shares a similar non-specific fragment. It suggests that it may have co-eluted with the oxidizing peptide. The co-eluted y9 of LC-MS pattern 3, y15 without a phosphate group, and y17 with a phosphate group were identified as phosphorylation at residue T52 (c). Patterns 4/6 and 5/7 were each paired with conformers. That is, the two phosphorylated peptides were inseparable. The fragments found in these patterns corresponded to phosphorylation at one of the S61 (e), T63 (f), and S64 (g) residues. MS / MS intensity was insufficient to identify these sites, but at least two of these three sites appeared to be phosphorylated in this sequence. In addition, a small amount of y9 fragment without phosphate group was found on the shoulder of pattern 7, which could be attributed to a small amount of phosphorylation at residue S56 (d). The data obtained from the PRM screening of the monophosphorylated tau sequence 88-126 (2N isoform) is shown. There were 6 phosphorylated sites in this sequence, and 6 LC-MS patterns were identified. The fragments found in patterns 1 and 2 corresponded to the phosphorylated peptides at residues T111 (d) and S113 (e), respectively. No specific fragment due to the phosphorylated peptide was found at residue T123 (f). Patterns 4 and 6 contained a low signal of the y29 fragment consistent with phosphorylation at residue T101 (b) or T102 (c), but specific fragments capable of distinguishing between them were detected. Was not done. Patterns 3 and 5 shared the fragments found in Patterns 4 and 6, but the amount was small and was at the position of the phosphorylated residue at the N-terminal residue G109. This may indicate the presence of additional phosphorylated peptides, preferably phosphorylated peptides at residue T95 (a), or massive conformers of the peptides found in patterns 4 and 6. be. A PRM scan of monophosphorylated tau sequences 68-87 with four phosphorylated sites is shown. Three LC-MS patterns were detected. Patterns 2 and 3 were consistent with phosphorylation at residue S68 (a) or residue T69 (b). Pattern 1 contained both fragments corresponding to the simultaneous elution and presence of two phosphorylated peptides at T71 (c) and T76 (d). A comparison of the y14 XIC with and without the phosphate group in pattern 1 shows that pT71 (c) is more than pT76 (d). The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. The detection of phosphorylation sites at the mid-domain and C-terminus of the brain p-tau protein is shown. Phosphorylated peptide profiles from tau sequence 195-209 (SEQ ID NO: 38) and tau sequence 212-221 (SEQ ID NO: 64) are variable between the soluble brain fraction, normal CSF, and the tau protein of AD CSF. Show that. The brain-soluble tau extract is diluted as indicated to roughly match the corresponding CSF tau level. Phosphorylated Peptides at 195-209: In brain lysates, one signal corresponding to the simultaneous elution of two phosphorylated peptides pS199 and pS202 is observed. Two additional signals are observed in the CSF. Fragment analysis allowed the signal on the left to be assigned to pT205. In the CSF of AD, these two signals were augmented to allow identification of specific fragments, and the signal on the right was assigned to pS208. Phosphorylated Peptide at 212-221: Two signals with similar MS intensities correspond to pT217 and pS214, which are identified in brain lysates. In CSF, the signal corresponding to pT217 is the strongest, but pS214 is close to the detection limit, indicating that their relative abundance has changed dramatically when compared to brain extracts. In AD CSF, pT217 is markedly increased by specific hyperphosphorylation. Phosphorylated peptide profiles from tau sequence 195-209 (SEQ ID NO: 38) and tau sequence 212-221 (SEQ ID NO: 64) are variable between the soluble brain fraction, normal CSF, and the tau protein of AD CSF. Show that. The brain-soluble tau extract is diluted as indicated to roughly match the corresponding CSF tau level. Phosphorylated Peptides at 195-209: In brain lysates, one signal corresponding to the simultaneous elution of two phosphorylated peptides pS199 and pS202 is observed. Two additional signals are observed in the CSF. Fragment analysis allowed the signal on the left to be assigned to pT205. In the CSF of AD, these two signals were augmented to allow identification of specific fragments, and the signal on the right was assigned to pS208. Phosphorylated Peptide at 212-221: Two signals with similar MS intensities correspond to pT217 and pS214, which are identified in brain lysates. In CSF, the signal corresponding to pT217 is the strongest, but pS214 is close to the detection limit, indicating that their relative abundance has changed dramatically when compared to brain extracts. In AD CSF, pT217 is markedly increased by specific hyperphosphorylation. Phosphorylated peptide profiles from tau sequence 195-209 (SEQ ID NO: 38) and tau sequence 212-221 (SEQ ID NO: 64) are variable between the soluble brain fraction, normal CSF, and the tau protein of AD CSF. Show that. The brain-soluble tau extract is diluted as indicated to roughly match the corresponding CSF tau level. Phosphorylated Peptides at 195-209: In brain lysates, one signal corresponding to the simultaneous elution of two phosphorylated peptides pS199 and pS202 is observed. Two additional signals are observed in the CSF. Fragment analysis allowed the signal on the left to be assigned to pT205. In the CSF of AD, these two signals were augmented to allow identification of specific fragments, and the signal on the right was assigned to pS208. Phosphorylated Peptide at 212-221: Two signals with similar MS intensities correspond to pT217 and pS214, which are identified in brain lysates. In CSF, the signal corresponding to pT217 is the strongest, but pS214 is close to the detection limit, indicating that their relative abundance has changed dramatically when compared to brain extracts. In AD CSF, pT217 is markedly increased by specific hyperphosphorylation. Phosphorylated peptide profiles from tau sequence 195-209 (SEQ ID NO: 38) and tau sequence 212-221 (SEQ ID NO: 64) are variable between the soluble brain fraction, normal CSF, and the tau protein of AD CSF. Show that. The brain-soluble tau extract is diluted as indicated to roughly match the corresponding CSF tau level. Phosphorylated Peptides at 195-209: In brain lysates, one signal corresponding to the simultaneous elution of two phosphorylated peptides pS199 and pS202 is observed. Two additional signals are observed in the CSF. Fragment analysis allowed the signal on the left to be assigned to pT205. In the CSF of AD, these two signals were augmented to allow identification of specific fragments, and the signal on the right was assigned to pS208. Phosphorylated Peptide at 212-221: Two signals with similar MS intensities correspond to pT217 and pS214, which are identified in brain lysates. In CSF, the signal corresponding to pT217 is the strongest, but pS214 is close to the detection limit, indicating that their relative abundance has changed dramatically when compared to brain extracts. In AD CSF, pT217 is markedly increased by specific hyperphosphorylation. Phosphorylated peptide profiles from tau sequence 195-209 (SEQ ID NO: 38) and tau sequence 212-221 (SEQ ID NO: 64) are variable between the soluble brain fraction, normal CSF, and the tau protein of AD CSF. Show that. The brain-soluble tau extract is diluted as indicated to roughly match the corresponding CSF tau level. Phosphorylated Peptides at 195-209: In brain lysates, one signal corresponding to the simultaneous elution of two phosphorylated peptides pS199 and pS202 is observed. Two additional signals are observed in the CSF. Fragment analysis allowed the signal on the left to be assigned to pT205. In the CSF of AD, these two signals were augmented to allow identification of specific fragments, and the signal on the right was assigned to pS208. Phosphorylated Peptide at 212-221: Two signals with similar MS intensities correspond to pT217 and pS214, which are identified in brain lysates. In CSF, the signal corresponding to pT217 is the strongest, but pS214 is close to the detection limit, indicating that their relative abundance has changed dramatically when compared to brain extracts. In AD CSF, pT217 is markedly increased by specific hyperphosphorylation. Phosphorylated peptide profiles from tau sequence 195-209 (SEQ ID NO: 38) and tau sequence 212-221 (SEQ ID NO: 64) are variable between the soluble brain fraction, normal CSF, and the tau protein of AD CSF. Show that. The brain-soluble tau extract is diluted as indicated to roughly match the corresponding CSF tau level. Phosphorylated Peptides at 195-209: In brain lysates, one signal corresponding to the simultaneous elution of two phosphorylated peptides pS199 and pS202 is observed. Two additional signals are observed in the CSF. Fragment analysis allowed the signal on the left to be assigned to pT205. In the CSF of AD, these two signals were augmented to allow identification of specific fragments, and the signal on the right was assigned to pS208. Phosphorylated Peptide at 212-221: Two signals with similar MS intensities correspond to pT217 and pS214, which are identified in brain lysates. In CSF, the signal corresponding to pT217 is the strongest, but pS214 is close to the detection limit, indicating that their relative abundance has changed dramatically when compared to brain extracts. In AD CSF, pT217 is markedly increased by specific hyperphosphorylation. It is shown that pT153, pT175, and pT231 phosphorylated peptides are identified in CSF. AQUA internal standard signals are shown for pT175 and pT231. The fragmentation pattern of pT153 is similar to that of the unmodified one. It is shown that pT153, pT175, and pT231 phosphorylated peptides are identified in CSF. AQUA internal standard signals are shown for pT175 and pT231. The fragmentation pattern of pT153 is similar to that of the unmodified one. It is shown that pT153, pT175, and pT231 phosphorylated peptides are identified in CSF. AQUA internal standard signals are shown for pT175 and pT231. The fragmentation pattern of pT153 is similar to that of the unmodified one. It is shown that pT153, pT175, and pT231 phosphorylated peptides are identified in CSF. AQUA internal standard signals are shown for pT175 and pT231. The fragmentation pattern of pT153 is similar to that of the unmodified one. It is shown that pT153, pT175, and pT231 phosphorylated peptides are identified in CSF. AQUA internal standard signals are shown for pT175 and pT231. The fragmentation pattern of pT153 is similar to that of the unmodified one. It is shown that pT153, pT175, and pT231 phosphorylated peptides are identified in CSF. AQUA internal standard signals are shown for pT175 and pT231. The fragmentation pattern of pT153 is similar to that of the unmodified one. It is shown that pT153, pT175, and pT231 phosphorylated peptides are identified in CSF. AQUA internal standard signals are shown for pT175 and pT231. The fragmentation pattern of pT153 is similar to that of the unmodified one. It is shown that pT153, pT175, and pT231 phosphorylated peptides are identified in CSF. AQUA internal standard signals are shown for pT175 and pT231. The fragmentation pattern of pT153 is similar to that of the unmodified one. It is shown that pT153, pT175, and pT231 phosphorylated peptides are identified in CSF. AQUA internal standard signals are shown for pT175 and pT231. The fragmentation pattern of pT153 is similar to that of the unmodified one. It shows that the phosphorylation abundance at T111 compared to S113 phosphorylation is higher in CSF than in the brain. In both the brain and CSF, the MS / MS fragment y18 common to all monophosphorylated peptides in tau sequences 103-126 is detected. The relative abundance of the y15 fragment from pS113 (b) is significantly lower in the CSF than in the brain extract. Conversely, the y15 fragment from pT111 (a) is abundant in CSF and undetectable in brain-soluble extracts diluted to match CSF tau levels in AD. We show that the relative abundance of taurine oxidation depends on the bioextract and varies across protein sequences. The abundance of taurine oxidation was compared by extracting from normal brain lysate, normal CSF, and CSF of AD by immunocapture using HJ8.5 and tau1 and measuring by MS. The area of the circle is proportional to the abundance of phosphorylated sites. Red and green indicate increases and decreases relative to the reference brain-soluble fraction profile (blue), respectively. Tau is truncated at the C-terminus in CSF, which explains the lack of detection of C-terminal clusters at the phosphorylation site. Phosphorylation at T205 and S208 is CSF-specific (brain-soluble fraction deficit X-upper). It shows that the taurine oxidation site is modified differently in the brain, normal CSF, and AD CSF. The measurement is the relative abundance of the phosphorylation signal relative to the corresponding non-phosphorylated site (HJ8.5 + tau 1 IP-MS). Brain results were obtained with lysates diluted 500x-8000x times to match CSF tau levels. Phosphorylation at T205 and S208 is undetectable in brain tissue. Phosphorylation at T111 is undetectable with 500-fold diluted lysates, but is detected with 10-fold diluted lysates, which corresponds to an abundance of 0.02%. Legend: ^** indicates significance at the p = 0.01 level, ^* indicates significance at the p = 0.05 level. The antibody effect on the phosphorylation ratio measured by IP-MS is shown. Antibodies to the tau N-terminal protruding domain (tau 13 or HJ8.5) or middomain (HJ8.7, tau 1, or tau 5) were used in brain lysate pools, non-AD (n = 1) pools, and AD. Immunoprecipitation was performed in parallel from the CSF (n = 1) pool. Radar plots of phosphorylation ratios (log ₁₀ scale) measured at major CSF sites, brain lysate (FIG. 14A, left panel), non-AD CSF (FIG. 14A, center panel), and AD CSF (FIG. 14A, right). Panel) is shown. Measurements of taurine oxidation ratios at pT181 / T181, pT231 / T231 are consistent across the antibodies tested. PS199 / S199 is reduced by using Tau 1 or Tau 1 + HJ8.5 compared to other antibodies. The antibody effect on the phosphorylation ratio measured by IP-MS is shown. Antibodies to the tau N-terminal protruding domain (tau 13 or HJ8.5) or middomain (HJ8.7, tau 1, or tau 5) were used in brain lysate pools, non-AD (n = 1) pools, and AD. Immunoprecipitation was performed in parallel from the CSF (n = 1) pool. The low recovery of pS199 by tau1 or tau1 + HJ8.5IP results in an underestimation of the measurement of the pS199 / S199 ratio compared to the other antibodies tested. Tau 13, HJ8.5, and HJ8.7 antibodies show that there is no significant change in pS199 phosphorylation ratio between the brain and CSF. CSF pS202 / S202 low phosphorylation compared to brain The antibody effect on the phosphorylation ratio measured by IP-MS is shown. Antibodies to the tau N-terminal protruding domain (tau 13 or HJ8.5) or middomain (HJ8.7, tau 1, or tau 5) were used in brain lysate pools, non-AD (n = 1) pools, and AD. Immunoprecipitation was performed in parallel from the CSF (n = 1) pool. Compared to the brain, pS202 / S202 low phosphorylation of CSF (Fig. 14C) and pT217 / pT217 high phosphorylation (Fig. 14D) are apparent regardless of the antibody used for IP-MS. The legends of FIGS. 14B to 14D are the same and shown in FIG. 14C-brain (blue), non-AD CSF (green), and AD CSF (red). The antibody effect on the phosphorylation ratio measured by IP-MS is shown. Antibodies to the tau N-terminal protruding domain (tau 13 or HJ8.5) or middomain (HJ8.7, tau 1, or tau 5) were used in brain lysate pools, non-AD (n = 1) pools, and AD. Immunoprecipitation was performed in parallel from the CSF (n = 1) pool. Compared to the brain, pS202 / S202 low phosphorylation of CSF (Fig. 14C) and pT217 / pT217 high phosphorylation (Fig. 14D) are apparent regardless of the antibody used for IP-MS. It is shown that the incubation of CSF does not affect the phosphorylation rate measurement in tau. We show that amyloid plaques are strongly correlated with tau hyperphosphorylation, but vary by site of phosphorylation. Receiver operating characteristics of total tau (blue, AUC = 0.62) and site-specific phosphorylation ratio when subjects were classified as having Aβ pathology based on Aβ PiB-PET (SUVR cutoff is 1.25). And p-T217 (yellow line) showed a near perfect association with Aβ pathology (AUC = 0.97), followed by p-T181 (AUC = 0.89), and p-T205 (AUC). = 0.74). We show that amyloid plaques are strongly correlated with tau hyperphosphorylation, but vary by site of phosphorylation. Standardized (z-score) phosphorylation ratios of p-T217 (FIG. 16B), p-T181, (FIG. 16C), p-S202 (FIG. 16D), p-T205 (FIG. 16E), and total tau (FIG. 16F) levels. Is indicated by the Aβ PiB-PET quadrant (n = 45, 47, 28, 30), highlighting site-specific differences in phosphorylation with elevated Aβ PiB-PET levels for mutant carriers. : P-T217 and p-T181 have the highest increase, first increasing the amount of Aβ PiB-PET, which slows down and keeps the level of Aβ PiB-PET at the highest level. On the other hand, p-T205 and tau totals demonstrate to continue to increase. For p-S202, there is a significant reduction in phosphorylation at the highest Aβ PiB-PET interquartile range compared to the lowest; ^***- P value <0.001, ^**- P value < 0.01, based on Wilcoxon two-sample test; midline represents median, top and bottom notches = median +/- 1.58 ^* interquartile range / square root (n-observed), top and bottom Lower whiskers = upper / lower hinges above / below maximum observed +1.58 ^* IQR. We show that amyloid plaques are strongly correlated with tau hyperphosphorylation, but vary by site of phosphorylation. Standardized (z-score) phosphorylation ratios of p-T217 (FIG. 16B), p-T181, (FIG. 16C), p-S202 (FIG. 16D), p-T205 (FIG. 16E), and total tau (FIG. 16F) levels. Is indicated by the Aβ PiB-PET quadrant (n = 45, 47, 28, 30), highlighting site-specific differences in phosphorylation with elevated Aβ PiB-PET levels for mutant carriers. : P-T217 and p-T181 have the highest increase, first increasing the amount of Aβ PiB-PET, which slows down and keeps the level of Aβ PiB-PET at the highest level. On the other hand, p-T205 and tau totals demonstrate to continue to increase. For p-S202, there is a significant reduction in phosphorylation at the highest Aβ PiB-PET interquartile range compared to the lowest; ^***- P value <0.001, ^**- P value < 0.01, based on Wilcoxon two-sample test; midline represents median, top and bottom notches = median +/- 1.58 ^* interquartile range / square root (n-observed), top and bottom Lower whiskers = upper / lower hinges above / below maximum observed +1.58 ^* IQR. We show that amyloid plaques are strongly correlated with tau hyperphosphorylation, but vary by site of phosphorylation. Standardized (z-score) phosphorylation ratios of p-T217 (FIG. 16B), p-T181, (FIG. 16C), p-S202 (FIG. 16D), p-T205 (FIG. 16E), and total tau (FIG. 16F) levels. Is indicated by the Aβ PiB-PET quadrant (n = 45, 47, 28, 30), highlighting site-specific differences in phosphorylation with elevated Aβ PiB-PET levels for mutant carriers. : P-T217 and p-T181 have the highest increase, first increasing the amount of Aβ PiB-PET, which slows down and keeps the level of Aβ PiB-PET at the highest level. On the other hand, p-T205 and tau totals demonstrate to continue to increase. For p-S202, there is a significant reduction in phosphorylation at the highest Aβ PiB-PET interquartile range compared to the lowest; ^***- P value <0.001, ^**- P value < 0.01, based on Wilcoxon two-sample test; midline represents median, top and bottom notches = median +/- 1.58 ^* interquartile range / square root (n-observed), top and bottom Lower whiskers = upper / lower hinges above / below maximum observed +1.58 ^* IQR. We show that amyloid plaques are strongly correlated with tau hyperphosphorylation, but vary by site of phosphorylation. Standardized (z-score) phosphorylation ratios of p-T217 (FIG. 16B), p-T181, (FIG. 16C), p-S202 (FIG. 16D), p-T205 (FIG. 16E), and total tau (FIG. 16F) levels. Is indicated by the Aβ PiB-PET quadrant (n = 45, 47, 28, 30), highlighting site-specific differences in phosphorylation with elevated Aβ PiB-PET levels for mutant carriers. : P-T217 and p-T181 have the highest increase, first increasing the amount of Aβ PiB-PET, which slows down and keeps the level of Aβ PiB-PET at the highest level. On the other hand, p-T205 and tau totals demonstrate to continue to increase. For p-S202, there is a significant reduction in phosphorylation at the highest Aβ PiB-PET interquartile range compared to the lowest; ^***- P value <0.001, ^**- P value < 0.01, based on Wilcoxon two-sample test; midline represents median, top and bottom notches = median +/- 1.58 ^* interquartile range / square root (n-observed), top and bottom Lower whiskers = upper / lower hinges above / below maximum observed +1.58 ^* IQR. We show that amyloid plaques are strongly correlated with tau hyperphosphorylation, but vary by site of phosphorylation. Standardized (z-score) phosphorylation ratios of p-T217 (FIG. 16B), p-T181, (FIG. 16C), p-S202 (FIG. 16D), p-T205 (FIG. 16E), and total tau (FIG. 16F) levels. Is indicated by the Aβ PiB-PET quadrant (n = 45, 47, 28, 30), highlighting site-specific differences in phosphorylation with elevated Aβ PiB-PET levels for mutant carriers. : P-T217 and p-T181 have the highest increase, first increasing the amount of Aβ PiB-PET, which slows down and keeps the level of Aβ PiB-PET at the highest level. On the other hand, p-T205 and tau totals demonstrate to continue to increase. For p-S202, there is a significant reduction in phosphorylation at the highest Aβ PiB-PET interquartile range compared to the lowest; ^***- P value <0.001, ^**- P value < 0.01, based on Wilcoxon two-sample test; midline represents median, top and bottom notches = median +/- 1.58 ^* interquartile range / square root (n-observed), top and bottom Lower whiskers = upper / lower hinges above / below maximum observed +1.58 ^* IQR. We show that amyloid plaques are strongly correlated with tau hyperphosphorylation, but vary by site of phosphorylation. A bivariate correlation of Aβ PiB-PET SUVR and site-specific phosphorylation between cortex and subcortex for asymptomatic mutant carriers (n = 139). Color indications indicate that the correlations are positive (yellow to red) and negative (blue); all correlations show statistically significant values over false positive rates (p < 0.05), arranged in descending order of correlation from top to bottom. We show that long-term changes in different phosphorylated tau sites are disease-specific stages and change in the opposite direction as AD progresses. (FIG. 17A) p-T217, (FIG. 17B) p-T181, (FIG. 17C) total tau, (FIG. 17D) p-T205, and (FIG. 17E) p-S202 individual, z-transform, long-term phosphorylation ratios. Changes were made for mutation carriers (black = asymptomatic mutation carriers, (n = 152), red = symptomatic mutation carriers (77)), and non-carriers (blue, (n = 141)). It is shown over the estimated number of years (EYO) until the onset of symptoms. The vertical dashed line is the expected time of onset of symptoms, and the green line is the model estimation time when the rate of change of each p-tau isoform is greater in mutant carriers than in non-carriers. show. We show that long-term changes in different phosphorylated tau sites are disease-specific stages and change in the opposite direction as AD progresses. (FIG. 17A) p-T217, (FIG. 17B) p-T181, (FIG. 17C) total tau, (FIG. 17D) p-T205, and (FIG. 17E) p-S202 individual, z-transform, long-term phosphorylation ratios. Changes were made for mutation carriers (black = asymptomatic mutation carriers, (n = 152), red = symptomatic mutation carriers (77)), and non-carriers (blue, (n = 141)). It is shown over the estimated number of years (EYO) until the onset of symptoms. The vertical dashed line is the expected time of onset of symptoms, and the green line is the model estimation time when the rate of change of each p-tau isoform is greater in mutant carriers than in non-carriers. show. We show that long-term changes in different phosphorylated tau sites are disease-specific stages and change in the opposite direction as AD progresses. (FIG. 17A) p-T217, (FIG. 17B) p-T181, (FIG. 17C) total tau, (FIG. 17D) p-T205, and (FIG. 17E) p-S202 individual, z-transform, long-term phosphorylation ratios. Changes were made for mutation carriers (black = asymptomatic mutation carriers, (n = 152), red = symptomatic mutation carriers (77)), and non-carriers (blue, (n = 141)). It is shown over the estimated number of years (EYO) until the onset of symptoms. The vertical dashed line is the expected time of onset of symptoms, and the green line is the model estimation time when the rate of change of each p-tau isoform is greater in mutant carriers than in non-carriers. show. We show that long-term changes in different phosphorylated tau sites are disease-specific stages and change in the opposite direction as AD progresses. (FIG. 17A) p-T217, (FIG. 17B) p-T181, (FIG. 17C) total tau, (FIG. 17D) p-T205, and (FIG. 17E) p-S202 individual, z-transform, long-term phosphorylation ratios. Changes were made for mutation carriers (black = asymptomatic mutation carriers, (n = 152), red = symptomatic mutation carriers (77)), and non-carriers (blue, (n = 141)). It is shown over the estimated number of years (EYO) until the onset of symptoms. The vertical dashed line is the expected time of onset of symptoms, and the green line is the model estimation time when the rate of change of each p-tau isoform is greater in mutant carriers than in non-carriers. show. We show that long-term changes in different phosphorylated tau sites are disease-specific stages and change in the opposite direction as AD progresses. (FIG. 17A) p-T217, (FIG. 17B) p-T181, (FIG. 17C) total tau, (FIG. 17D) p-T205, and (FIG. 17E) p-S202 individual, z-transform, long-term phosphorylation ratios. Changes were made for mutation carriers (black = asymptomatic mutation carriers, (n = 152), red = symptomatic mutation carriers (77)), and non-carriers (blue, (n = 141)). It is shown over the estimated number of years (EYO) until the onset of symptoms. The vertical dashed line is the expected time of onset of symptoms, and the green line is the model estimation time when the rate of change of each p-tau isoform is greater in mutant carriers than in non-carriers. show. We show that long-term changes in different phosphorylated tau sites are disease-specific stages and change in the opposite direction as AD progresses. Model-estimated long-term rate of change for each phosphorylation site, standardized for non-carrying rate of change, plotted over EYO with amyloid PET (red) and cognitive decline (yellow); The crushing circle represents the time when the rate of change of the holder is first different for the non-holder for each variable. This underscores the pattern of changes in p-tau isoforms over the course of the AD spectrum and the close association between amyloid plaque growth and increase in p-T217, where plaques begin to increase at -21 EYO. , P-T217 (black) hyperphosphorylation begins at -21EYO, and a decrease in phosphorylation rates at these two sites is accompanied by cognitive decline (yellow line). In contrast, p-T205 (purple) continues to increase throughout the progression of the disease, and total tau levels (gray) increase at an increasing rate near the time of onset of symptoms. We show that phosphorylated tau sites are differently associated with hypometabolism and atrophy of the brain. A bivariate correlation between cortical and subcortical atrophy and site-specific phosphorylation ratios in asymptomatic mutant carriers (n = 152) demonstrated increased phosphorylation of p-T205 and p-T217. For -T181, the association is smaller. Total tau levels are associated with more atrophy in multiple cortical and subcortical areas. We show that phosphorylated tau sites are differently associated with hypometabolism and atrophy of the brain. The bivariate correlation between cortical and subcortical brain metabolism and site-specific phosphorylation ratios measured by FDG-PET in asymptomatic mutant carriers (n = 143) is mostly due to increased phosphorylation of p-T205. Demonstrate that it is associated with a decrease in the cortex and subcortical region of the body, but not for other p-tau sites or tau. It is shown that a decrease in phosphorylation at p-T217, p-T181, and p-T205 is associated with dementia and cognitive decline. For mutant carriers, the individually estimated annual rate of change (y-axis) for p-tau isoforms and total tau was correlated with annual changes in general cognitive function. The straight line represents a simple linear regression and the shaded area represents a 95% confidence interval. Each point represents an individual level correlation between measurements. Linear regression was applied for no dementia (black, n = 47) and with dementia (red, n = 25). p-T217 (FIG. 19A), r = 0.43 (p = 0.02), p-T181 (FIG. 19B), r = 0.72 (p <.001), and p-T205 (FIG. 19C), A decrease in phosphorylation rate at r = 0.41 (p = 0.03) was associated with cognitive decline after the onset of symptoms (red). There was a tendency to suggest an inverse correlation with cognition for total tau (Fig. 19D), but this was not significant. It is shown that a decrease in phosphorylation at p-T217, p-T181, and p-T205 is associated with dementia and cognitive decline. For mutant carriers, the individually estimated annual rate of change (y-axis) for p-tau isoforms and total tau was correlated with annual changes in general cognitive function. The straight line represents a simple linear regression and the shaded area represents a 95% confidence interval. Each point represents an individual level correlation between measurements. Linear regression was applied for no dementia (black, n = 47) and with dementia (red, n = 25). p-T217 (FIG. 19A), r = 0.43 (p = 0.02), p-T181 (FIG. 19B), r = 0.72 (p <.001), and p-T205 (FIG. 19C), A decrease in phosphorylation rate at r = 0.41 (p = 0.03) was associated with cognitive decline after the onset of symptoms (red). There was a tendency to suggest an inverse correlation with cognition for total tau (Fig. 19D), but this was not significant. It is shown that a decrease in phosphorylation at p-T217, p-T181, and p-T205 is associated with dementia and cognitive decline. For mutant carriers, the individually estimated annual rate of change (y-axis) for p-tau isoforms and total tau was correlated with annual changes in general cognitive function. The straight line represents a simple linear regression and the shaded area represents a 95% confidence interval. Each point represents an individual level correlation between measurements. Linear regression was applied for no dementia (black, n = 47) and with dementia (red, n = 25). p-T217 (FIG. 19A), r = 0.43 (p = 0.02), p-T181 (FIG. 19B), r = 0.72 (p <.001), and p-T205 (FIG. 19C), A decrease in phosphorylation rate at r = 0.41 (p = 0.03) was associated with cognitive decline after the onset of symptoms (red). There was a tendency to suggest an inverse correlation with cognition for total tau (Fig. 19D), but this was not significant. It is shown that a decrease in phosphorylation at p-T217, p-T181, and p-T205 is associated with dementia and cognitive decline. For mutant carriers, the individually estimated annual rate of change (y-axis) for p-tau isoforms and total tau was correlated with annual changes in general cognitive function. The straight line represents a simple linear regression and the shaded area represents a 95% confidence interval. Each point represents an individual level correlation between measurements. Linear regression was applied for no dementia (black, n = 47) and with dementia (red, n = 25). p-T217 (FIG. 19A), r = 0.43 (p = 0.02), p-T181 (FIG. 19B), r = 0.72 (p <.001), and p-T205 (FIG. 19C), A decrease in phosphorylation rate at r = 0.41 (p = 0.03) was associated with cognitive decline after the onset of symptoms (red). There was a tendency to suggest an inverse correlation with cognition for total tau (Fig. 19D), but this was not significant. It is shown that tau PET is increased near the onset of symptoms in DIAD mutation carriers. The average cortex standardized unit value ratio (SUVR) for mutant carriers (red, n = 12) and non-carriers (blue, n = 9) is on the y-axis, the estimated years to onset of symptoms in these subjects (EYO). ) Is taken on the x-axis, and a long-term CSF evaluation is performed prior to the time of tau PET. The plot shows that for mutant carriers, there is little increase in tau PET until the time of onset of presumptive sign (EYO = 0). This figure shows that the neurofibrillary tangle (NFT) pathology detected by AV-1451 occurs much later than the increase in multiple soluble phosphotau sites, and these soluble tau markers appear to be markers of NFT pathology. However, it suggests that it may be a predisposing factor for the development of hyperphosphorylation-insoluble tau deposits characteristic of AD pathology. We show that long-term changes in tau and taurine oxidation sites are associated differently with neurofibrillary tau (tau PET) in dominantly inherited AD. The individual estimated rate of change of phosphorylation and total tau (y-axis) is shown relative to the tau-PET scan time (x-axis). The vertical line is the SUVR of 1.22, which represents a conservative estimate of the time at which NFT tau PET (composite of multiple cortical and marginal regions) is considered elevated compared to non-carriers. The plot shows that increased soluble tau and p-T205 are associated with higher levels of aggregated tau, while phosphorylation rates at p-T217 and p-T181 decrease as levels of aggregated tau increase. Suggest that. These findings indicate that different sites have different increases in tau and phosphorylation levels, and in some cases, soluble p-tau is isolated as the load of hyperphosphorylation aggregates increases with the spread of tau pathology. May indicate that it will be done. They also suggest that as aggregated tau increases, soluble tau levels also increase, which may represent either passive or active release, along with a heavier load of aggregated tau pathology. do. We show that long-term changes in tau and taurine oxidation sites are associated differently with neurofibrillary tau (tau PET) in dominantly inherited AD. The individual estimated rate of change of phosphorylation and total tau (y-axis) is shown relative to the tau-PET scan time (x-axis). The vertical line is the SUVR of 1.22, which represents a conservative estimate of the time at which NFT tau PET (composite of multiple cortical and marginal regions) is considered elevated compared to non-carriers. The plot shows that increased soluble tau and p-T205 are associated with higher levels of aggregated tau, while phosphorylation rates at p-T217 and p-T181 decrease as levels of aggregated tau increase. Suggest that. These findings indicate that different sites have different increases in tau and phosphorylation levels, and in some cases, soluble p-tau is isolated as the load of hyperphosphorylation aggregates increases with the spread of tau pathology. May indicate that it will be done. They also suggest that as aggregated tau increases, soluble tau levels also increase, which may represent either passive or active release, along with a heavier load of aggregated tau pathology. do. We show that long-term changes in tau and taurine oxidation sites are associated differently with neurofibrillary tau (tau PET) in dominantly inherited AD. The individual estimated rate of change of phosphorylation and total tau (y-axis) is shown relative to the tau-PET scan time (x-axis). The vertical line is the SUVR of 1.22, which represents a conservative estimate of the time at which NFT tau PET (composite of multiple cortical and marginal regions) is considered elevated compared to non-carriers. The plot shows that increased soluble tau and p-T205 are associated with higher levels of aggregated tau, while phosphorylation rates at p-T217 and p-T181 decrease as levels of aggregated tau increase. Suggest that. These findings indicate that different sites have different increases in tau and phosphorylation levels, and in some cases, soluble p-tau is isolated as the load of hyperphosphorylation aggregates increases with the spread of tau pathology. May indicate that it will be done. They also suggest that as aggregated tau increases, soluble tau levels also increase, which may represent either passive or active release, along with a heavier load of aggregated tau pathology. do. We show that long-term changes in tau and taurine oxidation sites are associated differently with neurofibrillary tau (tau PET) in dominantly inherited AD. The individual estimated rate of change of phosphorylation and total tau (y-axis) is shown relative to the tau-PET scan time (x-axis). The vertical line is the SUVR of 1.22, which represents a conservative estimate of the time at which NFT tau PET (composite of multiple cortical and marginal regions) is considered elevated compared to non-carriers. The plot shows that increased soluble tau and p-T205 are associated with higher levels of aggregated tau, while phosphorylation rates at p-T217 and p-T181 decrease as levels of aggregated tau increase. Suggest that. These findings indicate that different sites have different increases in tau and phosphorylation levels, and in some cases, soluble p-tau is isolated as the load of hyperphosphorylation aggregates increases with the spread of tau pathology. May indicate that it will be done. They also suggest that as aggregated tau increases, soluble tau levels also increase, which may represent either passive or active release, along with a heavier load of aggregated tau pathology. do. We show that long-term changes in tau and taurine oxidation sites are associated differently with neurofibrillary tau (tau PET) in dominantly inherited AD. The individual estimated rate of change of phosphorylation and total tau (y-axis) is shown relative to the tau-PET scan time (x-axis). The vertical line is the SUVR of 1.22, which represents a conservative estimate of the time at which NFT tau PET (composite of multiple cortical and marginal regions) is considered elevated compared to non-carriers. The plot shows that increased soluble tau and p-T205 are associated with higher levels of aggregated tau, while phosphorylation rates at p-T217 and p-T181 decrease as levels of aggregated tau increase. Suggest that. These findings indicate that different sites have different increases in tau and phosphorylation levels, and in some cases, soluble p-tau is isolated as the load of hyperphosphorylation aggregates increases with the spread of tau pathology. May indicate that it will be done. They also suggest that as aggregated tau increases, soluble tau levels also increase, which may represent either passive or active release, along with a heavier load of aggregated tau pathology. do. It is a figure which shows that the tau pathology develops through each different stage in Alzheimer's disease. By measuring four different soluble tau species and insoluble tau in a group of subjects with deterministic Alzheimer's disease mutations, we present disease stages and other measurements over a 35-year process (x-axis). It is shown that tau-related changes progress (y-axis) and differ based on possible biomarkers. A. The onset is the development of fibrotic amyloid pathology, where phosphorylation at positions 217 (purple) and 181 (blue) begins to increase. B. With an increase in neurological dysfunction (based on metabolic changes), phosphorylation at position 205 (green) begins to increase with soluble tau (orange). C. Finally, with the onset of neurodegeneration (based on brain atrophy and cognitive decline), tau PET tangles (red) begin to develop, while phosphorylation at 217 and 181 begins to decrease. Taken together, this underscores the dynamic and diverse pattern of soluble and aggregated tau over the course of the disease and its close association with amyloid pathology. The quantification of phosphorylated tau isoform in CSF is shown. It is the sum of the extracted ion chromatograms obtained from the parallel reaction monitoring (PRM) analysis of the taurine oxidized peptide and the corresponding unmodified peptide in CSF. T181 monitoring using a microLC system. Cps = number of counts per second. The quantification of phosphorylated tau isoform in CSF is shown. It is the sum of the extracted ion chromatograms obtained from the parallel reaction monitoring (PRM) analysis of the taurine oxidized peptide and the corresponding unmodified peptide in CSF. S199, S202, T205 (simultaneous elution with intraframe signal), and T217 monitoring using the nanoLC system. Intrinsic signal (blue solid line), 15N labeled peptide (red dotted line), AQUA peptide (green dotted line). Cps = number of counts per second. The quantification of phosphorylated tau isoform in CSF is shown. It is the sum of the extracted ion chromatograms obtained from the parallel reaction monitoring (PRM) analysis of the taurine oxidized peptide and the corresponding unmodified peptide in CSF. It showed specific PRM transitions and followed the Biemann nomenclature of peptide fragments, which allowed the identification of three co-eluted monophosphorylated peptides carrying pS199, pS202, or pT205. Cps = number of counts per second. We show that CSF taurine oxidation at T217 is associated with amyloidosis. T217 phosphorylation is significantly increased in subjects with amyloidosis (positive Aβ42 / 40 ratio of PiB-PET and CSF) compared to amyloid-negative controls with no or mild cognitive decline. We show that CSF taurine oxidation at T217 is associated with amyloidosis. ROC curve for diagnosing amyloid-positive subjects separately from amyloid-negative subjects using the phosphorylation rates of T217 and T181 by MS and the T181 phosphorylation rate by ELISA. We show that CSF taurine oxidation at T217 is associated with amyloidosis. Comparison of pT217 / T217 ratios demonstrates specific phosphorylation at T217 in subjects with amyloidosis. We show that CSF taurine oxidation at T217 is associated with amyloidosis. FIGS. 24D-E: Comparison of T217 phosphorylation measured by MS with Aβ42 / 40 change of CSF and comparison with amyloid plaque deposition measured by PiB-PET. The degree of T217 hyperphosphorylation does not correlate with a decrease in Aβ42 relative to Aβ40 in CSF. All five contradictory cases (orange triangle, PiB-PET and T217 high phosphorylation positive but CSF Aβ negative) were all slightly higher than the threshold 0.12 chosen to define the amyloid state. It is suggested that these may be due to the inadequate sensitivity of the CSF amyloid assay. We show that CSF taurine oxidation at T217 is associated with amyloidosis. FIGS. 24D-E: Comparison of T217 phosphorylation measured by MS with Aβ42 / 40 change of CSF and comparison with amyloid plaque deposition measured by PiB-PET. PiB-PET loading (FBP total cortex mean) correlates with T217 phosphorylation status in amyloid-positive subjects. The cutoff value that distinguishes amyloid-positive by PiB from amyloid-negative is 0.18. It is shown that CSF taurine oxidation at T217 is independent of cognitive status and is significantly modified in presymptomatic AD. Left panel: There is no correlation between T217 phosphorylation and the cognitive profile measured by the Clinical Dementia Severity Scale (CDR-SB). Right panel: Among subjects without cognitive decline (CDR-SB = 0), T217 is already significantly hyperphosphorylated in the amyloid-positive group.

Aggregation of tau protein into neurofibrillary tangles in the central nervous system contributes to certain neurodegenerative diseases, including Alzheimer's disease (AD). Although the mechanism of tau destabilization has not yet been fully elucidated, it is known that tau protein is hyperphosphorylated during tau aggregation. Applicants can follow the AD process longitudinally from its clinically asymptomatic to symptomatic stages by using certain methods of quantifying taurine oxidation at specific amino acid residues. I found that there is. FIG. 22 shows the dynamic patterns of taurine oxidation measurable at T181, T205, and T217 for years to onset of MCI due to AD and certain pathophysiological changes, created by Applicants' method. show. The present disclosure includes predicting the time to onset of mild cognitive impairment due to Alzheimer's disease, guiding treatment decisions, selecting subjects for clinical trials, and using methods to quantify taurine oxidation at specific amino acid residues. Includes methods for assessing the clinical efficacy of a particular therapeutic intervention. Other aspects and iterations of the invention will be described in more detail below.

I. Definitions Certain terms are defined first so that the present invention can be understood more easily. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which an embodiment of the invention belongs. Many modified or equivalent methods and materials, similar to those described herein, can be used to practice embodiments of the invention without undue experimentation, but are preferred. Materials and methods are described herein. In the description and claims of the present invention, the following terms are used according to the definitions given below.

The term "about", as used herein, refers to possible quantitative variations with respect to any quantifiable variable, such as those that can occur through typical measurement techniques and instruments, such as mass, as a variable. Volume, time, distance, and quantity include, but are not limited to. Moreover, given the solid and liquid handling procedures used in the real world, there is no particular intention that may be due to differences in the manufacture, raw materials, or component purity used in the preparation of the composition or the implementation of the method, etc. There are errors and variations. The term "about" also includes these variations, which can be up to ± 5%, but can also be ± 4%, 3%, 2%, 1%, etc. be. The claims, whether modified by the term "about" or not, include the amounts of equivalents described.

The antibody, as used herein, is a complete antibody as understood in the art, ie consisting of two heavy chains and two light chains, optionally having an antigen binding region. It can also be an antibody-like molecule of, and examples of the antibody-like molecule include antibody fragments such as Fab', Fab, F (ab') 2, single domain antibody, Fv, and single-stranded Fv. , Not limited to these. The term antibody also refers to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and humanized antibodies. Techniques for the preparation and use of various antibody system constructs and fragments are well known in the art. Means of antibody preparation and characterization are also well known in the art (see, eg, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; which is incorporated herein by reference in its entirety).

As used herein, the term "aptamer" refers to a polynucleotide having useful biological activity with respect to biochemical activity, molecular recognition, or binding properties, and is generally RNA or DNA. Usually, aptamers have molecular activity such as binding to a target molecule at a specific epitope (region). It is generally accepted that aptamers that are specific for binding to a polypeptide may be those synthesized and / or those identified by in vitro evolution. Means of aptamer preparation and characterization are well known in the art, including those by in vitro evolution. See, for example, US7,939,313, which is incorporated herein by reference in its entirety.

The term "Aβ" refers to a peptide derived from the carboxy-terminal region of a giant protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many forms of Aβ that may have toxic effects: Aβ peptides are typically 37-43 amino acid sequence lengths, but may have undergone truncations and modifications that alter their overall length. There is. Aβ peptides can be found intracellularly or extracellularly in the soluble and insoluble compartments in the monomeric, oligomeric, or aggregated forms and can complex with other proteins or molecules. The harmful or toxic effects of Aβ can be due to all of the above forms, as well as others not specifically described. For example, two such Aβ isoforms include Aβ40 and Aβ42; Aβ42 isoforms are particularly fibrotic or insoluble and are associated with disease state. The term "Aβ" typically refers to multiple Aβ species and does not distinguish between individual Aβ species. Specific Aβ species are identified by the size of the peptide, such as Aβ42, Aβ40, Aβ38 and the like.

As used herein, the term "Aβ42 / Aβ40 value" means the ratio of the Aβ42 concentration in the sample to the Aβ40 concentration in the sample obtained from the subject.

"Aβ amyloidosis" is clinically defined as evidence of Aβ deposition in the brain. Subjects clinically confirmed to have Aβ amyloidosis are referred to herein as "amyloid positive", while subjects clinically confirmed not to have β amyloidosis are referred to as "amyloid negative". .. "Aβ amyloidosis" appears to have existed before it became detectable by current techniques. Nevertheless, there are some recognized indicators of Aβ amyloidosis in the art. At the time of this disclosure, Aβ amyloidosis is typically reduced by Amyloid imaging (eg, PiB PET, florbetapill, or other imaging methods known in the art) or in cerebrospinal fluid (CSF). Alternatively, it is identified by a decrease in the Aβ42 / 40 ratio of CSF. ^{The [11} C] PIB-PET imaging method, which has an average cortical connectivity (MCBP) score of> 0.18, has a cerebrospinal fluid (CSF) Aβ42 concentration of approximately 1 ng / by immunoprecipitation and mass spectrometry (IP / MS). Since it is ml, it is an index of Aβ amyloidosis. Aβ amyloidosis can be clinically confirmed by using these values, or other values known in the art, alone or in combination. For example, Krunk WE et al. Ann Neurol 55 (3) 2004, Fagan A M et al. Ann Neurol, 2006, 59 (3), Patterson et. al, Annuals of Neurology, 2015, 78 (3): 439-453, or Johnson et al. , J. Nuc. Med. , 2013, 54 (7): 1011-1013, each of which is incorporated herein by reference in its entirety. Subjects with Aβ amyloidosis may or may not be symptomatic, and symptomatic subjects may or may not meet clinical criteria for diseases associated with Aβ amyloidosis. Examples of, but not limited to, symptoms associated with Aβ amyloidosis include cognitive dysfunction, behavioral changes, speech dysfunction, emotional dysregulation, seizures, dementia, and impaired nervous system structure or function. Diseases associated with Aβ amyloidosis include, but are not limited to, Alzheimer's disease (AD), cerebral amyloid angiopathy, Lewy body dementias, and inclusion body myositis. Subjects with Aβ amyloidosis are at increased risk of developing diseases associated with Aβ amyloidosis.

"Clinical signs of Aβ amyloidosis" refers to a measure of Aβ deposition known in the art. Clinical signs of Aβ amyloidosis include amyloid imaging (eg, PiB PET, florbetapill, or other imaging methods known in the art), or by a decrease in Aβ42 or Aβ42 / 40 ratio in cerebrospinal fluid (CSF). Aβ deposits identified can be mentioned, but are not limited to these. For example, Krunk WE et al. Ann Neurol 55 (3) 2004, and Fagan AM et al. See Ann Neurol 59 (3) 2006, each of which is incorporated herein by reference in its entirety. Clinical signs of Aβ amyloidosis include measurements of Aβ metabolism, specifically measurements of Aβ42 metabolism alone or comparisons with measurements of metabolism of other Aβ variants (eg, Aβ37, Aβ38, Aβ39, Aβ40, and / or total Aβ). Such measurements can be made as described in US Patent Serial Nos. 14 / 366,831, 14 / 523,148, and 14 / 747,453, respectively. The entire body is incorporated herein by reference in its entirety. Further methods are available from Albert et al. Alzheimer's & Dementia 2007 Vol. 7, pp. 170-179; McKhann et al. , Alzheimer's & Dementia 2007 Vol. 7, pp. 263-269; and Sperling et al. Alzheimer's & Dementia 2007 Vol. 7, pp. 280-292, each of which is incorporated herein by reference in its entirety. Importantly, subjects with clinical signs of Aβ amyloidosis may or may not have symptoms associated with Aβ deposition. However, subjects with clinical signs of Aβ amyloidosis are at increased risk of developing diseases associated with Aβ amyloidosis.

A "candidate for amyloid imaging" refers to a subject identified by a clinician as an individual for whom amyloid imaging may be clinically justified. As an example, but not limited to, candidates for amyloid imaging are subjects with one or more clinical signs of Aβ amyloidosis, one or more Aβ plaque-related symptoms, or one or more CAA-related symptoms, or a combination thereof. In some cases. Clinicians may recommend amyloid imaging for such subjects to direct clinical care for such subjects. As an example, but not by another limitation, a candidate for amyloid imaging may be a candidate subject (either a control group or a test group) for a clinical trial of a disease associated with Aβ amyloidosis.

"Aβ plaque-related symptoms" or "CAA-related symptoms" are any that are caused by, or associated with, the formation of amyloid plaques or CAA composed of regularly arranged fibrous aggregates called amyloid fibrils, respectively. Show symptoms. Examples of Aβ plaque-related symptoms include neuronal degeneration, cognitive dysfunction, memory deficits, behavioral changes, emotional dysregulation, seizures, impaired nervous system structure or dysfunction, and increased risk of developing or exacerbating Alzheimer's disease or CAA. It can, but is not limited to these. As neuronal degeneration, structural changes in neurons (molecular changes such as intracellular accumulation of toxic proteins, protein aggregates, etc., and changes in the shape or length of axons or dendrites, changes in myelin sheath composition, loss of myelin sheaths, etc. (Including changes at the macro level, etc.), neuronal functional changes, neuronal loss of function, neuronal death, or any combination thereof. Cognitive dysfunction includes, but is not limited to, memory, attention, concentration, language, abstract thinking, creativity, executive function, planning, and difficulty in organizing. Behavioral changes can include violent behavior or language, impulsivity, decreased inhibitory, indifference, reduced initiation, personality changes, alcohol, tobacco, or substance abuse, and other addiction-related behaviors. Not limited to these. Emotional dysfunction includes, but is not limited to, depression, anxiety, mania, irritability, and emotional incontinence. Epileptic seizures include, but are not limited to, systemic tonic-clonic seizures, complex partial epilepsy, and non-epileptic, psychogenic seizures. Disorders of nervous system structure or function include, but are not limited to, hydrocephalus, parkinsonism, sleep disorders, psychosis, and impaired balance and coordination. This disorder may include movement disorders such as paresis, hemiparesis, limb paresis, ataxia, ballismus, and tremor. This disorder can also include loss of sensation or dysfunction, including the sense of smell, touch, taste, sight, and hearing. Moreover, the disorders may include autonomic nervous system disorders such as intestinal and bladder dysfunction, sexual dysfunction, dysregulation of blood pressure and body temperature. Finally, this disorder includes hormonal disorders caused by hypothalamic and pituitary dysfunction, such as growth hormones, thyroid stimulating hormones, luteinizing hormones, follicular stimulating hormones, gonadotropin-releasing hormones, prolactin, and many other hormones. Modulatory deficiency and dysregulation can be mentioned.

As used herein, the term "subject" refers to mammals, preferably humans. Mammals include, but are not limited to, humans, primates, livestock, rodents, and pets. The subject may be awaiting medical care or treatment, may be receiving medical care or treatment, or may have received medical care or treatment.

As used herein, the term "healthy control group", "normal group", or "healthy" control-derived sample is referred to by the clinician as Aβ amyloidosis based on qualitative or quantitative test results. , Or a subject or group of subjects diagnosed as not suffering from a clinical disorder associated with Aβ amyloidosis, including but not limited to Alzheimer's disease. "Normal" subjects are usually about the same age as the individual being evaluated, and such subjects include subjects of the same age as the subject and subjects with an age difference of 5 to 10 years. Not limited to.

As used herein, the term "blood sample" refers to a biological sample derived from blood, preferably peripheral (or circulating) blood. The blood sample can be whole blood, plasma, or serum, but plasma is typically preferred.

The term "isoform" as used herein refers to any of several different forms of the same protein variant, selective splicing of mRNA encoding a protein, post-translational modification of a protein, of a protein. It results from proteolytic processing, genetic mutation, and somatic recombination. The terms "isoform" and "mutant" are used synonymously.

Unless otherwise stated herein, the term "tau protein" or "tau" includes all tau isoforms, whether full length, truncation type, or post-translational modification. In many animals, including but not limited to humans, non-human primates, rodents, fish, cattle, frogs, goats, and chickens, tau is encoded by the gene MAPT. In humans, there are six tau isoforms produced by alternative splicing of MAPT exons 2, 3, and 10. These isoforms range in length from 352 to 441 amino acids. Exons 2 and 3 encode 29 amino acid inserts (referred to as N) at the N-terminus, respectively, and the full-length human tau isoform has both inserts (2N) and one insert (1N). Or it is possible to have no insert fragment. All full-length human tau isoforms also have a triple repeat sequence (referred to as R) of the microtubule binding domain. If the exon 10 is included at the C-terminus, the fourth microtubule binding domain encoded by the exon 10 will be included. Thus, a full-length human tau isoform may contain a 4-fold repetitive sequence of microtubule-binding domains (containing exons 10) or a 3-fold repetitive sequence of microtubule-binding domains (exons 10 not included). It is possible. Hitotau may or may not be post-translationally modified. For example, it is known in the art that tau can be phosphorylated, ubiquitinated, glycosylated, and saccharified. Therefore, the term "human tau" refers to (2N, 3R), (2N, 4R), (1N, 3R), (1N, 4R), (0N, 3R), and (0N, 4R) isoforms, among them. Includes isoforms that are N-and / or C-terminal truncation type species, as well as all post-translational modified isoforms. Alternative splicing of the gene encoding tau occurs in other animals as well. In animals where the gene has not been identified as MAPT, homologues may be identified by methods well known in the art.

Diseases associated with tau deposits in the brain can be referred to as "tauopathy". Known tauopathy in the arts include progressive nuclear paralysis, boxer dementia, chronic traumatic brain damage, frontotemporal lobar dementia and Parkinsonism associated with chromosome 17, Lytico-Bodig disease, and Parkinsonism in Guam. Complex, tangle-dominant dementia, ganglion cell collagen and ganglion cell tumor, meningeal hemangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, nodular sclerosis, Hallervorden-Spatz disease, lipofustin deposits, Examples include, but are not limited to, Parkinsonism, basal nucleus degeneration of the cerebral cortex, silver granule disease (AGD), frontotemporal lobar degeneration, Alzheimer's disease, and frontotemporal lobar dementia.

"Significantly deviates from the mean" means at least one standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or even more preferably at least two standard deviations. Indicates a value higher or lower than the average.

The phrase "Aβ and tau remedies" refers to subjects at risk of developing Aβ amyloidosis or AD, subjects diagnosed with Aβ amyloidosis, subjects diagnosed with tauopathy, or diagnosed with AD. A collective representation of any contrasting or therapeutic agent intended for or used in a subject.

II. Measurement of total tau and taurine oxidation in isolated tau samples The methods of the present disclosure are to prepare isolated tau samples obtained from a subject and to measure taurine oxidation at one or more amino acid residues, and Includes measuring total tau at any option.

(A) Isolated Tau Sample An isolated tau sample, when used herein, represents a composition comprising tau, which was purified from blood or cerebrospinal fluid (CSF) obtained from the subject. It is a thing. The subject is a mammal, preferably a human. CSF can be obtained by lumbar puncture with or without an indwelling CSF catheter. Multiple blood or CSF samples collected simultaneously from the subject can be pooled. Blood can be collected by venipuncture with or without an intravenous catheter, or by fingertip puncture (or their equivalent). Once collected, blood or CSF samples can be processed by methods known in the art (eg, centrifugation to remove whole cells and cell debris, stabilizing and storing pre-analytical samples. Use of additives designed for). Blood or CSF samples may be used immediately or stored frozen indefinitely.

In the isolated tau samples of the present disclosure, the tau is either partially or completely purified from blood or CSF. Methods for purifying tau from blood or CSF are known in the art, and such methods include, but are not limited to, selective precipitation, size exclusion chromatography, ion exchange chromatography, and affinity purification. .. Both phosphorylated tau and non-phosphorylated tau are concentrated from blood or CSF by an appropriate method.

In an exemplary embodiment, the isolated tau sample of the present disclosure comprises a tau purified from blood or CSF by affinity purification. Affinity purification describes a method of purifying a protein of interest due to the specific binding properties of the protein of interest to a fixed ligand. Typically, the fixed ligand is a ligand attached to a solid support, such as beads, resin, tissue culture plates, and the like. Suitable ligands specifically bind to both phosphorylated and non-phosphorylated tau. In one example, a suitable ligand can bind to an epitope within the middomain of tau. In another example, a suitable ligand can bind to an epitope within the N-terminus of tau, preferably amino acids 1-35 of tau. In another example, a suitable ligand can bind to an epitope within the tau MTBR. In another example, a suitable ligand can bind to an epitope within the C-terminus of tau. In a further embodiment, the tau can be affinity purified from blood or CSF using two or more fixed ligands. In one example, one fixed ligand binds to an epitope within the N-terminus of tau and another fixed ligand binds to an epitope within the middomain of tau. In another example, one fixed ligand binds to an epitope within the tau's MTBR and another fixed ligand binds to an epitope within the tau's middomain. In another example, one fixed ligand binds to an epitope within the C-terminus of tau and another fixed ligand binds to an epitope within the middomain of tau. In another example, one fixed ligand binds to an epitope within the C-terminus of tau and another fixed ligand binds to an epitope within the N-terminus of tau. In another example, one fixed ligand binds to an epitope within the tau MTBR and another fixed ligand binds to an epitope within the N-terminus of tau. In another example, one fixed ligand binds to an epitope within the tau MTBR and another fixed ligand binds to an epitope within the C-terminus of tau. In each of the above embodiments, the ligand may be an antibody or an aptamer. An example, rather than a limitation of suitable antibodies, is shown in FIG.

Isolated tau samples may be used immediately or stored indefinitely by methods known in the art.

(B) Tau Phosphorylation at One or More Amino Acid Residues Phosphorylation at a particular amino acid (ie, the "site") in tau results in a phosphorylated tau (p-tau) isoform. The methods of the present disclosure provide a means for stoichiometrically measuring phosphorylation of tau at one or more specific amino acids. In some embodiments, the methods herein are at one or more residues selected from T111, S113, T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231. Includes measuring taurine oxidation. In some embodiments, the methods herein include measuring taurine oxidation at one or more residues selected from T111, T181, T205, S208, S214, T217, and T231. In other embodiments, the methods herein include measuring taurine oxidation at one or more residues selected from T181, S214, and T217. In other embodiments, the methods herein include measuring taurine oxidation at one or more residues selected from T181, T205, and T217. In other embodiments, the methods herein include measuring taurine oxidation at one or more residues, including S199. In other embodiments, the methods herein include measuring taurine oxidation at one or more residues, including S202. In other embodiments, the methods herein include measuring taurine oxidation at one or more residues, including S199. In other embodiments, the methods herein include measuring taurine oxidation at one or more residues, including T181. In other embodiments, the methods herein include measuring taurine oxidation at one or more residues, including T205. In other embodiments, the methods herein include measuring taurine oxidation at one or more residues, including T217. In other embodiments, the methods herein include measuring taurine oxidation at two or more residues, including T153 and T175. In other embodiments, the methods herein include measuring taurine oxidation at two or more residues selected from T181, T205, and T217. In other embodiments, the methods herein include measuring taurine oxidation at three or more residues, including T181, T205, and T217.

Applicants have developed a sensitive and specific mass spectrometry (MS) method using parallel reaction monitoring (PRM) to discover taurine sites, first in isolated tau proteins. The abundance of was quantified. However, the present disclosure is not limited to any one of the specific methods for quantitatively assessing site-specific phosphorylation of tau. Appropriate methods identify different tau isoforms only in the phosphorylated state of a single amino acid, identify p-tau isoforms phosphorylated with different amino acids, and identify them independently of the overall change in total tau. It must be possible to quantify the changes in phosphorylation that occur at the site. Three approaches to quantify the pharmacological changes in phosphorylation that occur at specific sites, independent of the overall change in total tau, will be described in detail by example: 1) Relative comparison between phosphorylated peptide isomers. , This can be used to estimate the relative abundance of each phosphorylated peptide that shares the same sequence; 2) normalize the phosphorylated peptide using any peptide derived from tau protein as a reference; and 3 ) Absolute quantification of each phosphorylated peptide and non-phosphorylated peptide using synthetic labeling internal criteria, with any absolute quantification value obtained for any peptide derived from tau protein. Normalize. All three approaches utilize internal normalization to compare relative phosphorylation changes at each site. Other methods known in the art are also available.

In an exemplary embodiment, tau site-specific phosphorylation is measured by high resolution mass spectrometry. Suitable types of mass spectrometers are known in the art. Such mass spectrometers include quadrupole, time-of-flight, ion trap and Orbitrap, and hybrid mass spectrometers that combine different types of mass spectrometers into a single structure (eg, Orbitrap from Thermo Fisher Scientific). Fusion (registered trademark) Tribrid (registered trademark) mass spectrometer), but is not limited thereto. Further processing of the isolated tau sample may be performed prior to MS analysis. For example, tau may be used for proteolytic digestion. Suitable proteases include, but are not limited to, trypsin, Lys-N, Lys-C, and Arg-N. When producing isolated tau samples using affinity purification, digestion may occur after the tau is eluted from the fixed ligand or while the tau is bound. After one or more cleanup steps, the digested tau peptide can be separated by a liquid chromatography system linked with a high resolution mass spectrometer. The chromatography system can be optimized by routine experiments to purify the desired LC-MS pattern. A variety of LC-MS techniques can be used to quantitatively analyze site-specific taurine oxidation. Examples, but not limited, include selective reaction monitoring, parallel reaction monitoring, selective ion monitoring, and data-independent analysis. As mentioned above, all quantitative assessments of site-specific taurine oxidation should explain the overall change in total taurine. In an exemplary embodiment, the mass spectrometry protocol outlined in the examples is used.

(C) Total Tau Amount "Tau Total", as used herein, refers to the total tau isoform in a given sample. Tau can be found intracellularly or extracellularly in a soluble and insoluble compartment, in a monomeric or aggregated form, in an ordered or disordered structure, and complex with other proteins or molecules. In some cases. Therefore, the raw material of the biological sample (eg, brain tissue, CSF, blood, etc.) and any downstream processing of the biological sample will affect the total amount of tauisoform in the given sample.

The total amount of tau can be measured by monitoring the abundance of unmodified tau peptide. For each phosphorylated tau site, the total tau level can be measured by preferentially using a tau peptide that shares an amino acid sequence in common with the phosphorylated peptide of interest, but any peptide derived from the tau sequence can be used. It is possible. Tau peptide measurements can be performed by mass spectrometry and the accuracy of the measurements can be improved by using labeled internal standards as a reference. Alternatively, total tau volume can be measured by immunoassay or other tau concentration quantification method.

III. A method of diagnosing a subject before the onset of MCI due to AD and diagnosing the stage of AD of the subject One aspect of the present disclosure is a method of diagnosing a subject at high risk of conversion to mild cognitive impairment due to Alzheimer's disease. It optionally includes methods of diagnosing or classifying the stage of interest with respect to the number of years to onset of MCI due to AD. Mild cognitive impairment (MCI) due to Alzheimer's disease (AD) represents the symptomatic pre-dementia stage of AD. This degree of cognitive impairment is not normal at that age and therefore does not meet constituent requirements such as age-related memory impairment and age-related cognitive decline. MCI by AD is a clinical diagnosis, and clinical criteria for diagnosing MCI by AD are known in the art. For example, Albert et al. See Alzheimer's & Dementia, 2011, 7 (3): 270-279. A cognitive function test is optimal for objectively assessing the degree of cognitive impairment in a subject. Cognitive function test scores for subjects with MCI are typically in culturally relevant normative data (ie, with respect to the disabled domain (s), if available) to the subject's age and education. 1-1.5 standard deviations lower than the mean for matching counterparts. MCI indications are often supported by a general rating of 0.5 on the Clinical Dementia Rating (CDR) scale. CDR is a numerical measure used to quantify the severity of symptoms of dementia. Other suitable cognitive function tests are also known in the art. Although there are appropriate tests to assess the severity of cognitive impairment, there is a need for tests in the field to identify subjects who develop MCI due to AD over a highly reliable number of years.

In one embodiment, the method of diagnosing a subject as having a high risk of conversion to MCI by AD is as follows: (a) Prepare an isolated tau sample obtained from the subject, and T111, S113 in the isolated tau sample. , T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231 to measure taurination at one or more amino acid residues selected from, and optionally to measure total tau. And (b) the measured phosphorylation levels (s) are significantly higher than the mean in the control population free of cerebral amyloid plaques as measured by PET imaging and / or by Aβ42 / 40 measurements in CSF. If deviating, it may include diagnosing the subject as having a high risk of conversion to MCI due to AD. In another embodiment, a method of diagnosing a subject at high risk of conversion to MCI by AD is as follows: (a) Prepare first and second isolated tau samples obtained from the subject and each isolated tau sample. Measuring taulin oxidation at one or more amino acid residues selected from T111, S113, T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231, and optionally. To measure total tau by selection; (b) to calculate site-specific changes in phosphorylation at each measured residue, and (c) to calculate changes in total tau by optional; and (c) calculated changes ( Subject to MCI by AD if (s) significantly deviate from the mean in the control population free of brain amyloid plaques as measured by PET imaging and / or by Aβ42 / 40 measurements in CSF. May include diagnosing a high conversion risk. "Significantly deviates from the mean" means at least one standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or even more preferably at least two standard deviations from the mean. Shows greater or lesser values by deviation (ie, 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is measured by PET imaging and / or Aβ42 / 40 measurements in CSF. The standard deviation defined by the normal distribution measured in the control population without brain amyloid plaques). In addition to using a threshold (eg, a value greater than or less than the mean by at least one standard deviation), some embodiments may use a degree of change above or below the mean to diagnose the subject. Isolated tau samples can be obtained from subjects that may or may not be asymptomatic. "Asymptomatic subject" refers to a subject who shows no signs or symptoms of AD. However, the subject may present with signs or symptoms of AD (eg, memory loss, misplacement, mood or behavioral changes, etc.) but have sufficient cognitive or dysfunction for clinical diagnosis of mild cognitive impairment. Not shown. In a further embodiment, the subject may carry one of the genetic mutations known to cause dominantly inherited Alzheimer's disease. In alternative embodiments, the subject may not carry one of the genetic mutations known to cause dominantly inherited Alzheimer's disease. Alzheimer's disease that does not have a specific familial relationship is referred to as sporadic Alzheimer's disease.

Another aspect of the disclosure includes a method of diagnosing a stage of Alzheimer's disease in a subject. In various embodiments, the "stage of AD" is the length of time that has elapsed since the onset of MCI due to AD (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, ... It can be defined as 12 months, etc.). Although clinical diagnostic criteria for AD exist, it is common that the clinical setting for the time of onset of symptoms for a given subject is unclear, or the diagnosis remains questionable as to whether it is MCI or AD. Therefore, in this field, a test for objectively diagnosing the stage of AD of a subject is required.

In one embodiment, the method of diagnosing the stage of AD in a subject is as follows: (a) Prepare an isolated tau sample obtained from the subject and in the isolated tau sample, T111, S113, T181, S199, S202, Measuring tau phosphorylation at one or more amino acid residues selected from S208, T153, T175, T205, S214, T217, and T231, and optionally measuring total tau; and (b). When the measured phosphorylation level (s) deviates significantly from the mean in the control population free of cerebral amyloid plaques as measured by PET imaging and / or by Aβ42 / 40 measurements in CSF. It may include diagnosing the stage of AD in the subject. In another embodiment, the method of diagnosing a subject before the onset of MCI by AD is as follows: (a) Prepare first and second isolated tau samples obtained from the subject, and T111 in each isolated tau sample. , S113, T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231 to measure tauphosphorylation at one or more amino acid residues selected from, and optionally total tau. (B) Calculating site-specific changes in phosphorylation at each measured residue, and optionally changes in total tau; and (c) Calculated changes (s) By the onset of MCI by AD, the subject is at the onset of MCI due to AD, when measured by PET imaging and / or when Aβ42 / 40 measurements in CSF deviate significantly from the mean in the control population free of cerebral amyloid plaques. It may include diagnosing the existence of a particular number of years. "Significantly deviates from the mean" means at least one standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or even more preferably at least two standard deviations from the mean. Shows greater or lesser values by deviation (ie, 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is measured by PET imaging and / or Aβ42 / 40 measurements in CSF. The standard deviation defined by the normal distribution measured in the control population without brain amyloid plaques). In addition to using a threshold (eg, a value greater than or less than the mean by at least one standard deviation), some embodiments may use a degree of change above or below the mean to diagnose the subject. Isolated tau samples can be obtained from subjects who may or may not have been diagnosed with MCI due to AD, dementia, or AD. In a further embodiment, the subject may carry one of the genetic mutations known to cause dominantly inherited Alzheimer's disease. In alternative embodiments, the subject may not carry one of the genetic mutations known to cause dominantly inherited Alzheimer's disease.

Phosphorylation levels (s) measured using site-specific tau phosphorylation measurements and optionally in combination with tau total tau total measurements, or in addition to this, in any of the above embodiments. The ratio calculated from, or the ratio calculated from the measured phosphorylation level (s) and total tau, may be used. Both approaches are described in detail with examples. Mathematical operations other than ratios may also be used. For example, in the example, site-specific taurine oxidation values are used in various statistical models (eg, linear regression, LME curve, LOESS curve, etc.) and other known biomarkers (eg APOEε4 status, age, gender, cognitive test score). , Functional test score, etc.). The specificity of the method can be maximized by optimizing the selection of measurements and the selection of mathematical operations. For example, the diagnostic accuracy can be evaluated by the area under the curve of the ROC curve, and in some embodiments, an AUC value of ROC of 0.7 or more is set as a threshold value (for example, 0.7, 0.75, etc.). 0.8, 0.85, 0.9, 0.95, etc.).

Human brain amyloid plaques are measured by amyloid-positron emission tomography (PET) as a routine method. For example, ¹¹ C-Pittsburgh compound B (PiB) PET imaging of cortical Aβ plaques is commonly used to detect Aβ plaque pathology. The standardized uptake value ratio (SUVR) of cortical PiB-PET reliably identifies significant cortical Aβ plaques and makes the subject PIB positive (SUVR ≥ 1.25) or negative (SUVR <1.25). Used to classify. Thus, in the above embodiments, the control population free of cerebral amyloid plaques as measured by PET imaging can indicate a control population with a cortical PiB-PET SUVR of less than 1.25. Analysis of other values of PiB binding (eg, mean cortical binding capacity) or regions of interest other than cortical regions can also be used to classify subjects as PIB positive or negative. Other PET contrast agents can also be used.

The control population without cerebral amyloid plaques as measured by Aβ42 / 40 measurements in CSF had 0. A population of controls that is less than 12 can be shown.

In an exemplary embodiment, the method of diagnosing a subject at high risk of conversion to MCI by AD, or diagnosing the AD stage of a subject, is (a) preparing an isolated tau sample obtained from the subject and isolating it. Measuring tauphosphorylation at one or more amino acid residues selected from T181, T205, and T217 in a tau sample, and optionally measuring total tau; and (b) measured. Subjects are AD if the phosphorylation level (s) deviates significantly from the mean in the control population free of cerebral amyloid plaque as measured by PET imaging and / or by Aβ42 / 40 measurements in CSF. It may include diagnosing a high risk of conversion to MCI by, diagnosing that there is a certain number of years before the onset of MCI by AD, or diagnosing the AD stage of the subject. FIG. 22 shows the dynamic patterns of taurine oxidation measurable at T181, T205, and T217 in isolated tau samples with respect to the number of years to onset of MCI by AD. Phosphorylation levels at T217 that deviated significantly from the mean first occurred approximately 21 years before the onset of MCI due to AD; phosphorylation at T181 that deviated significantly from the mean first occurred. Approximately 19 years before the onset of MCI due to AD; the first increase in total tau that deviates significantly from the average occurs approximately 17 years before the onset of MCI due to AD; and significantly deviates from the average. Phosphorylation levels at T205 first occur approximately 13 years before the onset of MCI due to AD. Upon onset of symptoms (eg, MCI by AD), phosphorylation levels at T217 and T181 are plateaus and then decrease.

As mentioned above, additional mathematical operations may be performed on the phosphorylation measurements at T181, T205, and / or T217, such as between the measured phosphorylation levels (s). The ratio and the ratio between the measured phosphorylation level (s) and the total amount of tau include, but are not limited to. The ratio calculated from the measured phosphorylation level (s) may be the ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217. .. The ratio calculated from the measured phosphorylation level (s) and total tau may be the ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. ..

In one example, the methods of the present disclosure prepare (a) isolated tau samples obtained from a subject, (i) T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and. Measuring taurine oxidation at T217; and (b) if taurine oxidation at T217 and / or T181 is greater than or equal to about 1.5σ and taurine oxidation at T205 is greater than or equal to about 1.5σ. Includes diagnosing that the onset of MCI by AD is about 10 to about 25 years, or about 10 to about 20 years, except that σ is measured by PET imaging and / or in CSF. A standard deviation defined by the normal distribution of taurine oxidation at T217 and T205, T181 and T205, or T181, T205, and T217 measured in a control population free of brain amyloid plaques by Aβ42 / 40 measurements. In various embodiments, taurine oxidation at T217 and / or taurine oxidation at T181 is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ. , About 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, taurine oxidation at T217 and / or taurine oxidation at T181 is about 1.85σ, about 1.9σ, about 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about. It may be 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In each of the above embodiments, taurine oxidation at T205 is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.51σ, about 1.55σ, about. It may be less than 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2.0σ, or 2.0σ. Alternatively, taurine oxidation at T205 is less than about 2.0σ, about 2.05σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or 2.5σ. In some cases. In a further example, taurine oxidation at T217 and / or taurine oxidation at T181 may be about 2σ or more, and taurine oxidation at T205 may be about 2σ or less. In addition to the use of thresholds (eg, at least one standard deviation above or below the mean), some embodiments may use a degree of change greater than or less than the mean to diagnose the subject. In a further embodiment, the measured levels of taurine oxidation at T205 and taurine oxidation at T181 and / or T217 can be used in various mathematical operations to improve predictive power compared to themselves. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In another example, the methods of the present disclosure prepare (a) isolated tau samples obtained from a subject and the total amount of tau and (i) T217 and T205, (ii) T181 and T205, or (iii) T181, Measuring taulin oxidation at T205 and T217; and (b) the ratio of taulin oxidation at T217 to total tau and / or the ratio of taulin oxidation at T181 to total tau is approximately 1.5σ or greater. If the ratio of taulin oxidation at T205 to total tau is about 1.5σ or less, the subject diagnoses that it is about 10 to about 25 years or about 10 to about 20 years before the onset of MCI due to AD. However, σ is the total amount of tau measured by PET imaging and / or in the control population free of cerebral amyloid plaques by Aβ42 / 40 measurements in CSF and T217 and T205, T181 and T205, Or the standard deviation defined by the normal distribution of taurin oxidation at T181, T205, and T217. In various embodiments, the ratio of taurine oxidation at T217 to total tau and / or the ratio of taurine oxidation at T181 to total tau is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ. , About 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, the ratio of taurine oxidation at T217 to total tau and / or the ratio of taurine oxidation at T181 to total tau is about 1.85σ, about 1.9σ, about 1.95σ, about 2σ, about. It may be 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In each of the above embodiments, the ratio of taurine oxidation at T205 to total tau is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.50σ, about 1.55σ, about. It may be less than 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2.0σ, or 2σ. Alternatively, the ratio of taurine oxidation at T205 to total tau is about 2.0σ, about 2.05σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or It may be less than 2.5σ. In a further example, the ratio of taurine oxidation at T217 to total tau and / or the ratio of taurine oxidation at T181 to total tau may be greater than or equal to about 2σ, and the ratio of taurine oxidation at T205 to total tau is , May be less than or equal to about 2σ. In addition to the use of thresholds (eg, at least one standard deviation above or below the mean), some embodiments may use a degree of change greater than or less than the mean to diagnose the subject.

In another example, the methods of the present disclosure prepare (a) isolated tau samples obtained from a subject, (i) T181 and T205, (ii) T217 and T205, or (iii) T181, T217, and. Measuring taurine oxidation at T205; as well as taurine oxidation at specific sites listed in (b) (a) (i), (a) (ii), or (a) (iii) is about 1.5σ In the above cases, the subject includes diagnosing that the onset of MCI due to AD is about 15 years or less, or about 10 years or less, except that σ is measured by PET imaging and / or. A standard deviation defined by the normal distribution of taurine oxidation at T217 and T205, T181 and T205, or T181, T205, and T217 measured in a control population free of cerebral amyloid plaques by Aβ42 / 40 measurements in CSF. .. In various embodiments, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is about 1.3σ, about 1.35σ, about. It may be 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is about 1.85σ, about 1.9σ, about. It may be 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In a further example, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) may be about 2σ or more. In addition to the use of thresholds (eg, at least one standard deviation above or below the mean), some embodiments may use a degree of change greater than or less than the mean to diagnose the subject. In a further embodiment, the measured levels of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) are used in various mathematical operations. , Each can improve the predictive power compared to itself. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In another example, the methods of the present disclosure prepare an isolated tau sample obtained from a subject and (i) total tau and (i) T181 and T205, (ii) T217 and T205, or (iii) T181, Measuring taulin oxidation at T217, and T205; and (b) taurine at specific sites listed in (a) (i), (a) (ii), or (a) (iii) relative to total tau. When the ratio of oxidation is about 1.5σ or more, the subject includes diagnosing that the onset of MCI by AD is about 15 years or less, or about 10 years or less, where σ is the PET image. Total tau as measured by method and / or measured in a control population free of cerebral amyloid plaques by Aβ42 / 40 measurements in CSF and tau phosphorylation at T217 and T205, T181 and T205, or T181, T205, and T217. The standard deviation defined by the normal distribution of. In various embodiments, the ratio of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) to total tau is about 1.3σ, about. It may be 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. .. In other embodiments, the ratio of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) to total tau is about 1.85σ, about. It may be 1.9σ, about 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In a further example, the ratio of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) to total tau may be greater than or equal to about 2σ. .. In addition to the use of thresholds (eg, at least one standard deviation above or below the mean), some embodiments may use a degree of change greater than or less than the mean to diagnose the subject.

In another example, the methods of the present disclosure prepare (a) first and second isolated tau samples obtained from a subject, where "first" and "second" are the order in which the samples were collected. To measure tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181, T217, and T205; (b) site-specific at each measured residue. Calculating changes in phosphorylation, and optionally changes in total tau; and (c) phosphorylation levels at T181 and / or T217 remain reduced or constant, and phosphorylation levels at T205 and optionally. Includes diagnosing the stage of AD in a subject when total tau is elevated. First and second isolated tau samples may be collected across dates, weeks, or months. Typically, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is also greater than or equal to about 1.5σ for both samples, however. , Σ at T217 and T205, T181 and T205, or T181, T205, and T217 measured by PET imaging and / or in control populations free of cerebral amyloid plaques by Aβ42 / 40 measurements in CSF. The standard deviation defined by the normal distribution of taurine oxidation in. In a further embodiment, the measured levels of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) are used in various mathematical operations. , Each can improve the predictive power compared to itself. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In another example, the methods of the present disclosure (a) prepare first and second isolated tau samples obtained from a subject, where "first" and "second" indicate the order in which the samples were collected. Show and measure tau total and (i) tau phosphorylation at T181 and T205, (ii) T217 and T205, or (iii) T181, T217, and T205; (b) site at each measured residue. Calculating changes in specific phosphorylation and changes in total tau; and (c) phosphorylation levels at T181 and / or T217 remain low or constant, and phosphorylation levels at T205 remain low or constant. , And diagnosing the stage of AD in the subject when the total amount of tau is increasing. First and second isolated tau samples may be collected across dates, weeks, or months. Typically, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is also greater than or equal to about 1.5σ for both samples, however. , Σ at T217 and T205, T181 and T205, or T181, T205, and T217 measured by PET imaging and / or in control populations free of cerebral amyloid plaques by Aβ42 / 40 measurements in CSF. The standard deviation defined by the normal distribution of taurine oxidation in. In a further embodiment, the measured levels of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) are used in various mathematical operations. , Each can improve the predictive power compared to itself. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

Methods for measuring taurine oxidation and total taurine are described in Chapter II and are incorporated herein by reference. For example, using the protocol detailed in Examples 5-9, taurine oxidation at T181, T205, and T217 was measured by PET imaging when measured with isolated tau samples purified from CSF. In the control population without cerebral amyloid plaque, the p-tau / tau ratio percentages were 21.7 ± 2.3, 0.34 ± 0.13, and 1.2 ± 0.66, respectively (21.7 ± 2.3, 0.34 ± 0.13, and 1.2 ± 0.66, respectively). See Table 2, Non-mutation carrier column). Therefore, twice the standard deviation (ie, 2σ) above the mean seen in non-mutant carriers is 43.4, 0 for p-T181 / T181, p-T205 / T205, and p-T217 / T217, respectively. .68 and 2.4. However, as those skilled in the art will know, absolute values can vary depending on the protocol and the raw materials / specifications of the internal standards used for absolute quantification.

In a preferred embodiment, the isolated tau sample comprises tau purified from blood or CSF by affinity purification, and taurine oxidation is measured by mass spectrometry. In another preferred embodiment, the isolated tau sample comprises a ligand that specifically binds to an epitope within the middomain of the tau, and optionally a secondary ligand that specifically binds to an epitope within the N-terminus of the tau. Including tau purified from blood or CSF by affinity purification, tauphosphory oxidation is measured by high resolution mass analysis. In another preferred embodiment, the isolated tau sample is a ligand that specifically binds to an epitope within the middomain of tau, and optionally a second that specifically binds to an epitope within the MTBR or C-terminus of tau. It contains tau purified from blood or CSF by affinity purification using a ligand, and tau phosphorylation is measured by high resolution mass analysis. In an exemplary embodiment, the mass spectrometry protocol outlined in the Examples is used.

IV. Therapeutic Methods Another aspect of the disclosure is a method for treating a subject in need of treatment. The terms "treat,""treating," or "treatment," as used herein, are trained or trained for subjects in need of medical care. Demonstrate the provision of medical care by qualified professionals. Medical care may be diagnostic tests, therapeutic treatments, and / or preventive or prophylactic measures. The purpose of therapeutic and prophylactic treatment is to prevent or slow down (reduce) unwanted physiological changes or diseases / disorders. Beneficial or desirable clinical outcomes of therapeutic and prophylactic treatment include alleviation of symptoms, reduction of the extent of the disease, stabilization of the condition of the disease (ie, does not worsen), slowing or slowing the progression of the disease, remission of the condition. Alternatively, mitigation and remission (whether in part or in whole) can be mentioned, but are not limited to these, and they may or may not be detectable. “Treatment” can also mean a longer survival rate when compared to what would be expected without treatment. Those in need of treatment include those who already have the disease, symptoms, or disability, and those who are prone to have the disease, symptoms, or disability, or who should prevent the disease, symptoms, or disability. .. In some embodiments, the subject to be treated is asymptomatic. "Asymptomatic subject" as used herein refers to a subject who shows no signs or symptoms of AD. In other embodiments, the subject may present with signs or symptoms of AD (eg, memory loss, misplacement, mood or behavioral changes, etc.) but are clinically diagnosed with mild cognitive impairment due to Alzheimer's disease. Does not show sufficient cognitive or dysfunction. The phrase "mild cognitive impairment due to Alzheimer's disease" is defined in Chapter IV. Both symptomatic and asymptomatic subjects may have Aβ amyloidosis; however, prior findings of Aβ amyloidosis are not a prerequisite for treatment. In a further embodiment, the subject may be diagnosed with AD. In any of the above embodiments, the subject may carry one of the genetic mutations known to cause dominantly inherited Alzheimer's disease. In alternative embodiments, the subject may not carry one of the genetic mutations known to cause dominantly inherited Alzheimer's disease.

In one embodiment, the method for treating a subject as described above is as follows: (a) Prepare an isolated tau sample obtained from the subject and in the isolated tau sample, T111, S113, T181, S199, S202. , S208, T153, T175, T205, S214, T217, and T231 to measure tau phosphorylation at one or more amino acid residues, and optionally to measure total tau; and (b). ) When the measured phosphorylation level (s) significantly deviates from the mean in the control population free of cerebral amyloid plaques as measured by PET imaging and / or by Aβ42 / 40 measurements in CSF. , May include administering the pharmaceutical composition to the subject. In another embodiment, the method for treating the subject as described above is as follows: (a) Prepare first and second isolated tau samples obtained from the subject, and T111, S113 in each isolated tau sample. , T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231 to measure tau phosphorylation at one or more amino acid residues, and optionally to measure total tau. To do; (b) calculate the change in site-specific phosphorylation at each measured residue, and optionally the change in total tau, and (c) the calculated change (s) are PET. Including administration of a pharmaceutical composition to a subject when measured by imaging and / or when Aβ42 / 40 measurements in CSF deviate significantly from the mean in a control population free of brain amyloid plaques. There is. "Significantly deviates from the mean" means at least one standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or even more preferably at least two standard deviations from the mean. Shows greater or lesser values by deviation (ie, 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is measured by PET imaging and / or Aβ42 / 40 measurements in CSF. The standard deviation defined by the normal distribution measured in the control population without brain amyloid plaques). In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean to diagnose the subject.

Ratio calculated from the measured phosphorylation levels (s) in any of the above embodiments, in lieu of or in addition to the use of site-specific tau phosphorylation measurements combined with optional tau total measurement. , Or the ratio calculated from the measured phosphorylation level (s) and total tau may be used. The ratio calculated from the measured phosphorylation level (s) may be the ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217. .. The ratio calculated from the measured phosphorylation level (s) and total tau may be the ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. .. Mathematical operations other than ratios may also be used. For example, in the example, site-specific taurine oxidation values are used in various statistical models (eg, linear regression, LME curve, LOESS curve, etc.) and other known biomarkers (eg, APOEε4 status, age, gender, cognitive test). Used in conjunction with scores, functional test scores, etc.).

Intended or intended for subjects at risk of developing Aβ amyloidosis or AD, subjects diagnosed with Aβ amyloidosis, subjects diagnosed with tauopathy, or subjects diagnosed with AD. Many contrasting agents or therapeutic agents used in such subjects target specific pathophysiological changes. For example, Aβ-targeted therapies are generally designed to reduce Aβ production, antagonize Aβ aggregation, or increase brain Aβ clearance; tau-targeted therapies generally modify tau phosphorylation patterns. Designed to antagonize tau aggregation or increase NFT clearance; various therapeutic agents are designed to reduce CNS inflammation or cerebral insulin resistance; etc. The effectiveness of these various agents is T111, S113, T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231 as measured by the methods disclosed herein. It may be ameliorated by administering the agent to subjects with specific taurine oxidation levels.

In an exemplary embodiment, for a subject at risk of developing Aβ amyloidosis or AD, a subject diagnosed with Aβ amyloidosis, a subject diagnosed with tauopathy, or a subject diagnosed with AD. The efficacy of contrast agents and therapeutic agents (collectively referred to herein as "Aβ and tau therapeutic agents") intended for or used in such subjects is disclosed herein and. For example, when measured by the method shown in FIG. 22, administration of Aβ and a tau therapeutic agent to a subject having a specific tauphosphory oxidation level of T181, T205, and / or T217 may improve. .. For example, when taurine oxidation at T217 is about 1.5σ or more and taurine oxidation at T181 and T205 is about 1.5σ or less, a suitable therapeutic agent is to prevent the subject from becoming amyloid positive. (For example, an amyloid-targeted therapeutic agent designed to antagonize Aβ aggregation, which reduces Aβ production). As another example, when taurine oxidation at T217 and / or T181 is greater than or equal to about 1.5σ and taurine oxidation at T205 is greater than or equal to about 1.5σ, amyloid deposition is increased as a suitable therapeutic agent. Can be mentioned as being designed to prevent or reduce the load on the subject's existing plaque. As another example, when taurine oxidation at T217, T181, and T205 is greater than or equal to about 1.5σ, the preferred therapeutic agent is to prevent increased amyloid deposits, reduce the pre-existing plaque load of the subject, tau. Examples include those designed to prevent agglomeration or target NFTs. As another example, taurine oxidation at T217, T181, and T205 is greater than or equal to about 1.5σ, and taurine oxidation at T217 or T181 is plateau or diminished, and at total tau and / or T205. Designed to prevent increased amyloid deposits, reduce the subject's existing plaque load, prevent tau aggregation, or target NFTs as suitable therapeutic agents when taurine oxidation is increased. Those, as well as those dedicated to subjects with AD can be mentioned. The details disclosed herein can also be used to administer therapeutic agents designed for other targets (eg, CNS inflammation, ApoE, etc.) as such targets: Examples include, but are not limited to, those identified in the paragraph.

In one example, the disclosure provides a method for treating a subject at high risk of conversion to MCI by AD, which method (a) prepares an isolated taurine sample obtained from the subject. i) Measuring taurine oxidation at T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and T217; and (b) taurine oxidation at T217 and / or T181 is about 1.5σ This includes administering the pharmaceutical composition to the subject when taurine oxidation at T205 is about 1.5σ or less, where σ is measured by PET imaging and / or CSF. A standard deviation defined by the normal distribution of taurine oxidation at T217 and T205, T181 and T205, or T181, T205, and T217 measured in a control population free of brain amyloid plaques by Aβ42 / 40 measurements in. In various embodiments, taurine oxidation at T217 and / or taurine oxidation at T181 is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ. , About 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, taurine oxidation at T217 and / or taurine oxidation at T181 is about 1.85σ, about 1.9σ, about 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about. It may be 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In each of the above embodiments, the taurine oxidation at T205 is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.51σ, about 1.55σ, about. It may be less than 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2.0σ, or 2.0σ. Alternatively, taurine oxidation at T205 is less than about 2.0σ, about 2.05σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or 2.5σ. There may be. In a further example, taurine oxidation at T217 and / or taurine oxidation at T181 may be greater than or equal to about 2σ and taurine oxidation at T205 may be greater than or equal to about 2σ. In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a therapeutic criterion for the subject. In a further embodiment, the measured levels of taurine oxidation at T205 and the measured levels of taurine oxidation at T181 and / or T217 are used in various mathematical calculations to improve predictive power compared to themselves. be able to. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In another example, the disclosure provides a method for treating a subject at high risk of conversion to MCI by AD, which method (a) prepares an isolated tau sample obtained from the subject and tau. Measuring the total amount and (i) Taulination at T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and T217; and (b) the ratio of taulination at T217 to the total amount of tau and / Or the pharmaceutical composition is administered to the subject when the ratio of taulin oxidation at T181 to the total amount of tau is about 1.5σ or more and the ratio of taulin oxidation at T205 to the total amount of tau is about 1.5σ or less. However, σ was measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques, total tau and T217 and T205, T181. And T205, or the standard deviation defined by the normal distribution of taulin oxidation at T181, T205, and T217. In various embodiments, the ratio of taurine oxidation at T217 to total tau and / or the ratio of taurine oxidation at T181 to total tau is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ. , About 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, the ratio of taurine oxidation at T217 to total tau and / or the ratio of taurine oxidation at T181 to total tau is about 1.85σ, about 1.9σ, about 1.95σ, about 2σ, about. It may be 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In each of the above embodiments, the ratio of taurine oxidation at T205 to total tau is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.50σ, about 1.55σ, about. It may be less than 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2.0σ, or 2σ. Alternatively, the ratio of taurine oxidation at T205 to total tau is about 2.0σ, about 2.05σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or It may be less than 2.5σ. In a further example, the ratio of taurine oxidation at T217 to total tau and / or the ratio of taurine oxidation at T181 to total tau may be greater than or equal to about 2σ, and the ratio of taurine oxidation at T205 to total tau is , May be less than or equal to about 2σ. In addition to using a threshold (eg, a value that is at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a therapeutic criterion for the subject.

In another example, the disclosure provides a method for treating a subject at high risk of conversion to MCI by AD, which method (a) prepares an isolated tau sample obtained from the subject (a). i) Measuring taurin oxidation at T181 and T205, (ii) T217 and T205, or (iii) T181, T217, and T205; and (b) (a) (i), (a) (ii), Alternatively, it comprises administering the pharmaceutical composition to the subject when the taurin oxidation at the specific site listed in (a) (iii) is about 1.5σ or more, where σ is by PET imaging. By normal distribution of taurination at T217 and T205, T181 and T205, or T181, T205, and T217, measured and / or measured by Aβ42 / 40 measurements in CSF in a control population free of brain amyloid plaques. The standard deviation defined. In various embodiments, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is about 1.3σ, about 1.35σ, about. It may be 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is about 1.85σ, about 1.9σ, about. It may be 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In a further example, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) may be about 2σ or more. In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a therapeutic criterion for the subject. In a further embodiment, the measured levels of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) are used in various mathematical operations. Therefore, the predictive power can be improved compared to each of them. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In another example, the disclosure provides a method of treating a subject at high risk of conversion to MCI by AD, which method (a) prepares an isolated tau sample obtained from the subject and provides total tau and tau. (I) Measuring taulin oxidation at T181 and T205, (ii) T217 and T205, or (iii) T181, T217, and T205; and (b) (a) (i), (a) relative to total tau. (Ii), or (a), including administering the pharmaceutical composition to the subject when the ratio of taulin oxidation at the specific site listed in (iii) is about 1.5σ or more, provided that σ Tau total and T217 and T205, T181 and T205, or T181, T205, and measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques. The standard deviation defined by the normal distribution of taurin oxidation at T217. In various embodiments, the ratio of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) to total tau is about 1.3σ, about. It may be 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. .. In other embodiments, the ratio of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) to total tau is about 1.85σ, about. It may be 1.9σ, about 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In a further example, the ratio of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) to total tau may be greater than or equal to about 2σ. .. In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a therapeutic criterion for the subject.

In another example, the disclosure provides a method for treating a subject with symptoms of AD, the method (a) preparing first and second isolated tau samples obtained from the subject. "First" and "second" indicate the order in which the samples were collected, measuring taur phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181, T217, and T205. To do; (b) measure site-specific phosphorylation changes at each measured residue, and optionally change total tau volume; and (c) phosphorylation levels at T181 and / or T217. It comprises administering the pharmaceutical composition to the subject when the phosphorylation level at T205 and optionally the total amount of tau is increased while remaining reduced or constant. First and second isolated tau samples may be collected across dates, weeks, or months. Typically, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is also greater than or equal to about 1.5σ in both samples. However, σ was measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques, T217 and T205, T181 and T205, or T181, T205, and The standard deviation defined by the normal distribution of taurine oxidation at T217. In a further embodiment, the measured levels of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) are used in various mathematical operations. Therefore, the predictive power can be improved compared to each of them. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In another example, the disclosure provides a method for treating a subject with symptoms of AD, the method (a) preparing first and second isolated tau samples obtained from the subject. "First" and "second" indicate the order in which the samples were collected, indicating the total amount of tau and tauline at (i) T181 and T205, (ii) T217 and T205, or (iii) T181, T217, and T205. Measuring oxidation; (b) calculating site-specific phosphorylation changes and changes in total tau at each measured residue; and (c) phosphorylation levels at T181 and / or T217 It comprises administering the pharmaceutical composition to the subject when the phosphorylation level at T205 remains low or constant, and the total amount of tau is increased. First and second isolated tau samples may be collected across dates, weeks, or months. Typically, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is also greater than or equal to about 1.5σ in both samples. However, σ was measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques, T217 and T205, T181 and T205, or T181, T205, and The standard deviation defined by the normal distribution of taurine oxidation at T217. In a further embodiment, the measured levels of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) are used in various mathematical operations. Therefore, the predictive power can be improved compared to each of them. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In each of the above embodiments, the pharmaceutical composition may include a contrast agent. Examples of, but not limited to, contrast agents include functional contrast agents (eg, fluorodeoxyglucose, etc.) and molecular contrast agents (eg, Pittsburgh compound B, florbetaben, florbetapill, flutemetamol, radionuclide-labeled antibodies, etc.).

Alternatively, the pharmaceutical composition may contain an active pharmaceutical ingredient. Examples are not limited to active pharmaceutical ingredients: cholineresterase inhibitors, N-methyl D-aspartic acid (NMDA) antagonists, antidepressants (eg, selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and Norepinephrine reuptake inhibitor, tricyclic antidepressant, etc.), gamma-secretase inhibitor, beta-secretase inhibitor, anti-Aβ antibody (including antigen-binding fragment, variant, or derivative thereof), anti-tau antibody (antigen-binding) Fragments, variants, or derivatives thereof), stem cells, dietary supplements (eg, lithium water, omega-3 fatty acids + lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin, and grape seed extract), serotonin receptors Body 6 antagonists, p38alphaMAPK inhibitors, recombinant granulocyte macrophage colony stimulators, passive immunotherapy, active vaccines (eg CAD106, AF20513, etc.), tau protein aggregation inhibitors (eg TRx0237, methylthioninium chloride, etc.), Therapeutic agents that improve blood glucose control (eg, insulin, exenatide, liraglutide pioglycazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma 1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptors Blocker, CB1 and / or CB2 endocannabinoid receptor partial agonist, β-2 adrenaline receptor agonist, nicotinic acetylcholine receptor agonist, 5-HT2A reverse agonist, alpha 2c adrenaline receptor antagonist, 5-HT1A and 1D receptor Body agonists, glutaminyl peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor activators, HMG-CoA reductase inhibitors, neuronutrition Agents, Muscarin M1 receptor agonists, GABA receptor modifiers, PPAR gamma agonists, microtubule protein modifiers, calcium channel blockers, antihypertensive agents, statins, and any combination thereof.

In another alternative form, the pharmaceutical composition may comprise a kinase inhibitor. Suitable kinase inhibitors are Thousand and One Amino Acid Kinases (TAOK), CDK, GSK-3β, MARK, CDK5, Fyn, 5'Adenosine Monophosphate Activated Protein Kinases (AMPK), Calcium Carmodulin Kinase II, Cycline Dependent Kinases. 5 (cdk5), casein kinase 1 (CK1), casein kinase 2 (CK2), cyclic AMP-dependent protein kinase (PKA), bispecific tyrosine phosphorylation-regulated kinase 1A (DYRK1A), glycogen synthase kinase-3 (GSK-3), JNK, LRRK2, Microtube Affinity Regulatory Kinase (MARK), MSK1, p35 / 41, p42 / p44 Division Promoter Activated Protein Kinase (ERKs1 / 2), p38 Division Promoter Activated Kinase (ERKs1 / 2) p38MAPK), p70S6 kinase, phosphorylase kinase, PKB / AKT, protein kinase C (PKC), protein kinase N (PKN), prostate-derived steryl 20-like kinase 1 alpha / beta, 90 kDa ribosome S6 kinase (RSK1 / 2) (PSK1 /) TAOK2), prostate-derived steryl 20-like kinase 2 (PSK2 / TAOK1), stress-activating protein kinase (SAPK) 1 gamma, SAPK2a, SAPK2b, SAPK3, SAPK4, SGK1, SRPK2, or tautubulin kinase 1/2 (TTBK1 / 2) may be inhibited.

In yet another alternative form, the pharmaceutical composition may comprise a phosphatase activator. By way of example, but not limited to, phosphatase activators may increase the activity of protein phosphatases 1, 2A, 2B, or 5.

Methods for measuring taurine oxidation and total tau are described in Chapter II and are incorporated herein by reference. In a preferred embodiment, the isolated tau sample comprises tau purified from blood or CSF by affinity purification, and taurine oxidation is measured by mass spectrometry. In another preferred embodiment, the isolated tau sample uses a ligand that specifically binds to an epitope within the middomain of tau and optionally a secondary ligand that specifically binds to an epitope within the N-terminus of tau. Containing tau purified from blood or CSF by affinity purification, tauphosphorylation is measured by high resolution mass analysis. In another preferred embodiment, the isolated tau sample uses a ligand that specifically binds to an epitope within the middomain of the tau and optionally a second that specifically binds to an epitope within the MTBR or C-terminus of the tau. Containing tau purified from blood or CSF by affinity purification in combination with two ligands, tauphosphorylation is measured by high resolution mass analysis. In an exemplary embodiment, the mass spectrometry protocol outlined in the Examples is used.

V. Clinical Trials Another aspect of the disclosure is a method for enrolling a subject in a clinical trial, specifically in a clinical trial of an Aβ or tau therapeutic agent, provided that all other criteria of the clinical trial are met. It is assumed that there is. In one embodiment, the method for enrolling a subject in a clinical study is as follows: (a) Prepare an isolated tau sample obtained from the subject and in the isolated tau sample, T111, S113, T181, S199, S202. , S208, T153, T175, T205, S214, T217, and T231 to measure tau phosphorylation at one or more amino acid residues, and optionally to measure total tau; and (b). ) When the measured phosphorylation level (s) significantly deviates from the mean in the control population free of cerebral amyloid plaques as measured by PET imaging and / or by Aβ42 / 40 measurements in CSF. May include enrolling the subject in a clinical trial. In another embodiment, the method for enrolling a subject in a clinical trial is as follows: (a) Prepare first and second isolated tau samples obtained from the subject and in each isolated tau sample, T111, S113. , T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231 to measure tau phosphorylation at one or more amino acid residues selected from, and optionally measure total tau. To do; (b) calculate site-specific changes in phosphorylation at each measured residue, and optionally change in total tau, and (c) the calculated changes (s) are PET. It may include enrolling the subject in a clinical trial if measured by imaging and / or Aβ42 / 40 measurements in CSF significantly deviate from the mean in a control population free of cerebral amyloid plaques. be. The phrase "control population free of cerebral amyloid plaques as measured by PET imaging and / or by Aβ42 / 40 measurements in CSF" is defined in Chapter IV. "Significantly deviates from the mean" means at least one standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or even more preferably at least two standard deviations from the mean. Shows greater or lesser values by deviation (ie, 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is measured by PET imaging and / or Aβ42 / 40 measurements in CSF. The standard deviation defined by the normal distribution measured in the control population without brain amyloid plaques). In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a registration criterion for the subject.

Ratio calculated from the measured phosphorylation levels (s) in any of the above embodiments, in lieu of or in addition to the use of site-specific tau phosphorylation measurements combined with optional tau total measurement. , Or the ratio calculated from the measured phosphorylation level (s) and total tau. The ratio calculated from the measured phosphorylation level (s) may be the ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217. .. The ratio calculated from the measured phosphorylation level (s) and total tau may be the ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. .. Mathematical operations other than ratios may also be used. For example, in the example, site-specific taurine oxidation values are used in various statistical models (eg, linear regression, LME curve, LOESS curve, etc.) and other known biomarkers (eg APOEε4 status, age, gender, cognitive test score). , Functional test score, etc.).

The planning of clinical trials of Aβ and tau therapeutics may be greatly assisted by the methods disclosed herein. Many clinical trials are designed to test the effectiveness of contrast or therapeutic agents that target specific pathophysiological changes that occur prior to the onset of AD symptoms. As mentioned above in Chapter V, the effectiveness of these various agents is applied to subjects with specific site-specific taurine oxidation levels as measured by the methods disclosed and exemplified herein. May be improved by administration of. Similarly, enrolling subjects with symptoms of AD in clinical trials (eg, after the onset of MCI by AD) also aims to determine if efficacy is associated with a particular stage of AD. It would benefit from being able to accurately stage the condition. Therefore, measuring taurine oxidation levels as described herein prior to enrolling a subject in a clinical trial, specifically in the treatment group of a clinical trial, reduces the size of the clinical trial and / or results. May result in improvement. In some cases, the methods described herein may be developed and used as companion diagnostics for therapeutic agents.

In an exemplary embodiment, the method for enrolling a subject in a clinical trial is (a) preparing an isolated tau sample obtained from the subject and selecting from T181, T205, and T217 in the isolated tau sample. Measuring tau phosphorylation at one or more amino acid residues, and optionally measuring total tau; and (b) measured phosphorylation levels (s) measured by PET imaging. And / or enrolling a subject in a clinical trial if Aβ42 / 40 measurements in CSF deviate significantly from the mean in a control population free of brain amyloid plaques. In another embodiment, the method for enrolling a subject in a clinical trial is as follows: (a) Prepare first and second isolated tau samples obtained from the subject and in each isolated tau sample, T181, T205. , And measuring tau phosphorylation at one or more amino acid residues selected from T217, and optionally measuring total tau; (b) site-specific phosphorus at each measured residue. Calculating changes in oxidation, and optionally changes in total tau; and (c) calculated changes (s) measured by PET imaging and / or by Aβ42 / 40 measurements in CSF. Enrollment of subjects in clinical trials may include enrollment of subjects if they deviate significantly from the mean in the control population without amyloid plaques. The phrase "control population free of cerebral amyloid plaques as measured by PET imaging and / or by Aβ42 / 40 measurements in CSF" is defined in Chapter IV. "Significantly deviates from the mean" means at least one standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or even more preferably at least two standard deviations from the mean. Shows greater or lesser values by deviation (ie, 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is measured by PET imaging and / or Aβ42 / 40 measurements in CSF. The standard deviation defined by the normal distribution measured in the control population without brain amyloid plaques). In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a registration criterion for the subject.

In one example, the disclosure provides a method for enrolling a subject in a clinical trial, the method (a) preparing an isolated tau sample obtained from the subject and in the isolated tau sample. To measure taurine oxidation at (i) T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and T217; and (b) taurine oxidation at T217 and / or T181 is about 1. Including administering the pharmaceutical composition to the subject when taurine oxidation at T205 is greater than or equal to 5σ and less than or equal to about 1.5σ, where σ is measured by PET imaging and / or A standard deviation defined by the normal distribution of taurine oxidation at T217 and T205, T181 and T205, or T181, T205, and T217 measured in a control population free of brain amyloid plaques by Aβ42 / 40 measurements in CSF. .. In various embodiments, taurine oxidation at T217 and / or taurine oxidation at T181 is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ. , About 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, taurine oxidation at T217 and / or taurine oxidation at T181 is about 1.85σ, about 1.9σ, about 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about. It may be 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In each of the above embodiments, the taurine oxidation at T205 is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.51σ, about 1.55σ, about. It may be less than 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2.0σ, or 2.0σ. Alternatively, taurine oxidation at T205 is less than about 2.0σ, about 2.05σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or 2.5σ. There may be. In a further example, taurine oxidation at T217 and / or taurine oxidation at T181 may be greater than or equal to about 2σ and taurine oxidation at T205 may be greater than or equal to about 2σ. In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a registration criterion for the subject. In a further embodiment, the measured levels of taurine oxidation at T205 and T181 and / or T217 can be used in various mathematical operations to improve predictive power compared to themselves. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In another example, the disclosure provides a method for enrolling a subject in a clinical trial, which prepares (a) isolated tau samples obtained from the subject, total tau and (i) T217. And T205, (ii) T181 and T205, or (iii) T181, T205, and T217; and (b) the ratio of tau phosphorylation at T217 to total tau and / or T181 to total tau. Includes administration of the pharmaceutical composition to the subject when the ratio of taulination at T205 to the total amount of tau is about 1.5σ or more and the ratio of taulination at T205 to total tau is about 1.5σ or less. However, σ is the total tau and T217 and T205, T181 and T205, or T181, T205 measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques. , And the standard deviation defined by the normal distribution of taulin oxidation at T217. In various embodiments, the ratio of taurine oxidation at T217 to total tau and / or the ratio of taurine oxidation at T181 to total tau is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ. , About 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, the ratio of taurine oxidation at T217 to total tau and / or the ratio of taurine oxidation at T181 to total tau is about 1.85σ, about 1.9σ, about 1.95σ, about 2σ, about. It may be 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In each of the above embodiments, the ratio of taurine oxidation at T205 to total tau is about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.50σ, about 1.55σ, about. It may be less than 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2.0σ, or 2σ. Alternatively, the ratio of taurine oxidation at T205 to total tau is about 2.0σ, about 2.05σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or It may be less than 2.5σ. In a further example, the ratio of taurine oxidation at T217 to total tau and / or the ratio of taurine oxidation at T181 to total tau may be greater than or equal to about 2σ, and the ratio of taurine oxidation at T205 to total tau is , May be less than or equal to about 2σ. In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a registration criterion for the subject.

In another example, the disclosure provides a method for enrolling a subject in a clinical trial, which prepares (a) isolated tau samples obtained from the subject and (i) T181 and T205, Measuring taurine oxidation at (ii) T217 and T205, or (iii) T181, T217, and T205; and (b) (a) (i), (a) (ii), or (a) (iii). ) Includes administration of the pharmaceutical composition to the subject when taurine oxidation at a particular site is greater than or equal to about 1.5σ, where σ is measured by PET imaging and / or A standard deviation defined by the normal distribution of taurine oxidation at T217 and T205, T181 and T205, or T181, T205, and T217 measured in a control population free of cerebral amyloid plaques by Aβ42 / 40 measurements in CSF. .. In various embodiments, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is about 1.3σ, about 1.35σ, about. It may be 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is about 1.85σ, about 1.9σ, about. It may be 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In a further example, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) may be about 2σ or more. In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a registration criterion for the subject. In a further embodiment, the measured levels of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) are used in various mathematical operations. Therefore, the predictive power can be improved compared to each of them. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In another example, the disclosure provides a method for enrolling a subject in a clinical trial, which prepares (a) isolated tau samples obtained from the subject, total tau and (i) T181. And measuring tau phosphorylation at T205, (ii) T217 and T205, or (iii) T181, T217, and T205; and (b) (a) (i), (a) (ii), with respect to total tau. Alternatively, it comprises administering the pharmaceutical composition to the subject when the ratio of taulin oxidation at the specific site listed in (a) (iii) is about 1.5σ or more, where σ is the PET image. Total tau measured by method and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques and tau phosphorylation at T217 and T205, T181 and T205, or T181, T205, and T217. The standard deviation defined by the normal distribution of. In various embodiments, the ratio of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) to total tau is about 1.3σ, about. It may be 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. .. In other embodiments, the ratio of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) to total tau is about 1.85σ, about. It may be 1.9σ, about 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ. In a further example, the ratio of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) to total tau may be greater than or equal to about 2σ. .. In addition to using a threshold (eg, at least one standard deviation greater than or less than the mean), some embodiments may use a degree of change above or below the mean as a registration criterion for the subject.

In another example, the disclosure provides a method for enrolling a subject in a clinical trial, the method (a) preparing first and second isolated tau samples obtained from the subject, "No. "1" and "2" indicate the order in which the samples were collected and measure tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181, T217, and T205. (B) Calculate site-specific phosphorylation changes at each measured residue, and optionally tau total change; and (c) Decreased levels of phosphorylation at T181 and / or T217. Includes enrolling the subject in a clinical trial if the phosphorylation level at T205 and optionally total tau is increased while remaining constant. First and second isolated tau samples may be collected across dates, weeks, or months. Typically, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is also greater than or equal to about 1.5σ in both samples. However, σ was measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques, T217 and T205, T181 and T205, or T181, T205, and The standard deviation defined by the normal distribution of taurine oxidation at T217. In a further embodiment, the measured levels of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) are used in various mathematical operations. Therefore, the predictive power can be improved compared to each of them. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

In another example, the disclosure provides a method for enrolling a subject in a clinical trial, the method (a) preparing first and second isolated tau samples obtained from the subject, "No. "1" and "2" indicate the order in which the samples were collected, indicating the total amount of tau and tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181, T217, and T205. To measure; (b) to calculate site-specific phosphorylation changes and changes in total tau at each measured residue; and (c) decreased phosphorylation levels at T181 and / or T217 or Includes enrolling a subject in a clinical trial if it remains constant, the phosphorylation level at T205 remains low or constant, and the total amount of tau is increasing. First and second isolated tau samples may be collected across dates, weeks, or months. Typically, taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) is also greater than or equal to about 1.5σ in both samples. However, σ was measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques, T217 and T205, T181 and T205, or T181, T205, and The standard deviation defined by the normal distribution of taurine oxidation at T217. In a further embodiment, the measured levels of taurine oxidation at specific sites listed in (a) (i), (a) (ii), or (a) (iii) are used in various mathematical operations. Therefore, the predictive power can be improved compared to each of them. For example, the ratio (s) can be calculated from the measured phosphorylation levels. Mathematical operations other than ratios can also be used.

Methods for measuring taurine oxidation and total tau are described in Chapter II and are incorporated herein by reference. For example, using the protocol detailed in Examples 5-9, taurine oxidation at T181, T205, and T217 was measured by PET imaging when measured with isolated tau samples purified from CSF. In the control population without cerebral amyloid plaque, 21.7 ± 2.3, 0.34 ± 0.13, and 1.2 ± 0.66, respectively (see Table 2, non-mutation carrier column). ). Therefore, twice the standard deviation (ie, 2σ) above the mean seen in non-mutant carriers is 43.4, 0.68, and 2., respectively, at p-T181, p-T205, and p-T217. 4. However, as those skilled in the art will know, the absolute value can vary depending on the protocol.

In a preferred embodiment, the isolated tau sample comprises tau purified from blood or CSF by affinity purification, and taurine oxidation is measured by mass spectrometry. In another preferred embodiment, the isolated tau sample uses a ligand that specifically binds to an epitope within the middomain of tau and optionally a secondary ligand that specifically binds to an epitope within the N-terminus of tau. Containing tau purified from blood or CSF by affinity purification, tauphosphorylation is measured by high resolution mass analysis. In another preferred embodiment, the isolated tau sample uses a ligand that specifically binds to an epitope within the middomain of the tau and optionally a second that specifically binds to an epitope within the MTBR or C-terminus of the tau. Containing tau purified from blood or CSF by affinity purification in combination with two ligands, tauphosphorylation is measured by high resolution mass analysis. In an exemplary embodiment, the mass spectrometry protocol outlined in the Examples is used.

In each of the above embodiments, the subject may be enrolled in a treatment group for clinical trials. "Treatment" is defined in Chapter V. Subjects enrolled in the treatment group of clinical trials may receive the pharmaceutical composition. In some embodiments, the pharmaceutical composition may include a contrast agent. Examples of, but not limited to, contrast agents include functional contrast agents (eg, fluorodeoxyglucose, etc.) and molecular contrast agents (eg, Pittsburgh compound B, florbetaben, florbetapill, flutemetamol, radionuclide-labeled antibodies, etc.). Alternatively, the pharmaceutical composition may contain an active pharmaceutical ingredient. Examples are not limited to active pharmaceutical ingredients: cholineresterase inhibitors, N-methyl D-aspartic acid (NMDA) antagonists, antidepressants (eg, selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and Norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), gamma secretase inhibitors, beta secretase inhibitors, anti-Aβ antibodies (including antigen-binding fragments, variants, or derivatives thereof), anti-tau antibodies (antigen-binding fragments, Variants or derivatives thereof), stem cells, dietary supplements (eg lithium water, omega-3 fatty acids + lipoic acid, long chain triglycerides, genistein, resveratrol, curcumin, and grape seed extract), serotonin receptor 6 Antagonists, p38alphaMAPK inhibitors, recombinant granulocyte macrophage colony stimulators, passive immunotherapy, active vaccines (eg CAD106, AF20513, etc.), tau protein aggregation inhibitors (eg TRx0237, methylthioninium chloride, etc.), blood glucose control Therapeutic agents that improve (eg, insulin, exenatide, liraglutide pioglycazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma 1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers , CB1 and / or CB2 endocannabinoid receptor partial agonist, β2 adrenaline receptor agonist, nicotinic acetylcholine receptor agonist, 5-HT2A reverse agonist, alpha 2c adrenaline receptor antagonist, 5-HT1A and 1D receptor agonist, Glutami Nylpeptide cyclotransferase inhibitor, selective inhibitor of APP production, monoamine oxidase B inhibitor, glutamate receptor antagonist, AMPA receptor agonist, nerve growth factor activator, HMG-CoA reductase inhibitor, neuronutrient, Muscarin M1 Receptor agonists, GABA receptor modifiers, PPAR-gamma agonists, microtubule protein modifiers, calcium channel blockers, antihypertensive agents, statins, and any combination thereof. In an exemplary embodiment, the pharmaceutical composition may comprise a kinase inhibitor. Suitable kinase inhibitors may inhibit Thousand and One Amino Acid Kinases (TAOK), CDK, GSK-3β, MARK, CDK5, or Fyn. In another exemplary embodiment, the pharmaceutical composition may comprise a phosphatase activator. By way of example, but not limited to, phosphatase activators may increase the activity of protein phosphatase 2A.

In each of the above embodiments, the subject may or may not be symptomatic. "Asymptomatic subject" as used herein refers to a subject who shows no signs or symptoms of AD. Alternatively, the subject may present with signs or symptoms of AD (eg, memory loss, misplacement, mood or behavioral fluctuations, etc.) but have sufficient cognition or function to make a clinical diagnosis of mild cognitive impairment. Does not indicate a failure. Both symptomatic and asymptomatic subjects may have Aβ amyloidosis; however, prior findings of Aβ amyloidosis are not a prerequisite for treatment. In a further embodiment, the subject may have AD. In any of the above embodiments, the subject may carry one of the genetic mutations known to cause dominantly inherited Alzheimer's disease. In alternative embodiments, the subject may not carry one of the genetic mutations known to cause dominantly inherited Alzheimer's disease.

The following examples are included to demonstrate preferred embodiments of the present invention. As will be appreciated by those skilled in the art, the techniques disclosed in the examples below represent techniques found by the inventors to be fully functional in the practice of the present invention. However, one of ordinary skill in the art can make changes to the particular embodiments disclosed in the light of the present disclosure without departing from the spirit and scope of the invention, and nevertheless obtain similar or similar results. You should know that you can. Therefore, all content described or shown in the accompanying drawings should be construed as an example and not in a limited sense.

The following examples illustrate various iterations of the invention.

Example 1 Phosphorylation site of tau protein in normal non-AD human brain To identify the phosphorylation site of tau in normal soluble brain, extracts from healthy controls were used for phosphorylated tau and non-phosphorylated tau. Purified by immunocapture with tau-1 and HJ8.5 tau antibodies, both of which are concentrated. Iso-type trypsin digestion of brain tau produced 27 unmodified peptides that were long, sufficiently hydrophobic and detectable by LC-MS (Barthelemy et al., 2016). Twenty-five peptides contained serine and / or threonine as potential phosphorylation sites. Several peptides contain multiple potential phosphorylation sites (4-9 sites), which allow us to co-elute different monophosphorylated peptides together during LC separation. I came to think that there is sex. After this, the PRM screening method described above was applied to these peptides.

Analysis of the projection domains (103-126) in the tau sequence revealed the presence of multiple p-tau peptides eluted in overlapping complex LC-MS / MS patterns (FIGS. 3-7). The present inventors have identified three phosphorylation sites from the 0N isotype, which is the shortest isotype of tau protein. The LC system was not capable of separating the three phosphorylated peptides in question, but using fragment identification and corresponding strength, one major phosphorylation site of residue S113 and the rest. It was clarified that the signal is composed of two secondary phosphorylation sites of the groups T111 and T123 (Fig. 3). In contrast, PRM screening of tau sequences from longer 1N and 2N isotypes simplifies the elution pattern of phosphorylated peptides, especially if multiple potential monophosphorylation sites are predicted within the same peptide sequence. Further chromatographic separation was required for the conversion (FIGS. 4-6). We were able to identify co-eluted, semi-specific MS / MS fragments containing differentially phosphorylated residues by LC separation. However, we have also identified a small number of signals that may be due to LC artifacts, and these signals are probably due to two conformations of the same peptide in solution. It seems (Fig. 5). This effect is important for peptide sequences (in the 0N isotype) containing amino acid residues 45-67 of both unmodified and phosphorylated peptides (FIG. 5), sequences 68-126 (1N) and 88. It was less common for ~ 126 (2N) (FIGS. 4 and 6).

Phosphorylation at residues S113, T111, and T123 was confirmed at peptide sequences 68-126 (1N) and 88-126 (2N). In all cases, the signal of S113 was the largest of the above three species, with a phosphorylation rate of 0.2-0.5% (FIGS. 4 and 6, Table 1). Phosphorylation at residues T50, T52, and S56 was clearly identified on peptide sequences 45-67 (shared by 1N and 2N isotypes). LC-MS signals showed that at least two of the three residues (S61, T63, and S64) were phosphorylated on the same peptide (FIG. 5). No specific signal was found to identify potential phosphorylation at residue S46. Phosphorylation at residues S68 and T69 in sequences 68-126 (1N) and 68-87 (2N) was supported, but no specific fragment identifying their LC-MS pattern was detected. On the same 1N and 2N peptide sequences, lower phosphorylation levels at T71 were found. Phosphorylation on T76, T101, and T102 residues, specific for 2N, was also identified (summarized in FIG. 8).

Introduction of trypsin missed cleave for screening of previously reported phosphorylated residues (sites T175, T181, S212, T231, and S396) located after the trypsin cleavage site (Hanger et al. 1998). Was also taken into consideration. By PRM screening by the present inventors, Hanger et al. Succeeded in detecting the LC-MS pattern corresponding to the previously reported phosphorylated tau peptide in normal brain tissue (T181, S199, S202, and S404, FIG. 8). The corresponding LC-MS signal is high, suggesting that the amount of previously reported p-tau peptide is likely to be highest in the normal human brain. A comparison of phosphorylation of S199 and S202 by the present inventors showed that phosphorylation in S202 was generally significantly higher (FIG. 8). Extraction of tau using an anti-Tau1 antibody and ligation with a non-phosphorylated epitope on the 192-199 amino acid sequence can explain the low recovery of S199 phosphorylation in the extract. The present inventors searched for the presence of diphosphorylated peptides derived from sequences 195-209 and 386-406 on the premise that phosphorylation of S202 and S404 occurs frequently in this extract. We correspond to two LC-MS patterns containing specific fragments corresponding to two-site phosphorylation in S202 / S199 and S202 / S198, and two corresponding phosphorylation in 396/404 1 Two LC-MS patterns were detected (Fig. 8).

Further screening of soluble tau in brain extracts demonstrated the presence of lower amounts of other monophosphorylated peptides corresponding to the phosphorylated residues of T175, S214, T217, T231, and S396. When the monophosphorylated peptide sequences 181-190 were screened, signals from fragments corresponding to the phosphorylated peptides in S184 or S185 were also detected at low levels (FIG. 8). A search for monophosphorylation on long sequences 407-438 found a pattern consistent with phosphorylation at residues S409 and S416 and at least one phosphorylation in the group of residues S412 / S413 / T414.

Overall, we used the PRM screening method to identify at least 29 unique phosphorylation sites detectable in the soluble tau fraction extracted from normal non-AD brain (Table). 1). Twenty-five of the above phosphorylation sites can be clearly attributed to a unique LC-MS signal, and four additional phosphorylation sites are not assigned to the exact LC-MS pattern, but their presence is present. Proven. These sites were located on three clusters. That is, at least 14 phosphorylation sites were located in the N-terminal projection domain, 10 sites were located on the proline-rich domain in the center of the sequence, and 6 phosphorylation sites were located on the C-terminal (Fig. 14).

Example 2 Phosphorylation site on tau protein in CSF In purification of CSF tau using tau-specific antibodies and digestion compared to brain tau digests, predominantly the middomain of the protein sequence (residue 150). A detectable peptide from ~ 221) was produced. Peptides derived from the N-terminus of tau were, to a lesser extent, detectable, and sequences derived from the microtubule-bound repeat (MTBR) domain or C-terminus were scarcely detectable (Barthelemy et al. , 2016; Sato et al., 2018). This difference in signal recovery may be due to tau truncation during release from neurons (Sato et al., 2018). This middomain peptide recovery was sufficient to monitor the corresponding small amount of phosphorylated isotypes using current PRM methods. Conversely, detection of phosphorylated peptides in the MTBR and C-terminal domains would require significant technological advances in MS methods.

PRM screening of tau phosphorylated peptides from normal control CSF revealed several phosphorylated sites at T181 (not shown), S199, S202, and T217 (FIG. 9), similar to soluble tau in the brain. Identified. A low signal corresponding to pS214 was also detected (FIG. 9). A specific fragmentation pattern corresponding to pT205 was identified in the CSF extract and chromatographically separated from the pS199 / pS202 signal. (Fig. 9).

We analyzed CSF pools from AD patients with mild to moderate dementia to increase the likelihood of detecting additional phosphorylation sites in the tau of CSF. We predicted that the CSF pool of AD would contain not only high concentrations of tau, but also high levels of phosphorylated tau. In the CSF of AD, phosphorylated residues similar to those found in normal CSF (T181, S199, S202, T217, and T231) were detected. Furthermore, signals corresponding to pS113 and pT175, which were previously detected in brain-derived tau and not in normal CSF, were detected in AD CSF (FIGS. 9 and 10). The LC-MS / MS pattern, which contained specific fragments derived from the S202 / S199 phosphorylated peptide and a retention time different from the others, allowed the identification of new peptides phosphorylated in S208. When we reviewed pS208 in normal CSF, we detected a corresponding small amount of signal. An LC-MS / MS pattern consistent with the new phosphorylated peptide at residue T153 was detected at levels close to those of the corresponding non-phosphorylated peptide (FIG. 10). Careful review of the brain extract data suggested that the signal corresponding to this site was abundant. Finally, we found a specific signal corresponding to phosphorylation on the 103-126 amino acid sequence from the 0N isotype in the CSF of AD, where T111 was on this peptide. While it is the major phosphorylation site, S113 shows little phosphorylation (FIG. 11). Overall, 12 phosphorylation sites were detected in the tau of the CSF, and 2 of them could not be detected in the brain lysate (Fig. 12).

Example 3 The measured value of the p-tau abundance of CSF is significantly different from the p-tau abundance in the brain. In the brain, S404 is the most highly phosphorylated among the phosphorylated sites examined. (PS404 / S404 = 110%, that is, 52% of S404 is phosphorylated). Therefore, more than half of the brain tau was phosphorylated in S404 compared to other highly phosphorylated sites, S202 and T181 (9.7% and 9.5%, respectively). 1.3% of S199 was phosphorylated, but this value was due to the inability of the Tau1 antibody used for extraction to bind to its corresponding phosphorylated epitope (Liu et al., 1993). It may be underestimated. Phosphorylation on the C-terminal site other than S404 was found to be about 1%. At the N-terminus, T52, S68 / T69, T50, and S113 were also phosphorylated in an amount in the range of about 0.5% to 2%. Phosphorylation levels at other detected sites appeared to be significantly lower (<0.5%).

In addition to the unique detection of pT205 and pS208 in the CSF tau and not in the brain tau, the relative phosphorylation abundance of certain other phosphorylation sites detected in the brain tau is , Changed compared to the control CSF tau (Fig. 13). Phosphorylation of tau in CSF was significantly lower at sites pS199, pS202, and pS214 compared to the brain (reduced to 1/6, p = 0.008; 1/5, p = 0, respectively). .016; and reduced to 1/2, p = 0.016). Conversely, CSF tau phosphorylation was significantly higher at 0N-derived sites pT217, pT231, and pT111 (4-fold increase, p = 0.016; 7-fold increase, p = 0.016; and, respectively. 16 times increase). The abundance of pT181 was similar in CSF and in the brain (about 10%). When measured in CSF of AD, pT175 remained low (0.1 to 0.2% compared to 0.1% in the brain).

Tau of the brain and tau of CSF have different truncation patterns. That is, the tau isotype of the brain is predominantly full length, while the tau isotype of CSF is truncated. The tau isoforms of the brain and the tau isoforms of the CSF and the corresponding peptides recovered after IP depend on the antibody used for immunoprecipitation (Sato et al., 2018). Similarly, the antibodies used to immunoprecipitate tau can affect the recovery of tau phosphorylation. To assess the effect of this p-tau on recovery, we in addition to the combination of tau 1 + HJ 8.5, as well as various antibodies (tau 13, HJ 8.5, HJ 8.7, tau 1, and tau 1). Using tau 5), the IP-MS results for the phosphorylation rate were compared (FIG. 14). When tau 1 or tau 1 + HJ8.5 was used in the brain and cerebrospinal fluid, a significant decrease in the pS199 / S199 ratio was observed. This suggests that CSF truncation between the N-terminus and the middomain may affect the measurement of pS199 phosphorylation as compared to the brain. The pS199 / S199 ratios measured with other antibodies in the brain and CSF appeared to be relatively similar. Interestingly, some reactivity was still observed for pS199 and less reactive in CSF, which means that tau 1 binding is non-selective, but phosphorylation of S199 has been reported. It is suggested that it is present at the end of the tau 1 epitope (192-199).

We are common in CSF and brain extracts because phosphatase activity may reduce tau phosphorylation measured in brain extracts during the postmortem period (PMI) to necropsy. The effect of PMI on the phosphorylation rate seen in was investigated. Ten brain samples (middle frontal gyrus) without tau pathology were analyzed from subjects with a PMI range of 5 to 16 hours. The PMI did not exceed 16 hours for any of the brain extracts analyzed initially. No significant association was found between PMI and phosphorylation rates measured at the T181, S199, S202, S214, T217, T231, and S404 sites (Spearman's test, 95% confidence interval, not shown). ..

Example 4 Changes in AD-Specific p-Tau in CSF The increase in p-tau in CSF, commonly reported in AD, has two potential effects: 1) Tau phosphorylation status. Regardless, it may be the result of an overall increase in tau, or 2) an increase in hyperphosphorylation at specific sites. Normalizing the signal or level of p-tau with non-phosphorylated tau results in a change in phosphorylation chemologic theory at a particular site (ie, normal CSF) that occurs independently of the overall change in total tau. High or low phosphorylation compared to tau or brain tau) can be quantified. As we have recently demonstrated, there is increased production of soluble tau in AD, which is correlated with amyloid plaques. Therefore, controlling not only the amount but also the rate of phosphorylation is the key to understanding. In the CSF of AD, pT181, pT217, pT231, pT205, pS208, and pS214 were hyperphosphorylated as well as 0N-specific pT111 (FIG. 13). pT153 and pT175 are not detected in non-AD and may be slightly increased in AD. Sites that showed significantly higher phosphorylation compared to non-AD were pT181 (p = 0.010), pS214 (p = 0.005), and pT217 (p = 0.003). pT181, pS214, and pT217 showed about 1.2-fold, 1.6-fold, and 4-fold increases in phosphorylation, respectively. Interestingly, not all monitored sites were hyperphosphorylated. That is, pS199 / S199 did not change significantly, and pS202 / S202 was significantly low (p = 0.030), decreasing to about 1 / 1.2.

The robustness of the phosphorylation ratio was evaluated by incubating CSF for 16 hours. Incubation had no significant effect on the observed difference in tau phosphorylation ratio between non-AD CSF and AD CSF (Fig. 15). Furthermore, the absence of phosphorylation of recombinant 15N-tau spiked in the CSF after incubation confirmed no kinase activity on the tau of the CSF (not shown).

Discussion on Examples 1-4 The technological advances in PRM in p-tau measurement-The most comprehensive qualitative and quantitative analysis of p-tau in normal brain tissue and CSF to date is described. Our approach contrasts with previous DDA studies of tau phosphorylation, which may not have captured potential sites where small amounts are phosphorylated. However, our approach uses targeted MS in sensitive PRM mode to detect and quantify small amounts of phosphorylation. The identification of phosphorylation sites relies on careful and manual interpretation of the LC-MS / MS pattern to identify the co-elution of specific ionic fragments of each phosphorylated peptide under test. In previous studies, brain tau phosphorylation was tested primarily in highly phosphorylated tau-rich insoluble extracts from PHF, and the most detailed studies to date have been in normal human tau protein. Nine phosphorylation sites have been reported in (Hanger et al. 2007). Detailed PRM data analysis by the present inventors detected more than 29 phosphorylated residues, most of which were in very low abundance. In fact, the nine previously reported sites were among the most abundant modifications in the protein. As a result of applying PRM analysis to the tau of normal CSF, which is present in a significantly smaller amount compared to the brain, 9 phosphorylation sites were first detected, and 3 more sites were detected in the CSF of AD. rice field.

In addition to a significant increase in the number of phosphorylation sites detected, we quantify tau phosphorylation rates or stoichiometry by sensitive PRM screening, independent of overall tau concentration. I showed how to make it possible to evaluate. This concept is often overlooked, but the absolute concentration of p-tau in CSF is solely due to an increase in the overall iso-type concentration of tau, not a relative change in the abundance of phosphorylated tau. It is important in assessing changes in AD when it is likely to increase in. For the first time by measuring the phosphorylation rate in this study, the phosphorylation rate throughout the protein, in various compartments (intracellular brain vs. extracellular CSF), and in various pathological conditions (AD vs. non-AD). It has become possible to compare the degree and distribution of changes in protein (high phosphorylation vs. low phosphorylation).

Some caveats of the PRM screening process are the low rate of detection and the risk of loss of p-tau species or other post-translational modifications (PTMs) not assumed in this study. Identification from MS data can be further used for broader validation or clinical cohorts to design scheduled LC-MS methods to improve multiplexing and processing speed (Gillette and Carr, 2013). Other proteases, such as AspN, may provide a different set of taupeptides and p-taupeptides than the tryptic digest, which allows for a wider range of tau, i.e. covering the C-terminal domain ( Hanger et al. 1998; Sato et al. 2018). The search can be further refined by further screening for two or three phosphorylated tau peptides not considered in this study, unless there is a biological adjustment of site phosphorylation. , The abundance of the phosphorylated tau peptide is very small.

Identifying clusters of phosphorylation sites on the projection domain of tau-We have previously reported clusters of phosphorylation residues on the N-terminal projection domain of tau that contain another splicing-dependent peptide. I found it. Interestingly, this domain was previously not known to be extensively phosphorylated in PHF in the insoluble fraction of the human brain (Funk et al., 2014; Hanger et al., 2007; Russel et. al., 2016; Thomas et al., 2012). This cluster was also not extensively characterized in a recent comprehensive study of mouse tau proteins in mouse brain (Morris et al., 2015). These discrepancies are attributed to the difficulty of characterizing complex mixtures of phosphorylated peptides that can only be identified by slight specific MS / MS fragments and / or subtle shifts in retention time on LC. there is a possibility. For example, S46 is the only previously reported phosphorylation site in this domain in normal human tau (Hanger et al. 1998). However, we have not detected a specific signal for this species, which is confused by search algorithms with adjacent T50 or T52 phosphorylation sites that share similar fragmentation patterns. It may have happened. In this regard, manual scrutiny is essential to clearly interpret and decipher the LC-MS / MS patterns corresponding to each phosphorylation site. Another possible explanation for this discrepancy is that the abundance of the N-terminal domain in the PHF is relatively low compared to the MTBR domain, mid-domain, and C-terminus (Mair et al., 2016). It may be.

This N-terminal projection domain was recently assigned as part of a major component of the repulsive barrier that prevents adjacent microtubules (connected to tau via the MTBR domain) from approaching each other (Chung et al. , 2016). This sequence contains a large number of acidic residues, and increased phosphorylation contributes to an increase in overall acidity, which may enhance the repulsion barrier. Tau phosphorylation, along with variable amounts of N-terminal elongation induced by alternative splicing of exons 2-3, may regulate tau / tau N-terminal interactions and intermolecular distances of microtubules.

Biological Significance of Different P-Tau Profiles in Brain vs. CSF-Tau is an intracellular protein that primarily functions for microtubule stability and has traditionally been released exclusively extracellularly during nerve injury or cell death. It was being considered. However, recent studies have suggested that tau is secreted in a regulated form under physiological and pathological conditions (Karch et al. 2012; Yamada et al. 2014). We recently compared the profile of soluble brain tau and CSF tau with the intracellular and extracellular tau profiles and metabolism in a neuronal model in parallel to tau and tau secretion truncated. It has been shown to be an active process with different turnover times of tau isotypes, including p-tau (Sato et al., 2018). Comparing p-tau profiles between the brain and CSF provides a possible insight into the pathogenesis of AD by understanding the association between this active secretion and tau phosphorylation.

We speculate that the isoform of p-tau concentrated in CSF has a low affinity for microtubules and is likely to be secreted. Conversely, p-tau isoforms diluted in CSF may be more likely to remain in neurons and / or avoid cleavage. Our results show that some phosphorylated residues such as T217, T231, T153, and T111 are significantly enriched in the CSF as compared to the brain extract. All are proline-oriented sites, potential substrates for GSK-3β protein kinases, and may be subject to kinase-dependent regulation. Unlike pT217, pS214 was not elevated during CSF compared to the brain. This extracellular enrichment of pT217 for pS214 is consistent with the dynamic differences we have previously identified intracellularly for these isotypes (Sato et al., 2018), and the turnover time of pT217 is non-existent. Shorter than phosphorylated tau and tau-pS214. Alternatively, the reduced phosphorylation compared to CSF observed for some of the tau sites in the brain extract may also be due to the partial dephosphorylation that occurs during PMI (Matsuo). et al., 1994). No such reduction was observed within the PMI range of 5 to 16 hours, but changes in phosphorylation ratio between mortality and this investigated period cannot be ruled out. Assessing the kinetics of dephosphorylation of these tau sites from brain biopsies by mass spectrometry will help address the impact of this phenomenon on future CSF / brain comparisons. By considering potential brain phosphatase biases that affect the measurement of brain tau phosphorylation, CSF tau phosphorylation is more neuronal in vivo than postmortem brain extracts. It will be supported that it is more likely to reflect the phosphorylated state of the tau inside. However, due to tau truncation of CSF, monitoring of tau phosphorylation in CSF in vivo will be limited primarily to sites detected in the middomain of tau.

Conversely, pS202 was significantly lower in CSF and pS199 and pS202 were not elevated in AD, which may be due to their phosphorylation promoting segregation of tau into neurons. It is shown that. T181 is phosphorylated equally in CSF and in the brain. pT205 and pS208 were detected exclusively in CSF tau, not in soluble tau protein in the brain. This means that phosphorylation at three sites in S202, T205, and S208 is a commonly used anti-AT8 antibody (Malia et al., 2016) to characterize the Brack stage of tau aggregation in the brain. ) Is recognized (Braak and Braak., 1995), which is interesting. In fact, pS208 was detected in PHF by MS (Hanger et al., 1998). In our study, pT205 and pS208 are absent in normal soluble brain tau, which is unlikely to detect the immune reactivity of AT8 in normal brain tau. confirmed. In addition, recent studies have suggested that pT205 and pS208 are associated with pS202 as phosphorylation patterns in multiple combinations leading to tau self-aggregation (Despres et al., 2017). In AD, the exclusive presence of pT205 and pS208 in CSF, as well as further increased phosphorylation rates at both sites, causes these p-tau species, which tend to be neuronal pathogenic, from cells. It may indicate a potential protective clearance mechanism to remove. When pS202 aggregates with pT205 and pS208 in AT8-positive neurofibrillary tangles enriched in the brain of AD, the simultaneous decrease of pS202 in the CSF of AD also corresponds to the increased isolation of associated isotypes. There may be. Alternatively, the absence of phosphorylation on pT205 and pS208 in brain extracts may be due to their specific degradation by phosphatases that occur during PMI. The rapid disappearance of AT8 reactivity (<3 hours) reported for tau taken from brain biopsy supports this idea (Matsuo et al., 1994). Therefore, such phosphatase activity that efficiently targets pT205 and pS208 can also be seen as a further mechanism that impedes the long half-life of AT8-positive substances in neurons. Therefore, the increase in pT205 and pS208 seen in the CSF of AD may indicate a potential pathological reduction in this protective mechanism. Finally, in AT8-positive neurofibrillary tangles enriched in the brain of AD, when pS202 aggregates with pT205 and pS208, a simultaneous decrease in pS202 in AD CSF leads to increased isolation of the associated isotype. May also be supported.

We were unable to confirmably detect phosphorylated residues from the MTBR domain in the soluble fractions from the normal brain. Phosphorylation sites on the MTBR domain have been shown to reduce the affinity of tau for microtubules (Biernat et al., 1993). The absence of such phosphorylation in normal tau may support the abnormalities of these modifications found in PHF (Hanger et al., 2007). Testing changes in phosphorylation in vivo by tau truncation of CSF, as the MTBR domain was likely degraded intracellularly after tau truncation and was therefore not recovered by the immunocapture method used in this study. Is restricted (Kanmert et al., 2015).

P-tau rate of CSF as an AD biomarker-In addition to a significant increase in the number of phosphorylation sites detected, we have a tau phosphorylation rate independent of the overall tau concentration. Alternatively, a method capable of quantifying stoichiometry was shown. This concept is often overlooked, but the absolute concentration of p-tau in CSF increases only due to an increase in the concentration of total tau (ie pT181) and not due to an increase in the relative phosphorylation rate itself. If possible, it is important in assessing changes in CSF tau phosphorylation in AD. For the first time by measuring the phosphorylation rate of this study, the phosphorylation rate throughout the protein, in various compartments (intracellular, brain vs. extracellular CSF), and in various pathological conditions (AD vs. non-AD). It has become possible to compare the degree and distribution of changes in protein (high phosphorylation vs. low phosphorylation). In the future, using this method, as recently performed using immunochemistry (Neddens et al. 2018), changes in the phosphorylation of the brain of AD in multiple sites throughout the brain region and the Brack stage. May be able to find out.

In this study, we have shown that tau in AD CSF is generally highly phosphorylated compared to non-AD CSF. However, the degree of high phosphorylation is site-dependent. T111, T205, S208, and T217 are more phosphorylated than T181, making T181 the most commonly measured target used as a p-tau biomarker for AD (Fagan et al. 2009). We have also identified the phosphorylation of T153 found exclusively in the CSF of AD. Interestingly, the sites found to be highly phosphorylated in AD CSF correspond to sites that are already significantly increased in normal CSF compared to the brain (ie,). T111, T205, S208, and T217, and to a lesser extent T231). This may indicate that AD pathology exacerbates the cellular mechanism that contributes to the enrichment of certain p-tau isotypes during the release of tau into CSF. Overall, due to this large change seen on these sites, we envision using these sites as sensitive biomarkers for AD detection. Our work relies on a limited number of CSF pools, and studies on larger CSF cohorts in the future will result in changes in p-tau and aggregation of brain amyloidosis and tau over the course of the disease. Will further establish the potential relationship of. It is also possible to ask if there is any particular p-tau profile change in non-AD tauopathy such as PSP, CBD, and FTD. Alternatively, this method can be used to track the activity of different kinases in vivo and in vitro, and may also provide a promising tool for assessing new drugs that target abnormal tau metabolism. be.

Methods of Examples 1-4 Brain soluble tau extraction-Subject-involved brain and CSF studies were approved by the Washington University Human Studios Committee and the General Clinical Research Center. Written informed consent was obtained from all subjects prior to joining the study. Soluble tau in the brain was extracted as previously reported (Sato et al., 2018). Briefly, the frozen human brain tissue derived from the control without the pathology of amyloid and tau described in the previous report (Sato et al., 2018) is used in Washington University School of Medical (St. Louis, MO) Knight Alz. Obtained from the Disease Research Center (ADRC). Sarcosyl soluble tau was separated and pooled by ultracentrifugation from the putative tau aggregates as reported in the literature (Hanger et al. 1998). Frozen human brain (200-400 mg, frontal region) on ice with Tris-HCl buffer (25 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 10 mM EGTA, 1 mM DTT, phosphatase inhibitor cocktail 3, Roche protease inhibitor, Homogenized in pH 7.4, finally 3.25 mL / mg-tissue). The homogenate was centrifuged at 11,000 xg at 4 ° C. for 60 minutes. The supernatant was solubilized in 1% sarcosyl for 60 minutes and centrifuged at 100,000 xg for 2 hours. The sarcosyl-soluble fraction was pooled, the 50 uL fraction was diluted 10-fold with 0.5% human plasma, followed by immunoprecipitation. For brain / CSF comparisons, brain lysate pools were diluted 500-8000 times prior to immunopurification to match CSF tau levels.

Extraction of tau of CSF-human CSF is amyloid-negative and cognitively normal (CDR = 0) control (n = 47, age over 60 years) and amyloid-positive and CDR> 0 AD patients (n = 33, age 60). Pooled from a cohort of 80 subjects, including (> age). Five and seven pools of 500 uL CSF aliquots were formed from the control and AD groups, respectively. At the time of the first collection, CSF was centrifuged at 1,000 xg for 10 minutes to remove cell debris and immediately frozen at -80 ° C. A protease inhibitor cocktail was added during the experiment. Tau was immunoprecipitated and desalted as previously reported (Sato et al., 2018) with some modifications. Briefly, CNBr-activated Sepharose beads (GE Healthcare 17-0430-01) were cross-linked separately from antibody tau 1 and HJ 8.5 at a concentration of 3 mg antibody per g bead. Samples are spiked per mL of sample with AQUA peptide (Thermo Fisher Scientific) corresponding to 10 fmol of phosphorylated tau and 100 fmol of non-phosphorylated tau for each sequence of interest. Tau and p-tau concentrations are calculated using these internal standards. Soluble tau was immunoprecipitated in a detergent (1% NP-40), a chaotropic agent (5 mM guanidine), and a protease inhibitor (Roche Complexe Protein Inhibitor Cocktail). The anti-tau 1 and HJ8.5 antibodies bound to the Sepharose beads were diluted 10-fold and 5-fold in inactivated Sepharose beads, respectively, and a 50% slurry of 30 uL antibody beads was spun with the above solution at room temperature for 90 minutes. rice field. The beads were washed 3 times in 25 mM triethylammonium bicarbonate buffer (TEABC, Fluka 17902). The bound tau was digested on beads with 400 ng of MS grade trypsin (Promega, V5111) at 37 ° C. for 16 hours. The digest was supported on TopTip C18 (Glygen, TT2C18.96), desalted and eluted according to the manufacturer's instructions. The eluted peptides were dried by vacuum centrifugation (CentriVap Concentrator Labconco) and resuspended in a solution of 25 uL of 2% acetonitrile and 0.1% formic acid in MS grade water.

Mass Spectrometry-MS analysis was performed by injecting a 5 uL aliquot of the peptide resuspension into nano-Acquiity LC. The nano-Acquiity LC (Waters Corporation, Milford, MA) was fitted with an HSS T3 75 um x 100 um, 1.8 um column, and peptides were separated using a gradient of solutions A and B at a flow rate of 0.5 uL / min. Solution A was composed of a solution of 0.1% formic acid in MS grade water and solution B was composed of a solution of 0.1% formic acid in acetonitrile. The peptide was eluted from the column with a gradient of 2% to 20% solution B for 28 minutes, then with a gradient of 20% to 40% solution B for an additional 13 minutes, and then elevated to 85% solution B in an additional 3 minutes. The column was washed. The Orbitrap Fusion Lumos was fitted with a Nanospray Flex electrospray ion source (ThermoFisher Scientist, San Jose, CA). Peptide ions sprayed into the ion source from a 10um Silicon Tip emitter (New Objective, Woburn, Ma) targeted the quadrupole and were separated at this quadrupole. Next, these were fragmented by HCD, and ion fragments were detected by Orbitrap (resolution 60,000, mass range 150 to 1200 m / z). Monitoring of hydrophilic peptides (SSRcal <9, all without leucine) for peptide profiling, HSS T3 300 um x 100 um, 1.8 mm column, 4 ul / min flow rate, 2% -12% solution B gradient. Performed with elution and spray running on a 30 mm Hydrotip emitter.

PRM Principles for P-Tau Peptide Detection-Digestion of proteins from tau-enriched biological extracts, such as soluble fractions of the human brain, to produce tau peptides containing the assumed phosphorylation sites. rice field. These peptides are screened by target MS analysis using a quadrupole-Orbitrap device such as Thermo Fisher Orbitrap Lumos. For each phosphorylated peptide, target MS parameters such as precursor mass, collision energy, or expected retention time were selected in Incilico and biophysics measured for the corresponding unmodified peptide with significantly higher abundance in the sample. It was refined using scientific properties. To detect the phosphorylated peptide, the quadrupole is set to select the mass window containing the assumed precursor mass. After collision of the precursors, all fragments produced over the entire chromatographic elution time are measured simultaneously in Orbitrap. Potentially phosphorylated peptides can be searched for by post-analysis of the obtained data. LC-MS / MS data was extracted using Skyline software (MacCoss Lab, University of Washington, WA). The hypothetical peptide being screened is detected as an LC-MS / MS fingerprint composed of exact co-elution of mass extracted from the predicted MS / MS fragment (Fig. 2). The advantage of this PRM method over conventional DDA or target MS methods is that the MS equipment can be used in the highest possible sensitivity configuration and the detection capability can be maintained.

The maximum sensitivity of the quadrupole-Orbitrap device during screening is essential for detecting small amounts of ion fragments, facilitating the identification of phosphorylated peptides as well as the location of phosphorylated sites. The sensitivity of PRM measurements depends primarily on the number of target-derived ions transferred into the Orbitrap analyzer. This number was increased by increasing the filling time used to obtain one MS / HRMS scan. This filling time corresponds to the time spent accumulating the target precursor mass from the ion beam to the ion trap device. The accumulated precursor is then fragmented and the resulting fragment is transferred into Orbitrap for analysis. Therefore, the fill time is a maximum value limited by the need for sufficient MS scan rates (ie, 8-15 scans per chromatographic peak) to obtain enough data points to describe the chromatographic signal. Is set to. The packing time for PRM screening for each phosphorylated peptide investigated was usually set to 1 second.

Filling time is also limited by the risk of trap saturation. This trap saturation occurs when too many ions are sampled in the same scan and are transferred to Orbitrap, resulting in inaccurate mass measurements due to the effects of space charge. To avoid such saturation, narrow precursor isolation (0.7 Da) is selected along with appropriate sample purification (immunopurification) to concentrate the target on the matrix to allow for potential near-isobaric interference (near-isobaric). The possibility of contribution of interference) was reduced.

The specificity of the PRM detection experiment depends on the resolution of the LC-Q Orbitrap system. The resolution can be improved by various analytical parameters and the ultimate goal is to obtain an LC-MS / MS fingerprint without interference of the target peptide. This limits the risk of ambiguity fragment attribution due to false positive signals. Purification of a sample that preferentially concentrates p-tau can also improve its specificity in detecting small amounts of phosphorylated tau peptide. High chromatographic peak volumes and resolution limit the possibility of co-elution. A narrow quadrupole separation window for the precursor reduces the probability of interference on the MS / MS spectrum and at the same time limits the risk of trap saturation described above. Orbitrap's high resolution and analyzer calibration allow accurate extraction of mass fragments during data processing, which limits the risk of transition interference (Gallien et al., 2012; Peterson et al., 2012). The choice of Orbitrap resolution (60k) is a balance between high resolution, which requires more acquisition time, and a reasonable scan rate that is compatible with chromatographic acquisition.

Quantitative Assessment of Tau Site-Specific Phosphorylation-We have detected each site to quantitatively assess the relative amount of phosphorylation of a particular site in brain and CSF-derived tau. The degree of phosphorylation was measured. The present inventors used the following three methods for this purpose. 1) Relative comparison between phosphorylated peptide isomers: Each phosphorylation site is identified by comparing signals derived from transition ions. This makes it possible to estimate the relative abundance of each phosphorylated peptide that shares the same sequence. 2) Non-phosphorylated peptide-based normalization of phosphorylated peptides: LC-MS / MS transitions specific for each phosphorylated peptide are compared with the corresponding transitions from non-phosphorylated peptides. The ratio of each phosphorylation site obtained can be compared over the entire protein sequence. This method can be biased by the difference in fragmentation efficiency between non-phosphorylated and phosphorylated peptides. This method is not applicable when the phosphorylation site is part of trypsin uncleaved. 3) Absolute quantification using labeled synthetic internal standards (eg, AQUA) for each phosphorylated and non-phosphorylated peptide: internal ratios are clarified using signals from phosphorylated and non-phosphorylated standards. This method takes into account the fragmentation specificity of each peptide to be compared, but requires peptide synthesis for each monitored species.

In this study, the phosphorylation rate was calculated using the second method described above, except for the phosphorylation site containing unclepsin. We also have a set of limited phosphorylation sites (ie, T175, T181, S199, S202,) that were present in both the brain and CSF extract when synthetic phosphorylated peptides were available. The third method (AQUA normalization) was also applied to T205, T217, and T231), and one site (S404) that existed only in the brain (Table 1). For AQUA measurements, the brain extract was diluted 500-8000 times to be comparable to the tau level of CSF. This dilution minimized the matrix effect that could result from significantly different ratios of the AQUA internal standard to tau peptide levels in the brain and CSF. The first method described above was used for early interpretation of complex LC-MS / MS patterns derived from p-tau sequences containing a large number of phosphorylated residues.

Statistics-Unless otherwise specified, data are expressed as mean ± SD. After confirming the normal distribution of the data, a post-hoc analysis (chuky test) was performed following the one-way ANOVA to compare the tau phosphorylation rates. Further statistical analysis can be found at GraphPad Software Inc. Completed using GraphPad version 8.0.1 (244). Statistical significance between related groups was determined by the two-sided, unpaired Mann-Whitney t-test. Significance was assessed at the 0.01 and 0.05 levels.

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Example 5 Pathophysiology of brain amyloid is associated with site-specific differences in tau hyperphosphorylation Cortical PiB-PET standardized uptake value ratio (SUVR) reliably identifies significant cortical Aβ plaques , Subjects are used to classify subjects as PIB positive (amyloid +, SUVR ≥ 1.25) or negative (amyloid-, SUVR <1.25). To investigate the relationship between amyloid plaques and soluble tau species, we used UVR of cortical PiB-PET to phosphorylate CSF tau at multiple sites (ie, non-tau tau). (Ratio of phosphorylated sites to phosphorylated sites)) (FIG. 16A). The sub-curve area (AUC) of tau phosphorylation (p-T217) in Thr217 to classify mutant carriers (MCs) as having cortical Aβ plaques (ie, amyloid +) was 97.2% (0. 95% confidence interval (CI) of 94, 0.99); AUC of tau phosphorylation at Thr181 (p-T181) is 89.1% (CI 0.83, 0.94); tau phosphorylation at Thr205 The AUC of (p-T205) was 74.5% (CI 0.69, 0.82); the total tau AUC was 72% (CI 0.65, 0.79). These data indicate that in the early stages of significant fibrotic Aβ-positive plaques, increased phosphorylation at specific sites has already begun and will eventually link these two processes. These data also show that an increase in the ratio of phosphorylation to non-phosphorylation of T217 serves as a sensitive diagnostic marker for fibrotic Aβ plaque pathology.

Next, in order to investigate the relationship between total Aβ plaque loading and phosphorylation, we investigated the average standardized phosphorylation ratio and total tau amount level of four phosphorylation sites by the PiB-PET SUVR quartile. Were compared (Fig. 16B). All phosphorylation sites except Ser202 (p-S202) showed increased phosphorylation with greater Aβ plaque pathology. We also found differences between the other three parts. p-T217 and p-T181 showed the greatest increase at the onset of plaque (quartiles 2-3), but the magnitude of these increases decreased with increasing plaque load; in contrast. , Phosphorylation and increased tau total levels at p-T205 continued to grow with Aβ plaques. These results suggest that the event that first leads to increased tau phosphorylation in AD may be associated with Aβ plaque pathology, presumably through the regulation of distinct kinases and phosphatases that are phosphorylation site-specific. Suggests that is high. In addition, the above data suggest that p-T217 may act as a surrogate biomarker for fibrotic Aβ plaque pathology and identify potentially unique signatures of Aβ-related tau treatment. Importantly, among non-mutation carriers (NC), the only subject who showed increased phosphorylation at T217 was PiB + (SUVR> 1.25, n = 4).

Next, we investigated the correlation between specific tau phosphorylation sites measured by PiB-PET SUVR and the cortical and subcortical regions of amyloid plaque deposits to investigate site-specific tau. We evaluated whether phosphorylation was associated with the anatomical distribution of Aβ plaque pathology in the brain (Fig. 16C). Phosphorylation at T217, T181, and T205 was positively correlated with Aβ plaques throughout the brain, whereas phosphorylation at S202 was negatively correlated. In the precuneus region of early amyloid plaque deposition, the correlation with tau isotypes was compared based on the intensity of bivariate regression that controls age, gender, and estimated age to onset (EYO). , Adjusted for multiple comparisons. The present inventors ^{have, p-T217 (β = 0.68} , p <10 -30), p-T181 (β = 0.46, p <10 -6), p-T205 (β = 0.41, We found the order of correlation with p < ^10-5 ) and total tau (β = 0.35, p <0.001), which had a positive correlation with Aβ plaque. In contrast, p-S202 had an inverse correlation (β = -0.47, p < ^10-7 ), suggesting that phosphorylation at this site decreases with increasing Aβ pathology. ing. These relationships exist in presymptomatic MC at a time when there is little evidence of neurodegeneration, and the increase in phosphorylated tau in CSF is not the result of passive release from dying neurons, but the presence of Aβ plaque pathology. It is likely to be an active process related to ²² .

Example 6 Stage and disease progression are associated with site-specific differences in tau hyperphosphorylation and long-term rate of change EYO (ie, by predictability of disease onset and symptom onset of DIAD) , Individual staging based on the age of the individual at the time of evaluation compared to the age of onset of other individuals with the mutation) 9, ^{10, 30} . Next, we determined whether there was a temporal difference in the pattern of phosphorylation of tau in CSF. This was done by estimating the time-dependent differences in the amount and rate of change of phosphorylation between MC and NC based on EYO. There were two important findings. First, there was evidence that total tau and increased phosphorylation at specific sites were inconspicuous. Phosphorylation of T217 occurred near -21EYO, followed by phosphorylation of T181 near -19EYO, followed by an increase in total tau near -17EYO, followed by phosphorylation of T205 near -13EYO (Fig. 17A). ~ F). The increase in phosphorylation in the early T217 and, to a lesser extent, the increase in T181 occurred at the same time (-19EYO) as the Aβ plaque began to increase. Second, the ratio of phosphorylation between T217 and T181 began to decrease significantly near the onset of symptoms, while phosphorylation at T205 remained elevated and total tau levels continued to increase. Notably, the concentration of all non-phosphorylated peptides increased with disease progression (data not shown), which means that the decrease in phosphorylation ratios of T217 and T181 was specific to these sites. It suggests that it is not the result of an imbalanced rise in non-phosphorylated peptides across. For S202, there was no significant change in phosphorylation over the course of the disease (Fig. 17E). These results indicate that tau phosphorylation occurs intermittently from site to site and increases or decreases at specific sites depending on the stage of the disease. These site-specific changes suggest that they are a chain of soluble tau changes that are more dynamic than previously recognized, and that the state or rate of tau phosphorylation does not increase monotonically. ing. The appearance of Aβ amyloid plaques and the onset of clinical exacerbations are some 20 years apart, indicating two important demarcations at the stage of altered phosphorylation of soluble tau.

Example 7 Neuroimaging markers for disease progression are associated with site-specific differences in high phosphorylation of tau In addition to estimating the onset of signs using family history (EYO), a variety of disease progression Disease progression of DIAD can also be estimated using neuroimaging measurements that track other factors, such as brain atrophy and decreased metabolism. These measurements vary during different pre-onset periods, with decreased brain metabolism (measured by [F18] fluorodeoxyglucose [FDG] -PET) occurring up to 18 years before the onset of symptoms and brain atrophy (measured by [F18] fluorodeoxyglucose [FDG] -PET). MRI) has been shown to occur up to 13 years before the onset of symptoms ^28,31-3 . This raises the question of whether these biomarkers also correlate with tau phosphorylation at specific sites. To investigate this, we present the phosphorylation site and total tau volume and imaging measurements of 34 cortical brain regions and 6 subcortical brain regions that control gender, age, and EYO. Bivariate cross-section correlation between was performed. We focused our analysis on asymptomatic MCs to identify any associations at the earliest stages of disease progression. The phosphorylation status of tau in S202 was not included in these analyzes, assuming that it did not change over disease progression.

MRI-hyperphosphorylation was inversely associated with cortical thickness in asymptomatic MC. That is, p-T205 was most strongly associated with reduced cortical and subcortical thickness throughout the brain (Fig. 18A), while p-T217 was less locally associated with total tau levels. The correlation was weak. The comprehensive correlation between hyperphosphorylation and cortical atrophy at p-T181 was the lowest, limited to the medial and lateral parietal lobes and the medial and medial frontal lobes. This suggests that the initial elevation of p-T205 in -13EYO may be related to the underlying pathogenesis of cortical atrophy, and we have previously found that cortical atrophy It has been revealed that it will start at approximately -13 EYO at the precuneus ²⁸ .

In addition to FDG PET-cortical atrophy, decreased glucose metabolism in neurons and glia is associated with disease progression in AD. Therefore, we tested whether there was a clear association between cortical or subcortical metabolic disorders and tau phosphorylation. In asymptomatic MC, phosphorylation at T205 was correlated with decreased glucose metabolism in the entire cortical and subcortical region as measured by FDG-PET (FIG. 18B). In asymptomatic MC, the association identified with respect to other p-tau sites or total tau levels was minimal.

Taken together, these results show that the latent processes in the progression of asymptomatic disease, which lead to neuropathy and neurodegeneration, as measured by neuroimaging, are most closely correlated with p-T205. ing. In a recent study, through the fyn kinase pathway, in response to the Aβ-induced postsynaptic cytotoxicity, the protective role against p-T205 in the postsynaptic terminal have been identified ^34. The associations found herein may represent a protective process that results in increased phosphorylation at T205 if synaptic function is impaired. However, over time, high phosphorylation can predispose to tau aggregation. On the other hand, phosphorylation at T181 and T217 appears to be more strongly associated with the presence of cortical Aβ plaque pathology, potentially increasing the levels of hyperphosphorylated and soluble non-phosphorylated tau of T205. It can occur upstream.

Example 8 Cognitive decline has a specific and differential association with site-specific differences in tau hyperphosphorylation In previous studies, AD dementia was associated with neocortical NFT pathology rather than neocortical Aβ pathology. Although closely related, ³⁵ , the relationship between soluble tau and cognition remains unclear. Accordingly, the present inventors have compared the clinical outcome was evaluated long-term changes in the phosphorylation ratio and tau total levels over time in soluble tau ^36. We found that long-term cognitive performance in neuropsychological composites as an outcome, as well as changes in tau measurements of CSF as predictors (derived from individual linear mixed-effects models), time, and their alternation. A mixed-effects model that included action was performed and adjusted for age, gender, education, and family relationships. We tested all MCs (symptomatic and asymptomatic) for this analysis and found the differential effect between phosphorylation sites and clinical and cognitive decline. Non-phosphorylated tau increased monotonically with cognitive deterioration, and phosphorylation at T217 and T181 decreased with cognitive deterioration, while pT205 showed no change in comparison with cognitive decline.

As the phosphorylation ratios of T217 and T181 decreased, cognitive decline accelerated (t-value 2.35, p = 0.02 and 2.11, p = 0.04) (Table 3 and FIG. 19). This suggests that the decrease in phosphorylation of T217 and T181 is greater than the increase in soluble tau and is an important marker of cognitive decline.

These findings disagree with the current paradigm that continuous elevation of CSF p-tau is associated with cognitive dysfunction. In this study, we have revealed two general patterns. That is, for some sites, phosphorylation decreased significantly as cognitive decline began, while for others, it showed a continuous increase or no change as the disease progressed. (See rate of increase and rate of decrease in FIG. 19). In addition, the process leading to tau phosphorylation in AD by the association of other disease progression markers (Aβ plaques, brain atrophy and metabolism) identified by us with tau total volume levels and phosphorylation at different sites. Is likely to be multifactorial, further emphasizing that they are not equivalent.

Example 9 Increase in non-phosphorylated tau correlates with cortical NFT by tau PET but not standard phosphorylated tau species by tau PET ( ¹⁸ F AV-1451, ie flotaucipir) for recent DIAD subjects. Studies suggest that an increase in NFT occurs only after the onset of clinical symptoms ^{37, 38} . We tested the hypothesis that soluble p-tau is a marker of NFT pathology, while soluble non-phosphorylated tau is passively released from dying neurons ^{14 as a measure of neurodegeneration.} We investigated the relationship between long-term changes in CSF tau and p-tau isoforms leading to the time of tau PET. A limited number of subjects (10 MCs and 4 NCs) underwent one tau PET scan within 72 hours of obtaining the CSF sample. For these individuals, CSF samples were also obtained during previous visits (within 1-3 years) to determine if tau PET levels could be predicted by long-term changes in soluble tau measurements. , The present inventors have become able to evaluate.

First, we confirmed that cortical NFT levels in MC increased only near the time of onset of symptoms (Fig. 20), which means that in DIAD MC, tau aggregates increased. It suggests that clinical deterioration begins when it begins. Second, we found that a long-term increase in non-phosphorylated tau of CSF was associated with an increase in cortical tau PET (p = 0.03) levels (Table 4). Increased phosphorylation of T205 tends to be associated with higher levels of tau PET, and conversely, decreased phosphorylation of T217, T181, and S202 is associated with increased levels of tau PET. There was a tendency that there was (Fig. 21). Taken together, these findings suggest that an increase in non-phosphorylated tau in CSF rather than p-tau is more closely linked to the spread of NFT pathology. In contrast, increased aggregated tau decreased soluble p-tau species, which may indicate that the isolation process is due to highly phosphorylated aggregates.

Discussion on Examples 5-9 Tau constitutes a characteristic of AD pathology and can be measured in aggregated or soluble form, but post-translational modification of this important neuroprotein is the development of NFT and neurodegeneration in humans. There remains a significant gap in our interpretation of how to connect to ^2. Here, we show how the pattern of tau phosphorylation in CSF changes during AD progression. In DIAD, the present inventors stated that the process of phosphorylation of tau and its release into the central nervous system is as follows: 1) Aβ plaque load (measured by PiB-PET) is established (several decades before onset). It begins and then develops over a period of nearly 20 years, during which different phosphorylation sites of tau protein are phosphorylated at separate stages with different markers of disease progression; and 2) cognitive decline and aggregated tau. Add proof to the existing clinical literature that it is a dynamic process that significantly decreases in a site-dependent manner near the time of ascent (measured by tau PET). Taken together, these results show that this method of quantifying soluble phosphorylated / non-phosphorylated tau peptides traces the AD process from preclinical to symptomatic stages and provides a signature of phosphorylated tau pathology in this disease. Indicates that it can be provided. Moreover, these results disagree with DIAD, and perhaps the role of tau / p-tau in AD in general, and are not the result of hyperphosphorylation of tau and the release of dying neurons in Aβ pathology. ^{Findings 21} , 23, 25, 39 from animal studies linked to active cellular release have been reproduced in humans.

Causality needs to be addressed in future studies, but the simultaneous increase in p-T217, p-T181, and PiB-PET is closely linked to the widespread levels of tau phosphorylation in AD with Aβ pathology. It suggests that. This hypothesis, p- tau isoform is ²² that is meant to be released from cells in the active process that increases in the presence of Aβ plaques, studies in recent AD transgenic mice ^{20,21,23,34 , 40} and consistent with studies in Stable Isotopic Labeling Dynamics (SILK). According to the results of the present inventors, Aβ pathology is associated with a clear change in soluble tau peptide concentration and phosphorylation pattern, and a significant increase in p-tau occurs in AD but not in other neurodegenerative tauopathy16 ^{. 17} is elucidated. These findings also provide important insights into potential therapeutic targets as well as biomarkers of early AD pathology before the onset of clinical manifestations.

Recent studies have shown that neurite tau aggregates (anti-spiral reticular fibers in dystrophic neurites) induced by NFT isolates from the brain of AD in Aβ-transfected mice, which occur long before somatic NFT colonization. ²⁰ increase and spread of) are revealed. The very early increase in p-T217 and p-T181 may reflect this "early" tau aggregation in response to Aβ plaques, which identified PiB PET as the inventors. It may be possible to explain the general association with these isotypes. Moreover, since the increase occurs years before significant neurodegeneration, the increase may better explain the absence of clinical symptoms during this early rise in p-tau. .. This study also proposes p-T217 as a very early biomarker with promising uses as an indicator of therapeutic response of therapeutic agents targeting amyloid. This may be particularly important in prophylactic studies where surrogate markers of disease progression are essential to identify effective therapeutic agents.

This specification casts doubt on some general assumptions about the diagnostic role of soluble tau and p-tau. ¹⁴ More specifically, in the diagnostic framework in the current of AD, specific and biomarkers representative of the non-specific pathology in AD (e.g., the A [beta], p-tau and tau) the presence of is highlighted. Within the framework of this diagnosis, soluble p-tau and non-phosphorylated tau are often assumed to be passively released from degenerating neurons, and p-tau is associated with aggregated NFT and non-phosphorylated. Oxidized tau is associated with axonal degeneration. Instead, our results in DIAD suggest that AD tauopathy may be defined as a change in the phosphorylation state (ratio of phosphorylation) of soluble tau. Furthermore, considering the timing of state changes at specific phosphorylation sites (21 to 13 years before the presumed onset of symptoms), the above results show that increased phosphorylation is a marker of pathology, but is not necessarily tau-related toxicity. It also suggests that it is not a marker for cell body NFT. In fact, our results show that for pT217 and pT181, when the NFT pathology is rapidly increasing, there is a dramatic decrease in phosphorylation rather than the ^{ever-increasing 37.} One possible explanation for this is similar to that observed for soluble / aggregated Aβ ⁴¹ , i.e., a dramatic increase in aggregated tau causes phosphorylated tau to be sequestered in the brain at CSF levels. Is to decrease. However, the decrease in tau due to the mechanism of protein homeostasis cannot be ruled out. In either case, our findings on the negative correlation between the phosphorylation ratio of T217 or T181 and long-term cognitive decline underscore the importance of this event in disease progression. .. Elucidation of the cause of this decline may provide a better understanding of the association between soluble tau and neuronal dysfunction and the use of CSF p-tau / tau in the prognosis of AD.

Given the role of kinases in the phosphorylation of tau, the enzymes are currently considered potential targets for treating AD agent ^43. Not all forms of p-tau are associated with markers of disease progression herein, and some may actually have the opposite effect. Certain kinase / phosphatase activities that lead to changes in p-tau may not be detrimental, at least early in the process of the disease.

In summary, we present here that in AD associated with autosomal dominant mutations, hyperphosphorylation of CSF tau occurs very early, and this hyperphosphorylation is site-specific at different stages. It has been demonstrated to show patterns of change. The mechanisms underlying these findings will have important implications for the understanding of the disease and for tau-oriented AD therapeutics.

Materials and Methods of Examples 5-9 Subjects-Subjects at least 50% at risk of inheriting a DIAD mutation from a family member with a confirmed gene mutation in PSEN1, PSEN2, or APP were the Dominontly Inherited Alzheimer Network Study (DIAN, NIA). It was registered in U19 AG032438) (dian.wustl.edu; clinicaltrials.gov number NCT000869817) ^44. All procedures were approved by the Institutional Review Board (IRB) of Washington University and were compliant with the IRB and Ethics Committees in the area where the study was conducted. The presence or absence of DIAD mutation was determined using appropriate exon PCR-based amplification followed by Sanger sequencing. Subjects underwent a comprehensive clinical assessment, cognitive test, neuroimaging test, and CSF survey each time they visited for study. However, each subject may not have completed all study procedures at the time of each visit. Details of the structure and evaluation of the study are described in ^{Publications 10, 44.} The follow-up interval was determined by the clinical status (normal or impaired) of each subject and the estimated number of years to onset of symptoms (EYO) and ranged from once a year to every three years. Data were taken from quality controlled data (annual quality assessment of irregular results and missing data from January 26, 2009 to June 30, 2017) and included 370 subjects (3 subjects). The subjects who underwent long-term CSF evaluation were n = 150, and the median interval between visits was 2.8 years)).

Estimated years to onset of symptoms (EYO) -There is almost 100% penetration in dominant hereditary AD, and the age of onset of symptoms in mutation carriers is relatively consistent for each mutation and within each family. This makes it possible to indicate the estimated number of years (EYO) until the onset of symptoms. EYO was defined as follows. That is, for each subject, the age at the earliest onset of symptoms of the parent was determined by a semi-structured interview. The age of parental onset for each mutation was then entered into a database consisting of a combination of published symptom onset values from the DIA and DIAD cohorts. Using these values, the average age of onset specific to each mutation was calculated ²⁹ . The age of onset specific to the mutation was subtracted from the age at the time of clinical evaluation of each subject to define the individual EYO. If the mean age of onset of a particular mutation is unknown, EYO was defined using the age of onset of the parent or surrogate ²⁹ . For subjects who were symptomatic at baseline, assessed by CDR> 0, EYO was defined by subtracting the reported age of actual onset of symptoms from the age of each clinical assessment.

Clinical Assessment-Each subject underwent a standardized clinical assessment, including the use of a study partner. The clinical dementia rating scale (CDR) was used to indicate the stage of dementia. Subjects were rated as cognitively normal (CDR = 0) or very mild dementia (CDR = 0.5), mild dementia (CDR = 1), or moderate dementia (CDR = 2) ^45. .. The genetic status was blinded to the assessing clinician. At each Oho, general cognitive function, memory, attention, executive function, visuospatial functions, and was a comprehensive neuropsychological battery for evaluating the language ^46. The present inventors, from these tests were developed cognitive composite to reliably detect a decrease in the overall range of EYO and CDR ^47. The composite represents an average z-score based on a test (Mini Mental State Examination) that includes episodic memory, complexity attention, and processing speed, as well as screening for general cognitive function.

Analysis of CSF tau-CSF was collected in two 13 ml polypropylene tubes by a standard lumbar puncture procedure (L4 / L5) with a non-traumatic Sprotte spinal needle (22Ga). The CSF was snap frozen on dry ice in an upright position. Samples collected in the United States were collected on dry ice at night at Washington University, St. Louis. While transported to the DIA biomarker core laboratory of Louis, MO, USA, samples collected at global sites were stored at -80 ° C and transported quarterly on dry ice. Upon arrival, each sample was thawed, grouped in a single polypropylene tube, and divided into polypropylene microcentrifuge tubes (Cat. No. 05-538-69C, Corning Life Science, Corning, NY, USA) (500 μl each). Then, these were rapidly frozen on dry ice again and stored at −80 ° C.

Each thawed CSF sample was mixed with a 25 ml solution containing 15N-441 tau internal standard (2.5 ng per sample), 50 mM guanidine, 10% NP-40, and 10X protease inhibitor cocktail (Roche). Tau is extracted by immunocapture using incubation under rotation for 2 hours at room temperature with 20 ml Sepharose beads crosslinked with Tau 1 (Tau epitopes 192-199) and HJ8.5 (Tau epitopes 27-35) antibodies. bottom. The beads were centrifuged and then rinsed 3 times with 1 ml of 25 mM TEABC. Samples were digested overnight at 37 ° C. with 400 ng of trypsin Gold (Promega, Madison, WI). AQUA peptides (Life Technologies, Carlsbad, CA) were spiked to give 5 fmol per labeled phosphorylated peptide and 50 fmol per labeled unmodified peptide in each sample. The peptide mixture was loaded onto a TopTip C18 chip, washed with 0.1% formic acid (FA) solution and eluted with 60% ACN 0.1% FA solution. The eluate was dried using Speedvac and the dried samples were stored at −80 ° C. and then analyzed. The sample was resuspended in 25 μl of 2% ACN 0.1% FA solution. Extracts were analyzed by NanoLC-MS / HRMS using Parallell Reaction Monitoring with HCD fragmentation. NanoLC-MS / MS experiments were performed using a nanoAccity UPLC system (Waters, Milford, Massachusetts) coupled with a Fusion Tribrid mass spectrometer (Thermo Scientific, San Jose, Calif.). 5 μl was injected for each sample. Peptide separation was performed on a Waters HSS T3 column (75 μm × 100 mm, 18 μm) at 60 ° C. for 24 minutes. The mobile phase was (A) 0.1% formic acid aqueous solution and (B) 0.1% formic acid acetonitrile solution. The gradient used was 0 min-: 0.5% B; 7.5 min-: 5% B; 22 min-: 18% B; then the column was rinsed with 95% B for 2 minutes. is. The flow rate was set to 700 ln / min for 7.5 minutes and then 400 ln / min for subsequent analysis. Data were obtained in cation mode with a spray voltage of 2200 V (Nanospray Flex Ion Source, Thermo Scientific) and an ion transfer tube set at 270 ° C. The S lens RF voltage was set to 60V. MS / HRMS transitions were extracted using Skyline software (MacCoss lab, University of Washington). Tau phosphorylation levels of CSF were calculated using the measured ratio between MS / HRMS transitions of the endogenous non-phosphorylated peptide to the protein internal standard 15N labeled peptide. The ratio of phosphorylation of T181, S202, T205, and T217 was measured using the ratio of MS / HRMS transitions from the phosphorylated peptide to the corresponding non-phosphorylated peptide. The ratio of each endogenous phosphorylated / non-phosphorylated peptide was normalized using the ratio measured on the corresponding AQUA phosphorylated / non-phosphorylated peptide internal standard MS / HRMS transition.

Brain Imaging-Amyloid Deposition, Glucose Metabolism, Tau (NFT) PET, and Cortical Thickness / Subcortical Volume are ¹¹ C-PiB-PET, ¹⁸ F-FDG-PET, ¹⁸ F-AV-1451 ( Also known as fludeoxypill), and volumetric T1-enhanced MRI scans were used for evaluation. ^{Standard procedure 28} was used to ensure consistency in data collection for all DIAN sites. ^{The 11} C-PiB-PET scan consists of a bolus injection of approximately 13 mCi of PiB followed by a 70 minute dynamic scan, which provides a standardized uptake value ratio (SUVR) of the region from a 40-70 minute timeframe. I asked. ^{The 18} F-FDG-PET scan started 30 minutes after the bolus injection of about 5 mCi and continued for 30 minutes. ¹⁸ F-AV-1451 data was taken from a window 80-100 minutes after bolus injection and converted to SUVR. The T1 MR sequence was performed with an acknowledged magnetization-preparated rapid acceleration with gradient echo (MPRAGE) acquired with a 3T scanner (parameters: TR = 23000, TE = 2.95, and 1.0 × 1.0 × 12 mm ^{3 resolution).} there were.

PIBs and FDG SUVRs were obtained from 34 cortices and 6 subcortical target areas (ROIs) using FreeSurf software (surfer.nmr.mgh.harvard.edu/). The SUVR was processed using cerebellar gray matter as the reference region, and the ROI data were corrected for the partial volume effect using the ^{region spread function (RSF) 48 in the geometric transfer matrix (GTM) framework.}

Statistical analysis-Subject baseline characteristics were summarized as mean ± SD for continuous variables and n (column percentage) for categorical variables. Baseline-defined P-values for comparing differences between asymptomatic MC, symptomatic MC, and NC provide logistic links for general linear mixed-effects models (LMEs) for continuous variables and categorical variables. Obtained using the generalized linear mixed-effects model provided. All models incorporated random family effects to account for the correlation of outcome measurements between subjects within the same family. Baseline cortical PiB PET SUVR cut points are selected such that the difference in long-term rate of change in cortical PiB PET between MC and NC is initially significantly different from zero.

PiB, FDG, and cortical thickness / subcortical at various phosphorylation sites of tau in all asymptomatic MCs (CDR = 0, n = 152) using a bivariate LME for each ROI. The cross-section correlation with the volume of was evaluated. The models included fixed effects of EYO, education, gender, and random interception at the family level. Bivariate LME, compared with the method of estimating the simple correlation (Pearson or Spearman correlation), not only can be tailored to covariates such EYO, it is also possible to consider the correlation within the family cluster ^{49 , 50} . The Benjamini-Hochberg method ⁵¹ was used to correct the P-value for testing the correlation to control the false positive rate due to the multiple test.

LME is used to estimate the best linear unbiased predictions for each biomarker with respect to the annual rate of change within the individual over long-term follow-up, and then plotted against baseline EYO to plot the biomarker trajectory. investigated. Then, where appropriate, a linear or linear spline mixed effect model was used to determine the baseline EYO points at which the baseline level and rate of change of each biomarker in MC would be significantly different from NC. Details of the linear spline mixed-effects model can be found in recent publications ⁹ . Linear or linear spline mixed effect models include mutant groups (MC or NC), baseline EYO, elapsed time from baseline, and all possible primary or secondary interactions between them. Includes fixed effects. Gender, years of education, and APOE ε4 status were considered covariates, but only significant effects were retained in the model. Random effects included in the model include random interception of family clusters, random interception of individuals, and random gradients in unstructured covariance matrices to account for intra-objective correlations due to frequent measurements. be. The adjusted difference in mean level at baseline and the difference in rate of change between MC and NC were then tested using a model-derived approximate t-test and the first EYO point where the difference was significant. Was judged.

To visualize the difference in rate of change between total tau, tau phosphorylation site, cortical PiB, and general cognitive function across the EYO range, MC measurements were first taken with NC mean and standard deviation. Was standardized using. The rate of change of each measured value for each MC was then calculated using LME and the LOESS curve was approximated to visually represent the trajectory of the standardized rate of change relative to EYO.

LME was used to assess baseline total tau and p-tau usefulness in predicting long-term cognitive decline. The fixation effects of the above model include the fixation effect of the mutant group, baseline age, gender, APOE ε4 status, time, and all possible primary or secondary interactions between them. Is done. Random effects in the above model include random interception of family clusters, random interception of individuals, and random gradients in unstructured covariance matrices.

Can linear regression be used to predict the tau PET SUVR and control the effect of age by the annual rate of change in the positions of MC and NC tau and phosphorylated tau leading to and including the time of tau PET? I checked. Family clusters were not included due to the limited number of subjects.

All analyzes were performed using SAS 9.4 (SAS Institute Inc., Cary, NC). A p-value <0.05 was considered statistically significant.

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Example 10
Alzheimer's disease (AD) is the leading cause of dementia worldwide. Diagnosis and treatment of AD is still extremely difficult in the absence of specific and early biomarkers. Recent developments in cerebrospinal fluid (CSF) biomarkers and positron emission tomography (PET) imaging to detect the pathophysiology of pathological AD with amyloid Aβ plaques and hyperphosphorylated tau changes in neurofibrils. A useful tool was provided. Currently, the CSF profile of AD patients is characterized by a decrease in amyloid beta 42 (Aβ42) and an increase in total tau and phosphorylated tau (p-tau 181) as measured by standard immunoassays. This profile can distinguish between AD and non-AD pathologies and detect the AD process many years before cognitive symptoms and complaints. However, changes in CSF tau and p-tau are not unique to AD. The increase in p-tau 181 has been interpreted as a result of high phosphorylation due to concentrate formation, but the p-tau in CSF increases at the same time as the total amount of tau. Although brain studies have shown tau phosphorylation at many sites, the diagnostic validity of these additional phosphorylation sites in CSF has not been adequately addressed. Mass spectrometry (MS) -based methods allow the independent quantification of phosphorylated peptides and their corresponding unmodified equivalents, thus allowing phosphorylation of specific sites rather than immunoassays. Suitable for assessing level changes independently of tau total level. Therefore, the rate of phosphorylation can be assessed by comparing the correlation between the gradients of phosphorylated and non-phosphorylated peptides. We use an innovative target high resolution MS (HRMS) method to target the phosphorylated peptide of tau in the middomain of the protein sequence to quantify the phosphorylated tauiso form in the CSF. board. The middomain is the most abundant domain in CSF and is phosphorylated at many sites in the aggregates of brain tau and AD tau.

We used a cohort of individuals with cognitively normal and patients with mild cognitive impairment stratified by CSF Aβ42 / 40 ratio and amyloid status based on PET-PIB imaging during CSF. Tau phosphorylation at T181, S199, S202, T205, and T217 was analyzed. This validation allows us to emphasize the potential of CSF pT217 for the diagnosis of AD and establish a correlation between pT217 and amyloidosis, which is the basis of tau modification in the early stages of the disease. became.

Subject-Patterson et al. From the Washington University in St. Louis ADRC study, reported by 2015 and available for CSF and amyloid PiB-PET data, 86 subjects with normal cognitive function (CDR = 0) or mild cognitive impairment I recruited. PiB-PET (considered positive above 0.18 used as cutoff) and Aβ42 / Aβ40 ratio of CSF measured by MS (considered pathological below 0.12 used as cutoff) The results showed that this cohort included 29 amyloid-positive and 47 amyloid-negative subjects, as well as 10 (5 PiB-) with conflicting results between PET-PIB and CSF profile. PET (+) / CSF (−) and 5 cases of PiB-PET (−) / CSF (+) amyloidosis profile) were included. Volatility was evaluated by extracting 7 CSF samples at n = 2 and 1 at n = 3.

Purification of CSF Tau Using Immunoprecipitation-800 μl of CSF supernatant obtained after -Aβ immunoprecipitation and storage at -80 ° C was used for tau analysis. The thawed supernatant was spiked with a 15 N tau internal standard (5 ng per sample) and extracted using tau 1 immunoprecipitation. To this sample was added 5 mM guanidine, 1% NP-40, and a protease inhibitor cocktail, and the sample was mixed with 20 μl of tau 1 antibody and crosslinked Sepharose beads for 3 hours at room temperature. The beads were precipitated and then rinsed with 0.5 M guanidine and 25 mM TEABC. The sample was digested with 400 ng of trypsin. AQUA peptides (Life Technologies, Carlsbad, CA) were spiked to give individual amounts of 10 fmol per labeled phosphorylated peptide and 100 fmol per labeled peptide per sample. AQUA TPSLPpTPPPTR (pT217) was used in place of the uncleaved peptides used in the discovery cohort. The trypsin-cleaving peptide was loaded onto a TopTip C18 chip, washed with 0.1% FA solution and eluted with 60% ACN 0.1% FA solution. The eluate was dried with Speedvac. The sample was stored at −80 ° C. Prior to LC-MS analysis, the sample was resuspended in 25 μl of 2% ACN 0.1% FA solution. Extracts were analyzed by nanoLC-MS / HRMS.

Quantification of Tau Peptides and Phosphorylated Peptides (Verification) Absolute levels of unmodified peptides were calculated using a stable isotope-diluted MS quantification method using -15N-labeled peptides. The phosphorylation rate of each site was calculated by one-point calibration comparing the area ratios obtained for the non-phosphorylated and phosphorylated peptide equivalents of AQUA to the area ratios measured for the corresponding endogenous peptides. Phosphorylated peptide levels were calculated by combining the counterparts of unmodified peptide levels with the phosphorylation rates of the corresponding sites.

Statistics-Statistical analysis, including comparison of regression line slopes, was performed using GraphPad Prism software (7.0). A nonparametric Mann-Whitney test was used to calculate the statistical significance between the numbers obtained from the entire study group. The nonparametric Spearman's rank correlation coefficient ρ was used to evaluate the correlation between two series of numbers. Statistical significance was defined as p <0.05.

Quantification of Tau Phosphorylated Peptide in CSF (Results) -A cohort of 86 subjects with no or mild cognitive impairment in Washington University in St. Louis' Knight AD Research Center (ADRC), T217 in AD. Increased tau phosphorylation was detected in. Subjects conflicted between 29 amyloid-positive, 47 amyloid-negative subjects, and PET-PIB and CSF profiles, consistent with the results of the Aβ42 / Aβ40 ratio of CSF measured by PiB-PET and MS. The results were stratified into 10 cases. Immunopurification with the Tau 1 antibody was performed to improve the sensitivity of the assay. Site phosphorylation rates were measured using isotope-labeled versions of both phosphorylated and non-phosphorylated peptides. All targeted phosphorylated peptides were detected in all samples, except for pS199-containing peptides that were not recovered by the Tau 1 antibody. In this cohort, we validated a positive diagnosis of the pT217 biomarker in the early stages of the disease. The pT217 / T217 ratio clearly separated the amyloid-positive and amyloid-negative groups (FIGS. 24B and 24C, AUC 0.999). Furthermore, the measured values of the pT181 / T181 ratio distinguished the amyloid-positive group from the amyloid-negative group (Fig. 24B, AUC 0.956). The phosphorylation ratios of T217 and T181 are also correlated (r = 0.524, p = 0.0002), suggesting that phosphorylation of these sites may be derived from a common pathway. doing. However, the diagnostic sensitivity of pT181 was lower than the diagnostic sensitivity of pT217. Both phosphorylation ratios were better discriminators than the p-tau (181) and t-tau levels measured by ELISA (AUC 0.874 and 0.932, respectively).

Correlation between T217 hyperphosphorylation, amyloid pathology, and cognitive status (results) -Next, we determined the correlation between this new biomarker and the amyloid process. The results of the ADRC cohort suggested a strong association between hyperphosphorylation of T217 and amyloid status. Importantly, the phosphorylation rate of T217 was significantly correlated with the degree of PiB-PET measured by FBP Total Cortical Mean (FIG. 24E, r = 0.60, p = 0.001). In addition, all five competing cases of the PiB-PET (+) / CSF (-) amyloidosis profile described above were highly phosphorylated at T217 (FIGS. 24D, E). In contrast, no significant correlation was found between T217 phosphorylation and CSF Aβ42 / Aβ40 ratio. Since the MS ratio of Aβ42 / Aβ40 in the corresponding CSF was close to the quantification threshold (0.12 ± 0.02, FIG. 24E), the discrepancy between PiB-PET and CSF Aβ42 / Aβ40 was due to the CSF amyloid. It may have been due to insufficient sensitivity of the assay. Of the five subjects with reverse PiB-PET (−) / CSF (+) amyloidosis described above, two showed hyperphosphorylated T217 (FIGS. 24D, E). This suggests that in these cases highlighted by high phosphorylation of tau, significant changes in Aβ of CSF may occur before amyloid plaque deposits are detected. .. Due to the combination of Aβ42 / Aβ40 ratio of CSF and phosphorylation rate of T217, there is no PiB-PET data, and the value of Aβ42 / Aβ40 ratio of CSF is slightly higher than the threshold value (0.12 ± 20%) in the medium range. Even in some cases, amyloid-positive subjects are reliably identified. Among subjects without cognitive complaints (CDR-SB = 0), amyloid-positive subjects (n = 9) were replaced by amyloid-negative subjects (n = 26, AUC 1.00, FIG. 25) by phosphorylation of T217. ), And further confirmed that this marker can reliably identify preclinical AD subjects. However, no correlation was observed between general cognitive performance measured by CDR-SB and the phosphorylation rate of T217 in this population (Fig. 25).

Discussion-We use an innovative target HRMS method to simultaneously measure low abundance of phosphorylated tau peptides and their unmodified counterparts in CSF, which occur simultaneously with amyloid changes and CSF. For the first time, we present direct evidence of changes in the phosphorylation rate of tau in CSF, which is different from the increase in tau concentration in CSF. This technique allows the study of AD-specific tau phosphorylation rates to assess high and low phosphorylation, which indicate the underlying abnormal metabolism.

The most striking results of this study are highly specific for T217 in tau of CSF from preclinical, mild, and moderate AD subjects investigated in two independent, well-characterized cohorts. High phosphorylation. The pathophysiology of amyloid, which is likely to occur before AD tauopathy, and the high phosphorylation of tau at T217 were strongly associated throughout the stage. This supports the hypothesis that amyloidosis may be associated with altered tau phosphorylation. In this study, the absence of subjects with Aβ-amyloidosis and normal phosphorylation of T217 suggests that this tau phosphorylation site may follow the amyloid process. High phosphorylation of T217 may play an important role in the pathophysiological processes of AD, a role different from other tau biomarkers. In our cohort, increased levels of tau or p-tau in CSF as assessed by ELISA were less effective than the pT217 / T217 ratio in identifying amyloidosis status. The specificity of these AD tau biomarkers may improve the prediction of the risk of cognitive decline in individuals who eventually progress to clinical AD. Therefore, detecting AD in combination with amyloidosis biomarkers in combination with pT217 / T217 ratio measurements should dramatically improve the diagnosis of preclinical AD. High phosphorylation of T217 correlates more with amyloid plaques measured under PiB-PET loading than with amyloid markers in CSF, but tau modification is not solely caused by the presence of fibrillar plaques. The above two subjects (FIG. 24E) with no cerebral amyloid load, high phosphorylation of T217, and low CSF Aβ42 / 40 were not detected by PiB-PET, but decreased CSF Aβ42 levels. May be a case of only contributing diffuse plaque or Aβ oligomer formation.

The widespread use of pT181 as an AD marker in clinics is likely due to a high level of detectability, not necessarily its high specificity. The pT181 measurements highlight a slight change in the stoichiometry of pT181 phosphorylation in AD compared to non-AD dementia. The increase in the phosphorylation rate of T181 was even more pronounced in the well-characterized amyloid-positive group examined in the validation cohort. In both studies, the combined ratio of t-tau and p-tau 181 by ELISA could not demonstrate the changes in phosphorylation ratio that occur at T181, which allows ELISA to accurately monitor these changes. The limits of that were emphasized. Therefore, the widely reported increase in pT181 in the CSF of AD is primarily due to the concomitant increase in tauiso-type levels, rather than a significant change in stoichiometry of T181 phosphorylation. The pathology of amyloid Aβ induces hyperphosphorylation at T181, but the resulting changes appear to be less specific or less significant compared to the relative increase observed at T217.

The changes in phosphorylation chemistries observed at S199, S202, and T205 in the CSF of AD may reflect certain changes in tau phosphorylation previously observed in the brain of AD. .. In fact, pS202 and pT205 are some of the double phosphorylated epitopes recognized by the AT8 antibody. AT8 binds to tau aggregates found in the anatomy of the brain of AD, and the degree of AT8 immunoreactive aggregates correlates with the severity of tauopathy (Brac stage). AT8 has no responsiveness to AD-related tau as measured in normal tau or CSF. Furthermore, the tau 1 antibody that binds to normal tau with a non-phosphorylated epitope containing S199 has no affinity for AD tau aggregates. Taken together, these findings support the high phosphorylation of co-occurring and abundant S199, S202, and T205 in AD brain-derived tau aggregates. However, there is controversy over the unique properties of the phosphorylated tauiso form that promote tau aggregation in AD. The reduction in the amount of phosphorylation of S199 and S202 observed in CSF with mild and moderate AD is consistent with the accumulation of corresponding phosphorylated tauiso forms in the aggregate. In addition, the detection of T205 phosphorylation primarily in moderate AD CSF suggests an important role for this site in the underlying pathological mechanism of amyloid-related tauopathy. Changes in S202 / T205 phosphorylation were not detected in a second cohort consisting of preclinical and mild AD subjects (data not shown).

This finding may suggest an interaction between AD amyloidosis and T217 and, to a lesser extent, high phosphorylation of tau at T181. Both of these sites are substrates for proline-directed serine / threonine kinase GSK-3, and activation of GSK-3 by Aβ oligomers has been proposed as a link between phosphorylation of amyloid peptides and tau. General and relatively highly correlated hyperphosphorylation at these two GSK-3 sites in patients with amyloidosis may be consistent with this mechanism. Further studies designed to compare tau aggregate-containing CSF and tau phosphorylation rates in the brain as measured by tau PET may provide new insights into the pathophysiology of AD and new treatments. May specify the method. These findings are directed to specific links between amyloid plaques and tauopathy in AD and provide potential links in the chain of molecular events leading to AD. Therefore, given the specificity of pT217 within the AD process, pT217 is an interesting target for future therapeutic drug development and for tracking the potential effects of anti-amyloid drugs that limit this aberrant tau metabolism. May represent a tool.

Claims (137)

A method of diagnosing a subject before the onset of Alzheimer's disease.
(A) Prepare isolated tau samples obtained from the subject and measure tauphosphorylation at (i) T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and T217; And (b) conversion of the subject to mild cognitive impairment due to Alzheimer's disease when the taurination at T217 or T181 is about 1.5σ or more and the taurination at T205 is about 1.5σ or less. Diagnose high risk, but σ was measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques, T217 and T205, T181 and The standard deviation defined by the normal distribution of taurination at T205, or T181, T205, and T217.
The method described above. The method of claim 1, wherein the subject is diagnosed when taurine oxidation at T217 is greater than 1.5σ and taurine oxidation at T205 is less than 1.5σ. (I) Taurine oxidation at T217 is greater than 1.75σ and taurine oxidation at T205 is less than 1.75σ, (ii) Taurine oxidation at T217 is greater than 1.8σ and at T205. The subject is diagnosed if taurine oxidation is less than 1.8σ, or (iii) taurine oxidation at T217 is greater than 1.9σ and taurine oxidation at T205 is less than 1.9σ. The method according to claim 2. The method of claim 2, wherein the subject is diagnosed when taurine oxidation at T217 is greater than 2σ and taurine oxidation at T205 is less than 2σ. The method of claim 1, wherein the subject is diagnosed when taurine oxidation at T181 is greater than 1.5σ and taurine oxidation at T205 is less than 1.5σ. (I) Taurine oxidation at T181 is greater than 1.75σ and taurine oxidation at T205 is less than 1.75σ, (ii) Taurine oxidation at T181 is greater than 1.8σ and at T205. The subject is diagnosed if taurine oxidation is less than 1.8σ, or (iii) taurine oxidation at T181 is greater than 1.9σ and taurine oxidation at T205 is less than 1.9σ. The method according to claim 5. The method of claim 5, wherein the subject is diagnosed when taurine oxidation at T181 is greater than 2σ and taurine oxidation at T205 is less than 2σ. The method of claim 1, wherein the subject is diagnosed when taurine oxidation at T181 and T217 is greater than 1.5σ and taurine oxidation at T205 is less than 1.5σ. (I) Taurine oxidation at T181 and T217 is greater than 1.75σ and taurine oxidation at T205 is less than 1.75σ, (ii) Taurine oxidation at T181 and T217 is greater than 1.8σ. And when the taurine oxidation at T205 is less than 1.8σ, or (iii) the taurine oxidation at T181 and T217 is more than 1.9σ and the taurine oxidation at T205 is less than 1.9σ, the above. The method of claim 8, wherein the subject is diagnosed. The method of claim 8, wherein the subject is diagnosed when taurine oxidation at T181 and T217 is greater than 2σ and taurine oxidation at T205 is less than 2σ. The method of any one of claims 1-10, wherein the diagnosis further comprises identifying the subject as developing mild cognitive impairment due to Alzheimer's disease within about 10 to about 25 years. The method of claim 11, wherein the diagnosis further comprises identifying the subject as developing mild cognitive impairment due to Alzheimer's disease within about 10 to about 20 years. A method of diagnosing a subject before the onset of Alzheimer's disease.
(A) Prepare an isolated tau sample obtained from the subject and measure the total amount of tau, and at (i) T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and T217. And (b) the ratio of taurin oxidation at T217 or T181 to total tau is greater than 2σ and the ratio of taurin oxidation at T205 to total tau is less than 2σ. Subjects are diagnosed with a high risk of conversion to mild cognitive impairment due to Alzheimer's disease, except that σ was measured by PET imaging and / or Aβ42 / 40 measurements in CSF were free of cerebral amyloid plaques. The standard deviation as defined by the total amount of tau and the normal distribution of taurin oxidation at T217 and T205, T181 and T205, or T181, T205, and T217, measured in the population.
The method described above. The subject is diagnosed if the ratio of taurine oxidation at T217 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is less than 1.5σ. , The method according to claim 13. (I) The ratio of taurine oxidation at T217 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is less than 1.75σ, (ii) at T217 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is less than 1.8σ, or (iii) the ratio of T217 taurine oxidation to total tau is 1. The method of claim 14, wherein the subject is diagnosed when the ratio of taurine oxidation at T205 to total tau is greater than 9σ and is less than 1.9σ. The subject is diagnosed if the ratio of taurine oxidation at T217 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is less than 2σ. The method described in. The subject is diagnosed if the ratio of taurine oxidation at T181 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is less than 1.5σ. , The method according to claim 13. (I) The ratio of taurine oxidation at T181 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is less than 1.75σ, (ii) at T181 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is less than 1.8σ, or (iii) the ratio of taurine oxidation at T181 to total tau is The method of claim 17, wherein the subject is diagnosed when the ratio of taurine oxidation at T205 to total tau is greater than 1.9σ and is less than 1.9σ. The subject is diagnosed if the ratio of taurine oxidation at T181 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is less than 2σ, claim 17 The method described in. The ratio of taurine oxidation at T181 to total tau and the ratio of taurine oxidation at T217 to total tau is greater than 1.5σ, and the ratio of taurine oxidation at T205 to total tau is less than 1.5σ. The method of claim 13, wherein the subject is diagnosed in this case. (I) The ratio of taurine oxidation at T181 to total tau and the ratio of taurine oxidation at T217 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is 1.75σ. Less than (ii) said ratio of taurine oxidation at T181 to total tau and said ratio of taurine oxidation at T217 to total tau is greater than 1.8σ and said ratio of taurine oxidation at T205 to total tau. Is less than 1.8σ, or (iii) said ratio of taurine oxidation at T181 to total tau and said ratio of taurine oxidation at T217 to total tau is greater than 1.9σ and at T205 to total tau. The method of claim 20, wherein the subject is diagnosed when the ratio of taurine oxidation is less than 1.9σ. The subject is diagnosed when the ratio of taurine oxidation at T181 to total tau and the ratio of taurine oxidation at T217 to total tau is greater than 2σ and the taurine oxidation at T205 is less than 2σ. 20. The method of claim 20. The method of any one of claims 13-22, wherein the diagnosis further comprises identifying the subject as developing mild cognitive impairment due to Alzheimer's disease within about 10 to about 25 years. 23. The method of claim 23, wherein the diagnosis further comprises identifying the subject as developing mild cognitive impairment due to Alzheimer's disease within about 10 to about 20 years. A method of diagnosing a subject before the onset of Alzheimer's disease.
(A) Prepare isolated tau samples obtained from the subject and measure tauphosphorylation at (i) T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and T217; And (b) (i) T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and T217 where taurin oxidation is about 1.5σ or more, the subject is subject to Alzheimer's disease. Diagnosing a high conversion risk to mild cognitive impairment, except that σ was measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of cerebral amyloid plaques. The standard deviation defined by the normal distribution of taurination at T217 and T205, T181 and T205, or T181, T205, and T217.
The method described above. 24. The method of claim 24, wherein the subject is diagnosed when taurine oxidation at T217 and T205 is greater than 1.5σ. 26. The method of claim 26, wherein the subject is diagnosed when taurine oxidation at T217 and T205 is greater than 1.75σ, greater than 1.8σ, or greater than 1.9σ. 26. The method of claim 26, wherein the subject is diagnosed when taurine oxidation at T217 and T205 is greater than 2σ. 25. The method of claim 25, wherein the subject is diagnosed when taurine oxidation at T181 and T205 is greater than 1.5σ. 29. The method of claim 29, wherein the subject is diagnosed when taurine oxidation at T181 and T205 is greater than 1.75σ, greater than 1.8σ, or greater than 1.9σ. 29. The method of claim 29, wherein the subject is diagnosed when taurine oxidation at T181 and T205 is greater than 2σ. 25. The method of claim 25, wherein the subject is diagnosed when taurine oxidation at T181, T205, and T205 is greater than 1.5σ. 32. The subject is diagnosed when taurine oxidation at T181, T205, and T217 is greater than 1.75σ, greater than 1.8σ, or greater than 1.9σ. the method of. 32. The method of claim 32, wherein the subject is diagnosed when taurine oxidation at T181, T205, and T217 is greater than 2σ. The method of any one of claims 25-34, wherein the diagnosis further comprises identifying the subject as developing mild cognitive impairment due to Alzheimer's disease within about 15 years. 35. The method of claim 35, wherein the diagnosis further comprises identifying the subject as developing mild cognitive impairment due to Alzheimer's disease within about 10 years. A method of diagnosing a subject before the onset of Alzheimer's disease.
(A) Prepare an isolated tau sample obtained from the subject and measure the total amount of tau, and at (i) T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and T217. And (b) the ratio of tau phosphorylation at (i) T217 and T205, (ii) T181 and T205, or (iii) T181, T205, and T217 to total tau is about 1.5σ. If this is the case, diagnose the subject as having a high risk of conversion to mild cognitive impairment due to Alzheimer's disease, except that σ is measured by PET imaging and / or by Aβ42 / 40 measurement in CSF. A standard deviation defined by total tau and the normal distribution of tau phosphorylation at T217 and T205, T181 and T205, or T181, T205, and T217, measured in a control population free of brain amyloid plaques.
The method described above. The subject is diagnosed if the ratio of taurine oxidation at T217 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.5σ. 37. The method of claim 37. (I) The ratio of taurine oxidation at T217 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is greater than 1.75σ, (ii) at T217 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.8σ. (Iii) The ratio of taurine oxidation at T217 to total tau is 1. 38. The method of claim 38, wherein the subject is diagnosed when the ratio of taurine oxidation at T205 to greater than .9σ and the total amount of taurine is greater than 1.9σ. The subject is diagnosed if the ratio of taurine oxidation at T217 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is greater than 2σ, claim 38. The method described in. The subject is diagnosed if the ratio of taurine oxidation at T181 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.5σ. 37. The method of claim 37. The ratio of taurine oxidation at T181 to total tau is greater than 1.75σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.75σ. (Ii) Taurine oxidation at T181 to total tau. The ratio of taurine oxidation at T205 to total tau is greater than 1.8σ, and (iii) the ratio of taurine oxidation at T181 to total tau is greater than 1.9σ. 41. The method of claim 41, wherein the subject is diagnosed when the ratio of taurine oxidation at T205 to total tau is greater than 1.9σ. The subject is diagnosed when the ratio of taurine oxidation at T181 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is greater than 2σ, claim 41. The method described in. The ratio of taurine oxidation at T181 to total tau is greater than 1.5σ, the ratio of taurine oxidation at T205 to total tau is greater than 1.5σ, and the ratio of taurine oxidation at T217 to total tau. 37. The method of claim 37, wherein the subject is diagnosed when is greater than 1.5σ. (I) The ratio of taurine oxidation at T181 to total tau is greater than 1.75σ, the ratio of taurine oxidation at T205 to total tau is greater than 1.75σ, and taurine oxidation at T217 to total tau. (Ii) The ratio of taurine oxidation at T181 to total tau is greater than 1.8σ, and the ratio of taurine oxidation at T205 to total tau is greater than 1.8σ. Yes, and the ratio of taurine oxidation at T217 to total tau is greater than 1.8σ, (iii) the ratio of taurine oxidation at T181 to total tau is greater than 1.9σ, at T205 to total tau. 44. The subject is diagnosed when the ratio of taurine oxidation is greater than 1.9σ and the ratio of taurine oxidation at T217 to total tau is greater than 1.9σ. the method of. The ratio of taurine oxidation at T181 to total tau is greater than 2σ, the ratio of taurine oxidation at T205 to total tau is greater than 2σ, and the ratio of taurine oxidation at T217 to total tau is greater than 2σ. 44. The method of claim 44, wherein the subject is diagnosed, if any. The method of any one of claims 37-46, wherein the diagnosis further comprises identifying the subject as developing mild cognitive impairment due to Alzheimer's disease within about 15 years. 47. The method of claim 47, wherein the diagnosis further comprises identifying the subject as developing mild cognitive impairment due to Alzheimer's disease within about 10 years. A method for treating a subject in need of treatment, wherein (a) an isolated taurine sample obtained from the subject is prepared, and (i) T217 and T205, (ii) T181 and T205, or (iii) T181. , T205, and T217; and (b) taurine oxidation at T217 or T181 is greater than or equal to about 1.5σ and taurine oxidation at T205 is greater than or equal to about 1.5σ. , Where the pharmaceutical composition is administered to the subject, where σ was measured by PET imaging and / or by Aβ42 / 40 measurement in CSF in a control population free of brain amyloid plaques, T217 and The standard deviation defined by the normal distribution of taurine oxidation at T205, T181 and T205, or T181, T205, and T217.
The method described above. 48. The method of claim 48, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T217 is greater than 1.5σ and taurine oxidation at T205 is less than 1.5σ. (I) Taurine oxidation at T217 is greater than 1.75σ and taurine oxidation at T205 is less than 1.75σ, (ii) Taurine oxidation at T217 is greater than 1.8σ and at T205. If taurine oxidation is less than 1.8σ, or (iii) taurine oxidation at T217 is greater than 1.9σ and taurine oxidation at T205 is less than 1.9σ, then the subject is a pharmaceutical composition. The method of claim 50, which is administered. The method of claim 50, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T217 is greater than 2σ and taurine oxidation at T205 is less than 2σ. 49. The method of claim 49, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181 is greater than 1.5σ and taurine oxidation at T205 is less than 1.5σ. (I) Taurine oxidation at T181 is greater than 1.75σ and taurine oxidation at T205 is less than 1.75σ, (ii) Taurine oxidation at T181 is greater than 1.8σ and at T205. If taurine oxidation is less than 1.8σ, or (iii) taurine oxidation at T181 is greater than 1.9σ and taurine oxidation at T205 is less than 1.9σ, then the subject is a pharmaceutical composition. The method of claim 53, which is administered. 53. The method of claim 53, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181 is greater than 2σ and taurine oxidation at T205 is less than 2σ. 49. The method of claim 49, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181 and T217 is greater than 1.5σ and taurine oxidation at T205 is less than 1.5σ. (I) Taurine oxidation at T181 and T217 is greater than 1.75σ and taurine oxidation at T205 is less than 1.75σ, (ii) Taurine oxidation at T181 and T217 is greater than 1.8σ. And when the taurine oxidation at T205 is less than 1.8σ, or (iii) the taurine oxidation at T181 and T217 is more than 1.9σ and the taurine oxidation at T205 is less than 1.9σ, the above. The method of claim 56, wherein the subject is administered a pharmaceutical composition. 56. The method of claim 56, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181 and T217 is greater than 2σ and taurine oxidation at T205 is less than 2σ. The treatment method for a subject in need of treatment is as follows: (a) Prepare an isolated tau sample obtained from the subject and measure the total amount of tau, and (i) T217 and T205, (ii) T181. And T205, or (iii) measuring taurin oxidation at T181, T205, and T217; and (b) the ratio of taurin oxidation at T217 or T181 to total tau is greater than 2σ and at T205 to total tau. Administering the pharmaceutical composition to said subject when the ratio of taurin oxidation is less than 2σ, except that σ is measured by PET imaging and / or by Aβ42 / 40 measurement in CSF for cerebral amyloid plaque. The standard deviation defined by the total amount of tau and the normal distribution of taurin oxidation at T217 and T205, T181 and T205, or T181, T205, and T217, as measured in the control population without.
The method described above. The subject is administered the pharmaceutical composition when the ratio of taurine oxidation at T217 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is less than 1.5σ. The method according to claim 59. (I) The ratio of taurine oxidation at T217 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is less than 1.75σ, (ii) at T217 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is less than 1.8σ, or (iii) the ratio of taurine oxidation at T217 to total tau is The method of claim 60, wherein the subject is administered a pharmaceutical composition when the ratio of taurine oxidation at T205 to total tau is greater than 1.9σ and is less than 1.9σ. Claim that the subject is administered a pharmaceutical composition when the ratio of taurine oxidation at T217 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is less than 2σ. 60. The subject is administered the pharmaceutical composition when the ratio of taurine oxidation at T181 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is less than 1.5σ. The method according to claim 59. (I) The ratio of taurine oxidation at T181 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is less than 1.75σ, (ii) at T181 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is less than 1.8σ, or (iii) the ratio of taurine oxidation at T181 to total tau is 63. The method of claim 63, wherein the subject is administered a pharmaceutical composition when the ratio of taurine oxidation at T205 to total tau is greater than 1.9σ and is less than 1.9σ. Claim that the subject is administered a pharmaceutical composition when the ratio of taurine oxidation at T181 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is less than 2σ. 63. The ratio of taurine oxidation at T181 to total tau and the ratio of taurine oxidation at T217 to total tau is greater than 1.5σ, and the ratio of taurine oxidation at T205 to total tau is less than 1.5σ. 59. The method of claim 59, wherein the subject is administered a pharmaceutical composition. (I) The ratio of taurine oxidation at T181 to total tau and the ratio of taurine oxidation at T217 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is 1.75σ. Less than (ii) said ratio of taurine oxidation at T181 to total tau and said ratio of taurine oxidation at T217 to total tau is greater than 1.8σ and said ratio of taurine oxidation at T205 to total tau. Is less than 1.8σ, or (iii) said ratio of taurine oxidation at T181 to total tau and said ratio of taurine oxidation at T217 to total tau is greater than 1.9σ and at T205 to total tau. The method of claim 66, wherein the subject is administered a pharmaceutical composition when the ratio of taurine oxidation is less than 1.9σ. If the ratio of taurine oxidation at T181 to total tau and the ratio of taurine oxidation at T217 to total tau is greater than 2σ and the taurine oxidation at T205 is less than 2σ, then the subject is a pharmaceutical composition. The method of claim 66, which is administered. A method for treating a subject in need of treatment, wherein (a) an isolated taurine sample obtained from the subject is prepared, and (i) T217 and T205, (ii) T181 and T205, or (iii) T181. , T205, and T217; and (b) (i) T217 and T205, (ii) T181 and T205, or (iii) taurine oxidation at T181, T205, and T217. Administering the pharmaceutical composition to said subject if greater than or equal to 5σ, where σ was measured by PET imaging and / or in a control population free of cerebral amyloid plaques by Aβ42 / 40 measurements in CSF. The measured standard deviation defined by the normal distribution of taurine oxidation at T217 and T205, T181 and T205, or T181, T205, and T217.
The method described above. The method of claim 69, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T217 and T205 is greater than 1.5σ. The method of claim 70, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T217 and T205 is greater than 1.75σ, greater than 1.8σ, or greater than 1.9σ. .. The method of claim 70, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T217 and T205 is greater than 2σ. The method of claim 69, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181 and T205 is greater than 1.5σ. 33. The method of claim 73, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181 and T205 is greater than 1.75σ, greater than 1.8σ, or greater than 1.9σ. .. The method of claim 73, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181 and T205 is greater than 2σ. The method of claim 69, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181, T205, and T217 is greater than 1.5σ. Claim 76, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181, T205, and T217 is greater than 1.75σ, greater than 1.8σ, or greater than 1.9σ. The method described. The method of claim 76, wherein the subject is administered a pharmaceutical composition when taurine oxidation at T181, T205, and T217 is greater than 2σ. The treatment method for a subject in need of treatment is as follows: (a) Prepare an isolated tau sample obtained from the subject and measure the total amount of tau, and (i) T217 and T205, (ii) T181. And T205, or (iii) measuring tau phosphorylation at T181, T205, and T217; and (b) (i) T217 and T205, (ii) T181 and T205, or (iii) T181, T205 with respect to total tau. And, when taurin oxidation at T217 is greater than or equal to about 1.5σ, administer the pharmaceutical composition to said subject, where σ is measured by PET imaging and / or Aβ42 / 40 in CSF. A standard deviation defined by total tau and the normal distribution of tau phosphorylation at T217 and T205, T181 and T205, or T181, T205, and T217, as measured in a control population free of cerebral amyloid plaques.
The method described above. The subject is administered the pharmaceutical composition when the ratio of taurine oxidation at T217 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.5σ. The method according to claim 79. (I) The ratio of taurine oxidation at T217 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is greater than 1.75σ, (ii) at T217 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.8σ. (Iii) The ratio of taurine oxidation at T217 to total tau is 1. The method of claim 80, wherein the subject is administered a pharmaceutical composition when the ratio of taurine oxidation at T205 to greater than .9σ and the total amount of taurine is greater than 1.9σ. Claim that the subject is administered a pharmaceutical composition when the ratio of taurine oxidation at T217 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is greater than 2σ. 80. The subject is administered the pharmaceutical composition when the ratio of taurine oxidation at T181 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.5σ. The method according to claim 79. The ratio of taurine oxidation at T181 to total tau is greater than 1.75σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.75σ. (Ii) Taurine oxidation at T181 to total tau. The ratio of taurine oxidation at T205 to total tau is greater than 1.8σ, and (iii) the ratio of taurine oxidation at T181 to total tau is greater than 1.9σ. The method of claim 83, wherein the subject is administered a pharmaceutical composition when the ratio of taurine oxidation at T205 to total tau is greater than 1.9σ. Claim that the subject is administered a pharmaceutical composition when the ratio of taurine oxidation at T181 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is greater than 2σ. 83. The ratio of taurine oxidation at T181 to total tau is greater than 1.5σ, the ratio of taurine oxidation at T205 to total tau is greater than 1.5σ, and the ratio of taurine oxidation at T217 to total tau. 79. The method of claim 79, wherein the subject is administered a pharmaceutical composition when is greater than 1.5σ. (I) The ratio of taurine oxidation at T181 to total tau is greater than 1.75σ, the ratio of taurine oxidation at T205 to total tau is greater than 1.75σ, and taurine oxidation at T217 to total tau. (Ii) The ratio of taurine oxidation at T181 to total tau is greater than 1.8σ, and the ratio of taurine oxidation at T205 to total tau is greater than 1.8σ. Yes, and the ratio of taurine oxidation at T217 to total tau is greater than 1.8σ, (iii) the ratio of taurine oxidation at T181 to total tau is greater than 1.9σ, at T205 to total tau. 26. The subject is administered a pharmaceutical composition when the ratio of taurine oxidation is greater than 1.9σ and the ratio of taurine oxidation at T217 to total tau is greater than 1.9σ. The method described. The ratio of taurine oxidation at T181 to total tau is greater than 2σ, the ratio of taurine oxidation at T205 to total tau is greater than 2σ, and the ratio of taurine oxidation at T217 to total tau is greater than 2σ. The method of claim 86, wherein the subject is administered a pharmaceutical composition in certain cases. A method for enrolling a subject in a clinical study, wherein (a) an isolated taurine sample obtained from the subject is prepared and (i) T217 and T205, (ii) T181 and T205, or (iii). Measuring taurine oxidation at T181, T205, and T217; and (b) when taurine oxidation at T217 or T181 is greater than or equal to about 1.5σ and taurine oxidation at T205 is greater than or equal to about 1.5σ. To enroll the subject in a clinical trial, where σ was measured by PET imaging and / or by Aβ42 / 40 measurements in CSF in a control population free of brain amyloid plaques, T217 and The standard deviation defined by the normal distribution of taurine oxidation at T205, T181 and T205, or T181, T205, and T217.
The method described above. The method of claim 89, wherein the subject is enrolled in the clinical trial when taurine oxidation at T217 is greater than 1.5σ and taurine oxidation at T205 is less than 1.5σ. (I) Taurine oxidation at T217 is greater than 1.75σ and taurine oxidation at T205 is less than 1.75σ, (ii) Taurine oxidation at T217 is greater than 1.8σ and at T205. If taurine oxidation is less than 1.8σ, or (iii) taurine oxidation at T217 is greater than 1.9σ and taurine oxidation at T205 is less than 1.9σ, the subject is in the clinical study. The method of claim 90, which is registered. The method of claim 90, wherein the subject is enrolled in the clinical trial when taurine oxidation at T217 is greater than 2σ and taurine oxidation at T205 is less than 2σ. The method of claim 89, wherein the subject is enrolled in the clinical trial when taurine oxidation at T181 is greater than 1.5σ and taurine oxidation at T205 is less than 1.5σ. (I) Taurine oxidation at T181 is greater than 1.75σ and taurine oxidation at T205 is less than 1.75σ, (ii) Taurine oxidation at T181 is greater than 1.8σ and at T205. If taurine oxidation is less than 1.8σ, or (iii) taurine oxidation at T181 is greater than 1.9σ and taurine oxidation at T205 is less than 1.9σ, the subject is in the clinical study. The method of claim 93, which is registered. The method of claim 93, wherein the subject is enrolled in the clinical trial when taurine oxidation at T181 is greater than 2σ and taurine oxidation at T205 is less than 2σ. The method of claim 89, wherein the subject is enrolled in the clinical trial when taurine oxidation at T181 and T217 is greater than 1.5σ and taurine oxidation at T205 is less than 1.5σ. (I) Taurine oxidation at T181 and T217 is greater than 1.75σ and taurine oxidation at T205 is less than 1.75σ, (ii) Taurine oxidation at T181 and T217 is greater than 1.8σ. And when the taurine oxidation at T205 is less than 1.8σ, or (iii) the taurine oxidation at T181 and T217 is more than 1.9σ and the taurine oxidation at T205 is less than 1.9σ, the above. The method of claim 96, wherein the subject is enrolled in the clinical trial. The method of claim 96, wherein the subject is enrolled in the clinical trial when taurine oxidation at T181 and T217 is greater than 2σ and taurine oxidation at T205 is less than 2σ. A method for enrolling a subject in a clinical trial, wherein (a) an isolated tau sample obtained from the subject is prepared and the total amount of tau is measured, and (i) T217 and T205, (ii). Measuring taurin oxidation at T181 and T205, or (iii) T181, T205, and T217; and (b) the ratio of taurin oxidation at T217 or T181 to total tau is greater than 2σ and T205 to total tau. Enroll the subject in a clinical trial if the ratio of taurin oxidation in is less than 2σ, except that σ is measured by PET imaging and / or by Aβ42 / 40 measurement in CSF for cerebral amyloid plaque. The standard deviation defined by the total amount of tau and the normal distribution of taurin oxidation at T217 and T205, T181 and T205, or T181, T205, and T217, as measured in the control population without.
The method described above. The subject is enrolled in the clinical trial if the ratio of taurine oxidation at T217 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is less than 1.5σ. The method according to claim 99. (I) The ratio of taurine oxidation at T217 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is less than 1.75σ, (ii) at T217 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is less than 1.8σ, or (iii) the ratio of taurine oxidation at T217 to total tau is The method of claim 100, wherein the subject is enrolled in the clinical study when the ratio of taurine oxidation at T205 to total tau is greater than 1.9σ and is less than 1.9σ. The subject is enrolled in the clinical trial if the ratio of taurine oxidation at T217 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is less than 2σ. The method according to 100. If the ratio of taurine oxidation at T181 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is less than 1.5σ, the subject is enrolled in the clinical trial. The method according to claim 99. (I) The ratio of taurine oxidation at T181 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is less than 1.75σ, (ii) at T181 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is less than 1.8σ, or (iii) the ratio of taurine oxidation at T181 to total tau is 10. The method of claim 103, wherein the subject is enrolled in the clinical study when the ratio of taurine oxidation at T205 to total tau is greater than 1.9σ and is less than 1.9σ. The subject is enrolled in the clinical trial if the ratio of taurine oxidation at T181 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is less than 2σ. 103. The ratio of taurine oxidation at T181 to total tau and the ratio of taurine oxidation at T217 to total tau is greater than 1.5σ, and the ratio of taurine oxidation at T205 to total tau is less than 1.5σ. The method of claim 99, wherein the subject is enrolled in the clinical trial. (I) The ratio of taurine oxidation at T181 to total tau and the ratio of taurine oxidation at T217 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is 1.75σ. Less than (ii) said ratio of taurine oxidation at T181 to total tau and said ratio of taurine oxidation at T217 to total tau is greater than 1.8σ and said ratio of taurine oxidation at T205 to total tau. Is less than 1.8σ, or (iii) said ratio of taurine oxidation at T181 to total tau and said ratio of taurine oxidation at T217 to total tau is greater than 1.9σ and at T205 to total tau. 10. The method of claim 106, wherein if the ratio of taurine oxidation is less than 1.9σ, the subject is enrolled in the clinical study. If the ratio of taurine oxidation at T181 to total tau and the ratio of taurine oxidation at T217 to total tau is greater than 2σ and the taurine oxidation at T205 is less than 2σ, the subject is included in the clinical trial. The method of claim 106, which is registered. A method for enrolling a subject in a clinical study, wherein (a) an isolated taurine sample obtained from the subject is prepared and (i) T217 and T205, (ii) T181 and T205, or (iii). Measuring taurine oxidation at T181, T205, and T217; and (b) (i) T217 and T205, (ii) T181 and T205, or (iii) taurine oxidation at T181, T205, and T217 is about 1 Enroll the subject in a clinical trial if ≥ The measured standard deviation defined by the normal distribution of taurine oxidation at T217 and T205, T181 and T205, or T181, T205, and T217.
The method described above. The method of claim 109, wherein the subject is enrolled in the clinical trial when taurine oxidation at T217 and T205 is greater than 1.5σ. The method of claim 110, wherein if taurine oxidation at T217 and T205 is greater than 1.75σ, greater than 1.8σ, or greater than 1.9σ, the subject is enrolled in the clinical trial. .. The method of claim 110, wherein the subject is enrolled in the clinical trial when taurine oxidation at T217 and T205 is greater than 2σ. The method of claim 109, wherein the subject is enrolled in the clinical trial when taurine oxidation at T181 and T205 is greater than 1.5σ. The method of claim 113, wherein if taurine oxidation at T181 and T205 is greater than 1.75σ, greater than 1.8σ, or greater than 1.9σ, the subject is enrolled in the clinical trial. .. The method of claim 113, wherein the subject is enrolled in the clinical trial when taurine oxidation at T181 and T205 is greater than 2σ. The method of claim 109, wherein the subject is enrolled in the clinical trial when taurine oxidation at T181, T205, and T217 is greater than 1.5σ. If taurine oxidation at T181, T205, and T217 is greater than 1.75σ, greater than 1.8σ, or greater than 1.9σ, the subject is enrolled in the clinical trial, claim 116. The method described. 11. The method of claim 116, wherein the subject is enrolled in the clinical trial if taurine oxidation at T181, T205, and T217 is greater than 2σ. A method for enrolling a subject in a clinical trial, wherein (a) an isolated tau sample obtained from the subject is prepared and the total amount of tau is measured, and (i) T217 and T205, (ii). Measuring taurin oxidation at T181 and T205, or (iii) T181, T205, and T217; and (b) (i) T217 and T205, (ii) T181 and T205, or (iii) T181, with respect to total tau. Enroll the subject in a clinical trial if the ratio of taurin oxidation at T205 and T217 is greater than or equal to about 1.5σ, where σ is measured by PET imaging and / or Aβ42 in CSF. A standard deviation defined by total tau and the normal distribution of taurination at T217 and T205, T181 and T205, or T181, T205, and T217, measured in a control population free of cerebral amyloid plaques by the / 40 measurement. ,
The method described above. The subject is enrolled in the clinical trial if the ratio of taurine oxidation at T217 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.5σ. The method according to claim 119. (I) The ratio of taurine oxidation at T217 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is greater than 1.75σ, (ii) at T217 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.8σ. (Iii) The ratio of taurine oxidation at T217 to total tau is 1. The method of claim 120, wherein the subject is enrolled in the clinical study when the ratio of taurine oxidation at T205 to total tau is greater than 1.9σ. Claim that the subject is enrolled in the clinical trial if the ratio of taurine oxidation at T217 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is greater than 2σ. 120. The subject is enrolled in the clinical trial if the ratio of taurine oxidation at T181 to total tau is greater than 1.5σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.5σ. The method according to claim 119. (I) The ratio of taurine oxidation at T181 to total tau is greater than 1.75σ, and the ratio of taurine oxidation at T205 to total tau is greater than 1.75σ, (ii) at T181 to total tau. The ratio of taurine oxidation to total tau is greater than 1.8σ and the ratio of taurine oxidation at T205 to total tau is greater than 1.8σ. (Iii) The ratio of taurine oxidation at T181 to total tau is 1. 12. The method of claim 123, wherein the subject is enrolled in the clinical study when the ratio of taurine oxidation at T205 to total tau is greater than 1.9σ. The subject is enrolled in the clinical trial if the ratio of taurine oxidation at T181 to total tau is greater than 2σ and the ratio of taurine oxidation at T205 to total tau is greater than 2σ. The method according to 123. The ratio of taurine oxidation at T181 to total tau is greater than 1.5σ, and the ratio of taurine oxidation at T205 to total tau is greater than 1.5σ, and the ratio of taurine oxidation at T217 to total tau. 119. The method of claim 119, wherein the subject is enrolled in the clinical study when the ratio is greater than 1.5σ. (I) The ratio of taurine oxidation at T181 to total tau is greater than 1.75σ, the ratio of taurine oxidation at T205 to total tau is greater than 1.75σ, and taurine oxidation at T217 to total tau. (Ii) The ratio of taurine oxidation at T181 to total tau is greater than 1.8σ, and the ratio of taurine oxidation at T205 to total tau is greater than 1.8σ. Yes, and the ratio of taurine oxidation at T217 to total tau is greater than 1.8σ, (iii) the ratio of taurine oxidation at T181 to total tau is greater than 1.9σ, at T205 to total tau. The subject is enrolled in the clinical study, claim 126, when the ratio of taurine oxidation is greater than 1.9σ and the ratio of taurine oxidation at T217 to total tau is greater than 1.9σ. The method described. The ratio of taurine oxidation at T181 to total tau is greater than 2σ, the ratio of taurine oxidation at T205 to total tau is greater than 2σ, and the ratio of taurine oxidation at T217 to total tau is greater than 2σ. The method of claim 126, wherein the subject is enrolled in the clinical trial, if any. The method of any one of claims 89-128, wherein the subject is enrolled in the treatment group of the clinical trial. 129. The method of claim 129, wherein the treatment group of the clinical trial comprises administering a pharmaceutical composition to the subject. The method of any one of claims 49-89 or 122, wherein the pharmaceutical composition comprises an Aβ or tau therapeutic agent. 13. The method of claim 131, wherein the Aβ or tau therapeutic agent is an amyloid beta targeted therapeutic agent, kinase, kinase inhibitor, or phosphatase. The method according to any one of claims 1 to 132, wherein the subject has a genetic mutation known to cause dominantly inherited Alzheimer's disease. The method according to any one of claims 1 to 132, wherein the subject does not have a genetic mutation known to cause dominantly inherited Alzheimer's disease. The method according to any one of claims 1 to 134, wherein the isolated tau sample comprises tau purified from blood or CSF by affinity purification. The isolated tau sample uses a ligand that specifically binds to an epitope in the middomain of tau, and optionally a second ligand that specifically binds to an epitope in the N-terminus of tau. The method of claim 135, comprising tau purified from blood or CSF by purification. The method according to any one of claims 1 to 135, wherein taurine oxidation is measured by mass spectrometry.

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