CSF phosphorylated tau and amyloid beta profiles as biomarkers for tauopathies

Abstract

The present disclosure provides methods to quantitate tau phosphorylation at specific amino acid residues, and optionally Ab species, to diagnose subjects, guide treatment decisions, and select subjects for clinical trials.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 169,193, filed March 31, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure encompasses the use of tau and amyloid-beta species in CSF to measure pathological characteristics and / or clinical symptoms of 3R- and 4R-tauopathies in order to diagnose and / or select appropriate treatments for a given disease. [Background technology]

[0003] Microtubule-associated protein tau (MAPT or tau) plays an essential role in neuronal morphology and physiology. Tau has six different full-length protein isoforms and undergoes numerous potential post-translational modifications, including acetylation, glycosylation and phosphorylation. Phosphorylation is important for regulating the normal function of tau in stabilizing axons and can occur at more than 80 different residues. However, hyperphosphorylation of tau appears to increase the probability that tau will aggregate into intracellular insoluble paired helical filaments (PHFs) and neurofibrillary tangles (NFTs), which are primarily composed of hyperphosphorylated tau.

[0004] Intracellular neurofibrillary tangles in the cerebral cortex are a defining pathological feature of Alzheimer's disease (AD) and correlate with the onset of clinical symptoms long after the appearance of extracellular amyloid-β (Aβ) plaques, which begin to appear up to 20 years before the onset of symptoms. In AD, soluble p-tau and nonphosphorylated tau are increased two-fold in the cerebrospinal fluid (CSF). These changes are proposed to reflect the effects of neuronal death (neurodegeneration) that passively releases tau and NFTs into the CSF. However, in other tauopathies with significant NFT pathology and neurodegeneration (e.g., progressive supranuclear palsy, frontotemporal lobar tau degeneration), CSF soluble p-tau and total tau levels are not increased.

[0005] Other tauopathies or neurodegenerative diseases, such as progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), and frontotemporal dementia (FTD), currently lack CSF or imaging biomarkers, and diagnosis primarily relies on clinical evaluation that is ultimately confirmed after brain autopsy. It remains unclear whether other tauopathies induce p-tau modifications in the absence of amyloid pathology. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there remains a need in the art for improved methods for reliable and accurate clinical diagnosis of tauopathies that impact the design and conduct of clinical trials. [Means for solving the problem]

[0007] Among various aspects of the disclosure, methods are provided for quantifying tau phosphorylation at specific amino acid residues, and optionally Aβ species, to diagnose subjects, guide treatment decisions, and select subjects for clinical trials.

[0008] One embodiment of the present disclosure encompasses a method of distinguishing MAPT R406W tauopathy, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species; and (b) quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample, where increased pT217 / T217 values ​​and normal Aβ42 / 40 values ​​distinguish MAPT R406W tauopathy from a healthy state.

[0009] Another embodiment of the present disclosure encompasses a method of distinguishing MAPT R406W tauopathy, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species; and (b) quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample, where a decrease in pT217 / T217 values ​​and an increase in Aβ42 / 40 values ​​distinguishes MAPT R406W tauopathy from Alzheimer's disease.

[0010] Another aspect of the present disclosure encompasses a method for distinguishing MAPT R406W tauopathy, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species and Aβ species; and (b) quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample, the pT217 / T217×Aβ42 / 40 value distinguishing MAPT R406W tauopathy from Alzheimer's disease, 4R-tauopathy, and healthy conditions. In some embodiments, an increase in the pT217 / T217×Aβ42 / 40 value quantified in step (b) compared to the pT217 / T217×Aβ42 / 40 value in a healthy control population distinguishes MAPT R406W tauopathy from healthy conditions. In some embodiments, an increase in pT217 / T217×Aβ42 / 40 values ​​quantified in step (b) distinguishes between MAPT R406W tauopathy and AD compared to pT217 / T217×Aβ42 / 40 values ​​in an Alzheimer's disease population. In some embodiments, an increase in pT217 / T217×Aβ42 / 40 values ​​quantified in step (b) distinguishes between MAPT R406W tauopathy and 4R-tauopathy compared to pT217 / T217×Aβ42 / 40 values ​​in a 4R-tauopathy population.

[0011] Another aspect of the present disclosure encompasses a method for distinguishing sporadic frontotemporal dementia (FTD), generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species; and (b) quantifying the pT181 / T181 value in the processed sample, the pT181 / T181 value distinguishing sporadic FTD from Alzheimer's disease, non-sporadic FTD tauopathy, and healthy state.In some cases, the decrease in the pT181 / T181 value quantified in step (b) compared with the pT181 / T181 value in Alzheimer's disease population distinguishes sporadic FTD from Alzheimer's disease. In some embodiments, a decrease in the pT181 / T181 value quantified in step (b) compared to the pT181 / T181 value in a healthy control population distinguishes between sporadic FTD and a healthy condition. In some embodiments, a decrease in the pT181 / T181 value quantified in step (b) compared to the pT181 / T181 value in a non-sporadic FTD tauopathy population distinguishes between sporadic FTD and a non-sporadic FTD tauopathy population.

[0012] Another aspect of the present disclosure encompasses a method of diagnosing a subject having symptoms of Alzheimer's disease, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and (b) diagnosing the subject as having frontotemporal dementia (FTD) or at increased risk for FTD if increased pT217 / T217 values ​​and normal Aβ42 / 40 values ​​are detected compared to pT217 / T217 and Aβ42 / 40 values ​​in a healthy control population. In some embodiments, the method further comprises determining a composite pT217 / T217×Aβ42 / 40 value, where an increased composite value compared to healthy controls indicates that the subject has FTD or at increased risk for FTD. In some embodiments, the subject is diagnosed as a MAPT R406W mutation carrier.

[0013] Another aspect of the present disclosure encompasses a method of diagnosing a subject having symptoms of Alzheimer's disease, generally comprising: (a) providing a processed CSF or blood sample obtained from the subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and (b) diagnosing the subject as having frontotemporal dementia (FTD) or at increased risk for FTD if a decreased pT217 / T217 value and an increased Aβ42 / 40 value are detected compared to the pT217 / T217 and Aβ42 / 40 values ​​in the Alzheimer's disease population. In some embodiments, the method further comprises determining a composite pT217 / T217×Aβ42 / 40 value, and an increased composite value compared to the Alzheimer's disease population indicates that the subject has FTD or at increased risk for FTD. In some embodiments, the subject is diagnosed as a MAPT R406W mutation carrier.

[0014] Another aspect of the present disclosure includes a method of diagnosing a subject having symptoms of Alzheimer's disease, generally comprising: (a) providing a processed CSF or blood sample obtained from the subject, where the CSF or blood sample is enriched in one or more p-tau species, and quantifying the pT181 / T181 value in the processed sample; and (b) diagnosing the subject as having or at increased risk for sporadic frontotemporal dementia (FTD) if a decrease in the pT181 / T181 value is detected compared to pT181 / T181 values ​​in a healthy control population.

[0015] Another aspect of the disclosure encompasses a method of diagnosing a subject having symptoms of Alzheimer's disease, generally comprising: (a) providing a processed CSF or blood sample obtained from the subject, where the CSF or blood sample is enriched in one or more p-tau species, and quantifying the pT181 / T181 value in the processed sample; and (b) diagnosing the subject as having sporadic frontotemporal dementia (FTD) or at increased risk for sporadic FTD if a decrease in the pT181 / T181 value is detected compared to pT181 / T181 values ​​in an Alzheimer's disease population.

[0016] Another aspect of the disclosure is a method for measuring MAPT R406W tauopathy disease progression in a subject, generally comprising: (a) providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the first processed sample; (b) providing a second processed CSF or blood sample obtained from the subject after the first sample, wherein the second CSF or blood sample is enriched in one or more p-tau and Aβ species, and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the first processed sample; providing a F or blood sample enriched in the same pTau and Aβ species as in step (a), and quantifying the pT217 / T217 and Aβ42 / 40 values ​​in the second processed sample; and (c) calculating a difference between the quantified pT217 / T217 and Aβ42 / 40 values ​​of the second sample and the first sample, wherein a statistically significant difference in the quantified pT217 / T217 and Aβ42 / 40 values ​​in the second sample is indicative of disease progression in the subject.

[0017] Another aspect of the disclosure is a method for measuring MAPT R406W tauopathy disease progression in a subject, generally comprising: (a) providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more pTau and Aβ species, and quantifying a composite pT217 / T217×Aβ42 / 40 value in the first processed sample; (b) providing a second processed CSF or blood sample obtained from the subject after the first sample, and quantifying a composite pT217 / T217×Aβ42 / 40 value in the first processed sample; providing a SF or blood sample enriched in the same pTau and Aβ species as in step (a), and quantifying a composite pT217 / T217×Aβ42 / 40 value in a second processed sample; and (c) calculating a difference between the quantified composite pT217 / T217×Aβ42 / 40 values ​​of the second sample and the first sample, wherein a statistically significant difference in the quantified composite pT217 / T217×Aβ42 / 40 value in the second sample is indicative of disease progression in the subject.

[0018] Another aspect of the disclosure encompasses a method for measuring sporadic frontotemporal dementia (FTD) disease progression in a subject, generally comprising: (a) providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more p-tau species, and quantifying a pT181 / T181 value in the first processed sample; (b) providing a second processed CSF or blood sample obtained from the subject after the first sample, the second CSF or blood sample being enriched in the same p-tau species as in step (a), and quantifying a pT181 / T181 value in the second processed sample; and (c) calculating a difference between the quantified pT181 / T181 values ​​in the second sample and the first sample, wherein a statistically significant difference in the quantified pT181 / T181 values ​​in the second sample is indicative of disease progression in the subject.

[0019] Another aspect of the disclosure encompasses a method for treating a subject in need thereof, generally comprising: (a) providing a processed CSF or blood sample obtained from the subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and (b) administering a pharmaceutical composition to the subject if increased pT217 / T217 and normal Aβ42 / 40 values ​​are detected compared to pT217 / T217 and Aβ42 / 40 values ​​in a healthy control population. In some embodiments, the pharmaceutical composition comprises an FTD therapy.

[0020] Another aspect of the disclosure encompasses a method for treating a subject in need thereof, generally comprising: (a) providing a processed CSF or blood sample obtained from the subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and (b) administering a pharmaceutical composition to the subject if a decrease in pT217 / T217 and an increase in Aβ42 / 40 values ​​are detected compared to pT217 / T217 and Aβ42 / 40 values ​​in an Alzheimer's disease population. In some embodiments, the pharmaceutical composition comprises an FTD therapy.

[0021] Another aspect of the disclosure encompasses a method for treating a subject in need thereof, generally comprising: (a) providing a processed CSF or blood sample obtained from the subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species; and quantifying a combined pT217 / T217×Aβ42 / 40 value in the processed sample; and (b) administering a pharmaceutical composition to the subject if an increase in the combined pT217 / T217×Aβ42 / 40 value is detected compared to the combined pT217 / T217×Aβ42 / 40 value in a healthy control population, an Alzheimer's disease population, or a 4R-tauopathy population. In some embodiments, the pharmaceutical composition comprises an FTD therapy.

[0022] Another aspect of the disclosure encompasses a method for treating a subject in need thereof, generally comprising: (a) providing a processed CSF or blood sample obtained from the subject, the CSF or blood sample being enriched in one or more p-tau species, and quantifying the pT181 / T181 value in the processed sample; and (b) administering a pharmaceutical composition to the subject if a decrease in the pT181 / T181 value is detected compared to the pT181 / T181 value in a healthy control population, an Alzheimer's disease population, or a non-sporadic FTD tauopathy population. In some embodiments, the pharmaceutical composition comprises a sporadic FTD therapy.

[0023] In each of the above aspects, in certain embodiments, the treatment does not include a therapeutic agent for reducing or preventing Aβ deposition and / or the treatment alters or stabilizes the amount of quantified p-tau species.

[0024] Another embodiment of the present disclosure encompasses a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched in one or more p-tau and Aβ species; (b) quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and (c) selecting the subject for clinical trials for FTD if the pT217 / T217 values ​​are increased and the Aβ42 / 40 values ​​are approximately the same as a healthy control population.

[0025] Another embodiment of the present disclosure encompasses a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched in one or more p-tau and Aβ species; (b) quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and (c) excluding the subject from clinical trials for AD or Ab therapy if the pT217 / T217 values ​​are increased and the Aβ42 / 40 values ​​are approximately the same as a healthy control population.

[0026] Another embodiment of the present disclosure encompasses a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched in one or more p-tau and Aβ species; (b) quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample; and (c) selecting the subject for clinical trials for FTD if the composite pT217 / T217×Aβ42 / 40 value is increased compared to a healthy control population.

[0027] Another aspect of the present disclosure encompasses a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched in one or more p-tau and Aβ species; (b) quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample; and (c) excluding the subject from clinical trials for AD if the composite pT217 / T217×Aβ42 / 40 value is increased relative to an AD population.

[0028] Another aspect of the disclosure encompasses a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched in one or more p-tau species; (b) quantifying the pT181 / T181 value in the processed sample; and (c) selecting the subject for clinical trials for sporadic FTD therapy if the pT181 / T181 value is decreased compared to a healthy control population.

[0029] Another aspect of the present disclosure encompasses a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched in one or more p-tau species; (b) quantifying the pT181 / T181 value in the processed sample; and (c) excluding the subject from clinical trials for AD if the combined pT181 / T181 value is decreased compared to an AD population.

[0030] Another aspect of the present disclosure encompasses a method of distinguishing Alzheimer's disease, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau species; and (b) quantifying pT153 / T153 values ​​in the processed sample, where the pT153 / T153 values ​​distinguish Alzheimer's disease from non-AD tauopathy and healthy conditions. In some embodiments, the increased pT153 / T153 values ​​distinguish Alzheimer's disease from non-AD tauopathy and healthy conditions compared to healthy control and non-Alzheimer's tauopathy populations.

[0031] Another aspect of the present disclosure includes a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched in one or more p-tau species; (b) quantifying the pT153 / T153 value in the processed sample; and (c) selecting the subject for clinical trials for AD therapy if the pT153 / T153 value is increased compared to a healthy control population or a non-AD tauopathy population.

[0032] Another aspect of the present disclosure encompasses a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched for one or more p-tau species; (b) quantifying the pT151 / T151 value in the processed sample; and (c) excluding the subject from clinical trials of non-AD tauopathy therapies if the pT153 / T153 value is increased compared to a non-AD tauopathy population.

[0033] Another aspect of the present disclosure encompasses a method of distinguishing Alzheimer's disease, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species; and (b) quantifying the pT111 / T111 value in the processed sample, where the pT111 / T111 value distinguishes Alzheimer's disease from non-AD tauopathy and healthy conditions. In some embodiments, the increased pT111 / T111 value distinguishes Alzheimer's disease from non-AD tauopathy and healthy conditions compared to a healthy control population and a non-Alzheimer's tauopathy population.

[0034] Another aspect of the present disclosure includes a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched in one or more p-tau species; (b) quantifying the pT111 / T111 value in the processed sample; and (c) selecting the subject for clinical trials for AD therapy if the pT111 / T111 value is increased compared to a healthy control population or a non-AD tauopathy population.

[0035] Another aspect of the disclosure encompasses a method for selecting subjects in clinical trials, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched for one or more p-tau species; (b) quantifying the pT111 / T111 value in the processed sample; and (c) excluding the subject from clinical trials of non-AD tauopathy therapies if the pT111 / T111 value is increased compared to a non-AD tauopathy population.

[0036] Another aspect of the present disclosure encompasses a method of distinguishing tauopathies, generally comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau species; and (b) quantifying the pT205 / T205 value in the processed sample, where the pT205 / T205 value distinguishes between tauopathies and healthy conditions. In some embodiments, an increase in pT205 / T205 value distinguishes between tauopathies and healthy conditions.

[0037] Another aspect of the present disclosure includes a method of distinguishing tauopathy, comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau species; and (b) quantifying the pT208 / T208 value in the processed sample, the pT208 / T208 value distinguishing between tauopathy and healthy conditions. In some embodiments, an increase in pT208 / T208 value distinguishes between tauopathy and healthy conditions.

[0038] These and other aspects and iterations of the invention are described more fully below.

[0039] The application file contains at least one photograph executed in color. Copies of this patent application publication with color photograph will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0040] [Figure 1]Figure 1A, Figure 1B, Figure 1C, Figure 1D, Figure 1E and Figure 1F show that MAPT R406W carriers had increased CSF pT217 / T217 without amyloid pathology. Figure 1A shows that all samples (n=252) were plotted in a quadrant analysis using the cutoffs calculated in the WashU-A cohort (CSF Aβ42 / 40 cutoff=0.086, CSF pT217 / T217 cutoff=4.76). Figure 1B-Figure 1E show pie charts showing the number of participants in each group clinically classified for each quadrant: II (Figure 1B), I (Figure 1C), III (Figure 1D), and IV (Figure 1E). CSF, cerebrospinal fluid. Figure 1F shows the flow chart of the cohort analyzed in this study. The WashU-A cohort includes AD and age-matched controls, including a subset with PiB PET imaging data (n=48). We used this subset and a PiB PET cutoff of 0.18 to determine amyloid positivity for immunoprecipitation and mass spectrometry (IP / MS) measurements of CSF Ab42 / 40 (cutoff 0.086). We then used all CSF from WashU-A to determine an IP / MS CSF pT217 / T217 cutoff of 4.76. The cutoffs for CSF concentrations of pT181, pT217, total tau, and the ratio of pT181 / T181 were 44.79, 222.4, 754.7, and 15.75, respectively. WashU-A (n=85), WashU-B (n=88), and Montpellier cohorts (n=79) were analyzed for IP / MS CSF Ab42 / 40 and pT217 / T217 in quadrant analysis (total n=252). Samples were classified into quadrants I-IV using determined amyloid and p-tau positivity cutoffs. AMC: age-matched controls, YNC: young normal controls, CBS: corticobasal syndrome, PSP: progressive supranuclear palsy, bvFTD: behavioral frontotemporal dementia. [Diagram 2]Figure 2A, Figure 2B, Figure 2C and Figure 2D show CSF Aβ42 / 40, CSF pT217 / T217 and composite biomarkers for AD and MAPT R406W mutation carrier diagnosis. Figure 2A shows that CSF Aβ42 / 40 is significantly decreased in AD (ANOVA, p<0.0001). Figure 2B shows that CSF pT217 / T217 is significantly increased in AD (ANOVA, p<0.0001). Figure 2C shows that CSF pT217 / T217 × CSF Aβ42 / 40 is significantly increased in AD compared to controls and the 4R tauopathy group (which may include FTD-TDP, FTD-FUS, and 3R tauopathy, ANOVA, p<0.0001), which is mostly composed of 4R tauopathies (PSP, CBS, FTD-MAPTP301L) and a subset of sporadic bvFTD. It is also significantly increased in MAPT R406W carriers compared to controls (ANOVA, p<0.0001) and the 4R tauopathy group (ANOVA, p=0.0001). Figure 2D shows that CSF pT217 / T217 divided by CSF Aβ42 / 40 is significantly increased in AD (ANOVA, p<0.0001). CSF, cerebrospinal fluid; AD, Alzheimer's disease; PSP, progressive supranuclear palsy; CBS, corticobasal syndrome; bvFTD, behavioral frontotemporal dementia. [Diagram 3]Figure 3A, Figure 3B, Figure 3C, Figure 3D, Figure 3E, Figure 3F, Figure 3G, Figure 3H, Figure 3I, Figure 3J, Figure 3K, and Figure 3L show ROC analysis of IP / MS CSF Aβ42 / 40, CSF tau and p-tau. Figure 3A shows that CSF Aβ42 / 40 ratio is significantly increased in amyloid PET-PiB+ participants in the Wash-A cohort (n=48). Figure 3B shows that CSF Aβ42 / 40 can distinguish amyloid PET-PiB+ from PiB- individuals (AUC=0.9207, p<0.0001). (Figure 3C, Figure 3E, Figure 3G, Figure 3I, Figure 3K) CSF pT217 / T217 ratio, CSF pT217, CSF T181, CSF total tau concentration, and CSF pT181 / T181 ratio are significantly increased in amyloid+ participants as defined by CSF Aβ42 / 40 ratio in the Wash-A cohort (n=85). (Figure 3D, F, H, J, L) CSF pT217 / T217 ratio, CSF pT217, CSF T181, CSF total tau concentration, and CSF pT181 / T181 ratio can distinguish CSF Aβ42 / 40 positive (amyloid+) from CSF Aβ42 / 40 negative (amyloid-) individuals with AUC=0.983, 0.949, 0.816, 0.693, and 0.934, respectively. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4] FIG. 4A shows a quadrant analysis by diagnosis. A pie chart showing the quadrant (I, II, III, IV) in which the participants of each disease group are located. FIG. 4B shows a quadrant analysis by diagnosis. A pie chart showing the quadrant (I, II, III, IV) in which the participants of each disease group are located. FIG. 4C shows a quadrant analysis by diagnosis. A pie chart showing the quadrant (I, II, III, IV) in which the participants of each disease group are located. FIG. 4D shows a quadrant analysis by diagnosis. A pie chart showing the quadrant (I, II, III, IV) in which the participants of each disease group are located. FIG. 4E shows a quadrant analysis by diagnosis. A pie chart showing the quadrant (I, II, III, IV) in which the participants of each disease group are located. FIG. 4F shows a quadrant analysis by diagnosis. A pie chart showing the quadrant (I, II, III, IV) in which the participants of each disease group are located. [Diagram 5]Figure 5A, Figure 5B, and Figure 5C show amyloid and tau PET imaging by quadrant. Figure 5A shows that amyloid PET imaging measured by PiB SUVR increases significantly and gradually in quadrants II>III>IV (ANOVA, p<0.0001). Figure 5B shows that amyloid PET imaging measured by AV45 SUVR increases significantly and gradually in quadrants II>III>IV (ANOVA, p<0.0001). Figure 5C shows that tau PET imaging measured by AV1451 SUVR increases only in quadrant II (ANOVA, p<0.0001). No participants had imaging data in quadrant I. ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 6] Figure 6 graphically depicts the correlation of CSF Aβ42 / 40 and CSF pT217 / T217 in quadrants III and IV. CSF pT217 / T217 is negatively correlated with CSF Ab42 / 40 in quadrants III and IV below the cutoff for p-tau positivity (Pearson correlation r=0.556, p<0.0001). [Figure 7] Figure 7A, Figure 7B, Figure 7C, and Figure 7D graphically depict the diagnostic subcategories shown in Figure 2. Figure 7A shows that CSF Aβ42 / 40 is significantly decreased in AD and localized AD. Figure 7B shows that CSF pT217 / T217 is significantly increased in AD and localized AD. Figure 7C shows that CSF pT217 / T217 x CSF Aβ42 / 40 is significantly increased in AD and MAPT R406W mutation carriers. Figure 7D shows that CSF pT217 / T217 divided by CSF Aβ42 / 40 is significantly increased in AD and localized AD. ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 8]Figure 8A, Figure 8B, Figure 8C, and Figure 8D graphically show quadrant analysis using CSF Ab42 / 40 and CSF total tau and p-tau. CSF concentrations of pT217 (Figure 8A), pT181 (Figure 8B), and total tau (Figure 8C) do not separate MAPT R406W mutation carriers or other groups. Figure 8A shows that CSF pT181 / T181 ratio does not separate MAPT R406W mutation carriers. However, sporadic bvFTD may have reduced CSF pT181 / T181 in quadrant IV. [Figure 9] Figure 9A, Figure 9B, Figure 9C, and Figure 9D graphically show IP / MS CSF total tau and p-tau in tauopathy subgroups. Figure 9A and Figure 9B show that CSF pT217 and pT181 concentrations were significantly increased in AD. Figure 9C shows that total CSF tau is significantly increased in AMC, AD, localized AD, and CBS PSP continuum compared to YNC. Figure 9D shows that sporadic bvFTD, including FTLD-tau, FTLD-TDP, and FTLD-FUS, have significantly lower CSF pT181 / T181 than R406W (*p<0.05), AMC (***p<0.001), and AD / localized AD (****p<0.0001). [Figure 10]Figure 10A and Figure 10B show that sporadic bvFTD, including FTLD-tau, FTLD-TDP, FTLD-FUS, can be separated from controls, AD, and other tauopathies with respect to CSF ​​pT181 / T181. Figure 10A shows that sporadic bvFTD (bvFTD), including FTLD-tau, FTLD-TDP, FTLD-FUS, has a higher CSF Aβ42 / 40 ratio and lower CSF pT181 / T181 than controls, AD, and tauopathies, including CBS, PSP, and MAPT-FTD (P301L and R406W mutation carriers). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 10B shows the diagnostic value of CSF pT181 / T181 in separating bvFTD from other cohorts. Sporadic bvFTD can be separated from controls (AUC=0.752) and from PSP, CBS, and MAPT-FTD (AUC=0.707). [Figure 11] FIG. 11 shows that the vast majority of participants identified as CBD or PSP had higher pT217 / T217 than was observed in the AD samples. [Figure 12] FIG. 12 shows pT181 / T181 levels in AD and non-AD tauopathies. [Figure 13] FIG. 13 shows pT205 / T205 values ​​in AD and non-AD tauopathies. [Figure 14] FIG. 14 shows pS208 / S208 levels in AD and non-AD tauopathies. [Figure 15] FIG. 15 shows pT111 / T111 values ​​in AD and non-AD tauopathies. [Figure 16] FIG. 16 shows pT153 / T153 levels in AD and non-AD tauopathies. [Figure 17]Figure 17A shows that sporadic FTD, including FTD-tau, FTD-ALS, FTD-FUS, can be separated from controls, AD, and other tauopathies with respect to CSF ​​pT181 / T181. In Figure 17B, sporadic FTD (sFTD) shows higher CSF Ab42 / 40 ratio and lower CSF pT181 / T181 than other cohorts in quadrant analysis. In Figure 17C, sFTD shows significantly lower CSF pT181 / T181 than R406W (*p<0.05), AMC (***p<0.001), and AD / localized AD (****p<0.0001). In Figure 17D, sFTD has significantly lower CSF pT181 / T181 than controls, AD and CBS, PSP, and familial FTD-MAPT (fFTD). sFTD can be separated from controls (AUC=0.75) and from PSP, CBS, and fFTD (AUC=0.72). [Figure 18] Figure 18A shows that MAPT R406W carriers have increased CSF pT217 / T217 without amyloid pathology, and sporadic FTD has decreased pT181 / T181. In Figure 18B, all samples (n=265) were subjected to quadrant analysis with CSF Ab42 / 40 ratio on the x-axis and CSF tau measurements on the y-axis. In Figure 18C, CSF pT217 / T217 is increased in MAPT R406W mutation carriers. In FIG. 18D, (CSF Aβ42 / 40 cutoff=0.086, CSF pT217 / T217 cutoff=4.755. CSF pT217 absolute concentration does not separate MAPT R406W mutation carriers. In FIG. 18E, CSF pT181 / T181 is decreased in sporadic FTD. CSF pT181 absolute concentration does not separate sporadic FTD. Total tau does not separate any of the groups. [Figure 19]Figure 19A shows that pT217 / T217 is increased in MAPT R406W carriers without amyloid pathology, and in Figure 19B, pT181 / T181 is decreased in sporadic FTD. In Figure 19C, pT217 / T217 is better than pT217 concentration alone to separate MAPT R406W carriers. In Figure 19D, pT181 / T181 is better than pT181 concentration alone to separate sporadic FTD without MAPT R406W and P301L carriers. In Figure 19E, age of different disease cohorts - in Figure 19F, CSF total tau is partially age dependent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Tau protein aggregation into neurofibrillary tangles in the central nervous system is responsible for the pathology of certain neurodegenerative disorders, including Alzheimer's disease (AD). Although the mechanism of tau destabilization is still not fully understood, tau protein has been found to be hyperphosphorylated in tau aggregates. The present disclosure encompasses the use of methods to diagnose tauopathy, to quantify tau phosphorylation at specific amino acid residues, to guide treatment decisions, to select subjects for clinical trials, and to evaluate the clinical efficacy of certain therapeutic interventions. These and other aspects and iterations of the present invention are described more fully below.

[0042] I. Definition In order to make the present invention easier to understand, certain terms are defined first.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention relates.Many methods and materials similar to, modified or equivalent to those described herein can be used in the practice of the present invention without undue experimentation, and preferred materials and methods are described herein.In describing and claiming the present invention, the following terms are used according to the definitions set forth below.

[0043] The term "about" as used herein refers to the variation of quantity for any quantifiable variable, including but not limited to mass, volume, time, distance and amount, which may occur, for example, through typical measuring techniques and instruments. Furthermore, given the solid and liquid manipulation procedures actually used, there are a certain number of inadvertent errors and variations that may occur through differences in the manufacture, source or purity of the components used to make the composition or carry out the method, etc. The term "about" also encompasses these variations, which may be up to ±5%, but may also be ±4%, 3%, 2%, 1%, etc. Whether or not modified by the term "about", the claims include the equivalents for the quantities.

[0044] As used herein, antibody refers to a complete antibody, i.e., composed of two heavy chains and two light chains, as understood in the art, or any antibody-like molecule having an antigen-binding region, including, but not limited to, fragments such as Fab', Fab, F(ab')2, single domain antibodies, Fv and single chain antibodies Fv. The term antibody also refers to polyclonal antibodies, monoclonal antibodies, chimeric antibodies and humanized antibodies. Techniques for preparing and using various antibody-based constructs and fragments are known in the art. Means for preparing and characterizing antibodies are also known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which is incorporated herein by reference in its entirety).

[0045] As used herein, the term "aptamer" refers to a polynucleotide, generally RNA or DNA, that has useful biological activity in terms of biochemical activity, molecular recognition or binding properties. Usually, aptamers have molecular activity, such as binding to a target molecule at a specific epitope (region). It is generally accepted that aptamers are specific in binding to polypeptides and can be synthesized and / or identified by in vitro evolution methods. Means for preparing and characterizing aptamers are known in the art, including in vitro evolution methods. See, for example, US 7,939,313, which is incorporated herein by reference in its entirety.

[0046] The term "Aβ" refers to a peptide derived from a region in the carboxy terminus of a larger protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many different forms of Aβ that can be toxic, and although Aβ peptides are typically 37-43 amino acid sequences long, they can have truncations and modifications that change their overall size. They can be found inside or outside the cell, in monomeric, oligomeric and aggregate forms, in soluble and insoluble compartments, and can be complexed with other proteins or molecules. The deleterious or toxic effects of Aβ can result from any or all of the above forms, as well as others not specifically mentioned. For example, two such Aβ isoforms include Aβ40 and Aβ42, with the Aβ42 isoform being particularly fibrillogenic or insoluble and associated with disease states. The term "Aβ" typically refers to multiple Aβ species without distinction between the individual Aβ species. Specific Aβ species are identified by the size of the peptide, e.g., Aβ42, Aβ40, Aβ38, etc.

[0047] As used herein, the term "Aβ42 / Aβ40 value" refers to the ratio of the amount of Aβ42 in a sample obtained from a subject compared to the amount of Aβ40 in the same sample.

[0048] "Aβ amyloidosis" is defined as clinically abnormal Aβ deposits in the brain. Subjects who are determined to have Aβ amyloidosis are referred to herein as "amyloid positive", while subjects who are determined not to have Aβ amyloidosis are referred to herein as "amyloid negative". Accepted indicators of Aβ amyloidosis exist in the art. At the time of this disclosure, Aβ amyloidosis is typically measured directly by amyloid imaging (e.g., PiB PET, fluorbetapir, or other imaging methods known in the art) or indirectly by reducing cerebrospinal fluid (CSF) Aβ42 or reducing CSF Aβ42 / 40 ratio. [11C]PIB-PET images with a mean cortical binding potential score >0.18 are indicative of Aβ amyloidosis, as is a cerebrospinal fluid (CSF) Aβ42 concentration of approximately 1 ng / ml by immunoprecipitation and mass spectrometry (IP / MS). Alternatively, a cutoff ratio of CSF Aβ42 / 40 that maximizes the predictive accuracy of amyloid positivity as determined by PIB-PET can be used. Values ​​such as these, or others known in the art and / or used in the examples, can be used alone or in combination to clinically confirm Aβ amyloidosis. See, for example, Klunk WE et al. Ann Neurol 55(3) 2004, Fagan AM et al. Ann Neurol, 2006, 59(3), Patterson et al, Annals 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 a disease associated with Aβ amyloidosis.Non-limiting examples of symptoms associated with Aβ amyloidosis can include cognitive dysfunction, behavioral changes, language dysfunction, emotional dysregulation, seizures, dementia, and nervous system structure or function disorders.Diseases associated with Aβ amyloidosis include, but are not limited to, Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia, and inclusion body myositis.Subjects with Aβ amyloidosis have an increased risk of developing diseases associated with Aβ amyloidosis.

[0049] "Clinical signs of Aβ amyloidosis" refers to the measure of Aβ deposition known in the art.Clinical signs of Aβ amyloidosis can include, but are not limited to, Aβ deposition identified by amyloid imaging (e.g., PiB PET, fluorbetapir, or other imaging methods known in the art) or by reduction in cerebrospinal fluid (CSF) Aβ42 or Aβ42 / 40 ratio.See, for example, Klunk WE et al. Ann Neurol 55(3) 2004, and Fagan AM et al. Ann Neurol 59(3) 2006, each of which is incorporated herein by reference in its entirety. As described in U.S. Patent Nos. 14 / 366,831, 14 / 523,148 and 14 / 747,453, each of which is incorporated by reference in its entirety, clinical indicia of Aβ amyloidosis can also include measurements of Aβ metabolism, particularly Aβ42 metabolism, either alone or compared to measurements of the metabolism of other Aβ mutants (e.g., Aβ37, Aβ38, Aβ39, Aβ40 and / or total Aβ). Further methods are described in 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.Subjects with clinical signs of Aβ amyloidosis also have an increased risk of developing diseases associated with Aβ amyloidosis.

[0050] "Candidate for amyloid imaging" refers to a subject identified by a clinician as an individual for whom amyloid imaging is clinically warranted. As a non-limiting example, a candidate for amyloid imaging may be a subject with one or more clinical signs of Aβ amyloidosis, one or more symptoms associated with Aβ plaques, one or more symptoms associated with CAA, or a combination thereof. A clinician may recommend amyloid imaging for such a subject to direct their clinical care. As another non-limiting example, a candidate for amyloid imaging may be a potential participant (either a control subject or a test subject) in a clinical trial for a disease associated with Aβ amyloidosis.

[0051] "Aβ plaque-associated symptoms" or "CAA-associated symptoms" refer to any symptoms caused by or associated with the formation of amyloid plaques or CAA, respectively, which are composed of aggregates of regularly arranged fibers called amyloid fibrils. Representative Aβ plaque-associated symptoms can include, but are not limited to, neuronal degeneration, cognitive impairment, memory impairment, behavioral changes, emotional dysregulation, seizures, impaired nervous system structure or function, and increased risk of developing or worsening Alzheimer's disease or CAA. Neuronal degeneration can include changes in neuronal structure (including molecular changes, e.g., intracellular accumulation of toxic proteins, protein aggregation, etc., and macro-level changes, e.g., changes in axon or dendrite shape or length, changes in myelin sheath composition, loss of myelin sheath, etc.), changes in neuronal function, loss of neuronal function, neuronal death, or any combination thereof. Cognitive impairment can include, but is not limited to, difficulties with memory, attention, concentration, language, reasoning, creativity, executive function, planning, and organization. Behavioral changes may include, but are not limited to, physical or verbal aggression, impulsivity, reduced inhibitions, apathy, reduced spontaneity, personality changes, alcohol, tobacco or drug abuse, and other addiction-related behaviors. Emotional dysregulation may include, but is not limited to, depression, anxiety, mania, irritability and affective incontinence. Seizures may include, but are not limited to, generalized tonic-clonic seizures, complex partial seizures, and non-epileptic psychogenic seizures. Disorders of nervous system structure or function may include, but are not limited to, hydrocephalus, parkinsonism, sleep disorders, psychosis, disorders of posture and coordination. This may include movement disorders such as monoparesis, hemiparesis, quadriparesis, ataxia, ballismus and tremor. This may also include sensory loss or dysfunction, including smell, touch, taste, vision, and hearing. Additionally, this can include autonomic nervous system disorders such as bowel and bladder dysfunction, sexual dysfunction, dysregulation of blood pressure and temperature.Finally, this can include hormonal disorders resulting from dysfunction of the hypothalamus and pituitary gland, such as deficiencies and dysregulation of growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, gonadotropin-releasing hormone, prolactin and countless other hormones and regulators.

[0052] As used herein, the term "subject" refers to a mammal, preferably a human. Mammals include, but are not limited to, humans, primates, livestock, rodents and pets. A subject may be waiting for medical care or treatment, may be undergoing medical care or treatment, or may have undergone medical care or treatment.

[0053] As used herein, the terms "control population," "normal population," or samples from "healthy" subjects refer to a subject or group of subjects who have been clinically determined to not have a tauopathy or Aβ amyloidosis, or a clinical disease associated with Aβ amyloidosis (including but not limited to Alzheimer's disease) based on qualitative or quantitative test results.

[0054] 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, although plasma is typically preferred.

[0055] The term "isoform" as used herein refers to any of several different forms of the same protein variant that arise due to alternative splicing of the mRNA encoding the protein, post-translational modification of the protein, proteolytic processing of the protein, genetic mutation and somatic recombination. The terms "isoform" and "variant" are used interchangeably.

[0056] The term "tau" refers to multiple isoforms encoded by the gene MAPT (or its homologues), as well as species that are C-terminally truncated in vivo, N-terminally truncated in vivo, post-translationally modified in vivo, or any combination thereof.As used herein, the terms "tau", "tau protein" and "tau species" can be used interchangeably.In many animals, including but not limited to humans, non-human primates, rodents, fish, cows, frogs, goats and chickens, tau is encoded by the gene MAPT.In animals where the gene is not identified as MAPT, homologues can be identified by methods well known in the art.

[0057] In humans, there are six isoforms of tau, generated by alternative splicing of exons 2, 3 and 10 of MAPT. These isoforms range from 352 to 441 amino acids in length. Exons 2 and 3 each encode a 29 amino acid insert at the N-terminus (referred to as N), and full-length human tau isoforms may have both inserts (2N), one insert (1N), or no inserts (0N). All full-length human tau isoforms also have three repeats of the microtubule-binding domain (referred to as R). Inclusion of exon 10 at the C-terminus results in the inclusion of a fourth microtubule-binding domain encoded by exon 10. Thus, full-length human tau isoforms may consist of four repeats of the microtubule-binding domain (including exon 10: R1, R2, R3 and R4) or three repeats of the microtubule-binding domain (excluding exon 10: R1, R3 and R4). Human tau may or may not be post-translationally modified. For example, it is known in the art that tau can be phosphorylated, ubiquitinated, glycosylated and glycated.Human tau can also be proteolytically processed or not in vivo at C-terminus, N-terminus, or C-terminus and N-terminus.Thus, the term "human tau" includes 2N3R, 2N4R, 1N3R, 1N4R, 0N3R, and 0N4R isoforms, as well as species that are truncated at C-terminus in vivo, species that are truncated at N-terminus in vivo, species that are post-translationally modified in vivo, or any combination thereof.Alternative splicing of the gene encoding tau occurs in other animals as well.

[0058] As used herein, the term "MTBR tau" refers to a tau protein, or multiple tau proteins, that contain two or more amino acids of the microtubule-binding region (MTBR) of tau (e.g., amino acids 244-368 of tau-441).

[0059] Diseases associated with tau deposition in the brain are referred to herein as "tauopathies." The term "tau deposition" includes all forms of pathological tau deposition, including, but not limited to, tau aggregates in neurofibrillary tangles, neuropil threads, and dystrophic neurites. Tauopathies known in the art include, but are not limited to, progressive supranuclear palsy, dementia pugilistica, chronic traumatic encephalopathy, frontotemporal dementia and parkinsonism linked to chromosome 17, Ritiko-Bodig disease, Parkinson's dementia of Guam, neurofibrillary tangle senile dementia, ganglioneuroma and neurocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Haravorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration (CBD), argyrophilic grain disease (AGD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), and frontotemporal dementia (FTD).

[0060] Tauopathies are classified according to the predominance of tau isoforms found in pathological tau deposits. Tauopathies with tau deposits predominantly composed of tau with three MTBRs are referred to as "3R tauopathies". Pick's disease is a non-limiting example of a 3R tauopathy. For clarity, the pathological tau deposits of some 3R tauopathies may be a mixture of 3R and 4R tau isoforms, with the 3R isoforms predominating. It is believed that intracellular neurofibrillary tangles (i.e., tau deposits) in the brains of Alzheimer's disease patients generally contain approximately equal amounts of 3R and 4R isoforms. Tauopathies with tau deposits predominantly composed of tau with four MTBRs are referred to as "4R tauopathies". PSP, CBD, and AGD are non-limiting examples of 4R tauopathies, as are some forms of FTLD. Notably, pathological tau deposits in the brain of some subjects with genetically confirmed FTLD cases, such as some V334M and R406W mutation carriers, display a mixture of 3R and 4R isoforms.

[0061] A clinical sign of a tauopathy may be aggregates of tau in the brain, including but not limited to neurofibrillary tangles. Methods for detecting and quantifying tau aggregates in the brain are known in the art (e.g., tau-specific ligands, e.g., 18 F]THK5317,[ 18 F]THK5351,[ 18 F]AV1451,[ 11 C]PBB3, 18 F]MK-6240,[ 18 F]RO-948,[ 18 F]PI-2620,[ 18 F]GTP1, 18 F]PM-PBB3 and [ 18 F]JNJ64349311,[ 18 F]JNJ-067) and other Tau PET.

[0062] The terms "treat", "treating" or "treatment" as used herein refer to the provision of medical care by a trained and licensed professional to a subject in need thereof. Medical care may be diagnostic testing, therapeutic treatment, and / or prophylactic or preventative measures. The purpose of therapeutic and prophylactic treatment is to prevent or slow (alleviate) undesired physiological changes or diseases / disorders. Beneficial or desired clinical results of therapeutic or prophylactic treatment include, but are not limited to, alleviation of symptoms, attenuation of the extent of disease, stabilized (i.e., not worsening) disease state, delayed or slowed disease progression, amelioration or reduction of disease state, and remission (either partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with a disease, condition or disorder, as well as those prone to having the disease, condition or disorder, or those in whom the disease, condition or disorder is to be prevented. Thus, a subject in need of treatment may or may not have symptoms or clinical signs of a disease.

[0063] The phrase "tau therapy" collectively refers to any imaging agent, therapeutic treatment, and / or prophylactic or preventative measures contemplated for or used with subjects at risk of developing a tauopathy or clinically diagnosed as having a tauopathy. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radiolabeled tau-specific ligands, radionuclide-labeled antibodies, etc.).Non-limiting examples of therapeutic agents include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., 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 (including antigen-binding fragments, variants or derivatives thereof), stem cells, dietary supplements (e.g., lithium water, omega-3 fatty acids with lipoic acid, long chain triglycerides, genistein, resveratrol, curcumin and grape seed extract, etc.), antagonists of serotonin receptor 6, p38 alpha MAPK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g., TRx0237, methylthionimium chloride, etc.), treatments to improve glycemic control (e.g. insulin, exenatide, liraglutide pioglitazone, 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 agonists, beta-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT1A and 1D receptor agonists, glutaminyl-peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents agents), muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensives, statins, and any combination thereof.

[0064] The phrase "Aβ therapy" collectively refers to any imaging or therapeutic agent contemplated for or for use with a subject at risk of developing Aβ amyloidosis or AD, a subject diagnosed with Aβ amyloidosis, or a subject diagnosed with AD.

[0065] "Significantly deviating from the mean" refers to a value that is 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 2 standard deviations above or below the mean.

[0066] II. Measurement of tau phosphorylation and Aβ in biological samples The methods of the disclosure include providing a biological sample obtained from a subject, isolating tau and / or Aβ, and measuring phosphorylation of tau at one or more amino acid residues, Aβ40, Aβ42, and optionally one or more proteins.

[0067] Suitable biological samples include blood samples or cerebrospinal fluid (CSF) samples obtained from a subject. In some embodiments, the subject is a human. The human subject may be awaiting medical treatment or treatment, or may be undergoing medical treatment or treatment, or may have undergone medical treatment or treatment. In various embodiments, the human subject may be a healthy subject, a subject at risk of developing a neurodegenerative disease, a subject with signs and / or symptoms of a neurodegenerative disease, or a subject diagnosed with a neurodegenerative disease. In further embodiments, the neurodegenerative disease may be a tauopathy. In certain examples, the tauopathy may be Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD). In other embodiments, the subject is an experimental animal. In further embodiments, the subject is an experimental animal genetically engineered to express human tau and optionally one or more additional human proteins (e.g., human Aβ, human ApoE, etc.).

[0068] CSF may be obtained by lumbar puncture, with or without an indwelling CSF catheter. Multiple blood or CSF samples collected simultaneously from a subject may be pooled. Blood may be collected by venipuncture, with or without an intravenous catheter, or by finger stick (or equivalent). Once collected, blood or CSF samples may be processed according to methods known in the art (e.g., centrifugation to remove whole cells and cellular debris; use of additives designed to stabilize and preserve specimens prior to analytical testing, etc.). Blood or CSF samples may be used immediately or may be frozen and stored indefinitely. Prior to use in the methods disclosed herein, biological samples may be modified, if necessary or desired, to include protease inhibitors, isotope-labeled internal standards, detergents and chaotropic agents, and / or to deplete other analytes (e.g., proteins, peptides, metabolites, etc.).

[0069] The size of the sample used can or will vary depending on the type of sample, the health status of the subject from whom the sample was obtained, and the analytes (in addition to tau) to be analyzed. The volume of the CSF sample may be about 0.01 mL to about 5 mL, or about 0.05 mL to about 5 mL. In certain examples, the size of the sample may be about 0.05 mL to about 1 mL of CSF. The amount of the plasma sample may be about 0.01 mL to about 20 mL.

[0070] The isotopic label can be used as an internal standard to account for variation across sample processing and calculate absolute concentrations where appropriate. Generally, the isotopic label internal standard is added prior to any significant sample processing, but can be added multiple times if necessary.

[0071] Several isotopically labeled internal standards are described herein. All incorporate a heavy isotope label into at least one amino acid residue. One or more full-length tau and / or Aβ isoforms may be used. Alternatively, or in addition, isoforms and / or peptide fragments with post-translational modifications may be used, as known in the art. Generally speaking, the incorporated labeled amino acid residue should increase the mass of the peptide without affecting its chemical properties, and the mass shift caused by the presence of the isotope label should be sufficient to allow mass spectrometry to distinguish between the internal standard (IS) and the endogenous analyte signal. As shown herein, suitable heavy isotope labels include, but are not limited to: 2 H, 13 C, and 15 N. Typically, about 1-10 ng of an internal standard is usually sufficient.

[0072] As used herein, an isolated tau sample refers to a composition comprising tau purified from blood or cerebrospinal fluid (CSF) obtained from a subject. In the isolated tau sample of the present disclosure, 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 these methods include, but are not limited to, selective precipitation, size exclusion chromatography, ion exchange chromatography, and affinity purification. A suitable method enriches both phosphorylated and non-phosphorylated tau from blood or CSF.

[0073] The method of the present disclosure may comprise depleting one or more proteins from a sample. The term "deplete" means to reduce in amount or number. Thus, a protein-depleted sample may have any amount of protein that is measurably less than the amount in the original sample, including no amount of protein.

[0074] Proteins can be depleted from samples by methods that specifically target one or more proteins, such as affinity depletion, solid-phase extraction, or other methods known in the art. Targeted depletion of a protein or proteins may be used in situations where downstream analysis of the protein is desired (e.g., identification, quantification, analysis of post-translational modifications, etc.). For example, Aβ peptides can be identified and quantified by methods known in the art after affinity depletion of Aβ with a suitable epitope binding agent. As another non-limiting example, apolipoprotein E (ApoE) status can be determined by methods known in the art following affinity depletion of ApoE and identification of ApoE isoforms. Targeted depletion can also be used to isolate other proteins for subsequent analysis, including, but not limited to, apolipoprotein J, synuclein, soluble amyloid precursor protein, alpha-2 macroglobulin, S100B, myelin basic protein, interleukins, TNF, TREM-2, TDP-43, YKL-40, VILIP-1, NFL, prion protein, pNFH, and DJ-1.

[0075] In some embodiments, target depletion may occur by affinity depletion. Affinity depletion refers to a method of depleting a protein of interest from a sample by utilizing its specific binding properties for a molecule. Typically, the molecule is a ligand (referred to as an immobilized ligand) attached to a solid support, such as a bead, resin, tissue culture plate, etc. The immobilization of the ligand to the solid support may occur after the interaction of the ligand with the protein has occurred. Suitable ligands include antibodies, aptamers, and other epitope binding agents. The molecule may be a polymer or other material that selectively absorbs the protein of interest. As a non-limiting example, polyhydroxymethylene substituted with fatty oxoethylated alcohols (e.g., PHM-L LIPOSORB, Sigma Aldrich) can be used to selectively absorb lipoproteins (including ApoE) from serum. Two or more affinity depletion agents can be combined to sequentially or simultaneously deplete multiple proteins.

[0076] Alternatively, samples can be depleted of proteins by more conventional methods, such as ultrafiltration or protein precipitation with acids, organic solvents or salts. Generally, these methods are used to ensure the reduction of high abundance and high molecular weight proteins, resulting in the enrichment of low molecular weight and / or low abundance proteins and peptides (e.g., tau, Aβ, etc.).

[0077] In some embodiments, proteins can be depleted from a sample by precipitation. Briefly, precipitation involves adding a precipitating agent to the sample and mixing thoroughly, incubating the sample with the precipitating agent to precipitate proteins, and separating the precipitated proteins by centrifugation or filtration. The resulting supernatant can then be used in downstream applications. The amount of reagent required can be determined experimentally by methods known in the art. Suitable precipitating agents include perchloric acid, trichloroacetic acid, acetonitrile, methanol, and the like. In an exemplary embodiment, proteins are depleted from a sample by acid precipitation. In a further embodiment, proteins are depleted from a sample by acid precipitation using perchloric acid.

[0078] Two or more methods from either or both of the above approaches can be combined to sequentially or simultaneously deplete multiple proteins. For example, one or more proteins can be selectively depleted (targeted depletion), followed by depletion of high abundance / molecular weight proteins. Alternatively, high abundance / molecular weight proteins can be depleted first, followed by targeted depletion of one or more proteins. In yet another alternative, high abundance / molecular weight proteins can be depleted first, followed by a first round of targeted depletion of one or more proteins, followed by a second round of targeted depletion of one or more different proteins that are different from the first round target. Other iterations will be readily apparent to those skilled in the art.

[0079] In an exemplary embodiment, the isolated tau sample of the present disclosure includes tau purified from blood or CSF by affinity purification. Affinity purification refers to a method of purifying a protein of interest by utilizing specific binding properties to an immobilized ligand. Typically, the immobilized ligand is a ligand attached to a solid support, such as a bead, a resin, a tissue culture plate, etc. A suitable ligand specifically binds to both phosphorylated and non-phosphorylated tau. In one example, a suitable ligand may bind to an epitope within the mid-domain of tau. In another example, a suitable ligand may bind to an epitope within the N-terminus of tau, preferably within amino acids 1-35 of tau. In another example, a suitable ligand may bind to an epitope within the MTBR of tau. In another example, a suitable ligand may bind to an epitope within the C-terminus of tau. In still further embodiments, tau may be affinity purified from blood or CSF using two or more immobilized ligands. In one example, the immobilized ligand binds to an epitope within the N-terminus of tau, and another immobilized ligand binds to an epitope within the intermediate domain of tau. In another example, the immobilized ligand binds to an epitope within the MTBR of tau, and another immobilized ligand binds to an epitope within the intermediate domain of tau. In another example, the immobilized ligand binds to an epitope within the C-terminus of tau, and another immobilized ligand binds to an epitope within the intermediate domain of tau. In another example, the immobilized ligand binds to an epitope within the C-terminus of tau, and another immobilized ligand binds to an epitope within the N-terminus of tau. In another example, the immobilized ligand binds to an epitope within the MTBR of tau, and another immobilized ligand binds to an epitope within the N-terminus of tau. In another example, the immobilized ligand binds to an epitope within the MTBR of tau, and another immobilized 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.

[0080] In each of the above embodiments, the epitope binding agent may comprise an antibody or an aptamer. In some embodiments, the epitope binding agent that specifically binds to amyloid beta is HJ5.1 or is an epitope binding agent that binds to the same epitope as HJ5.1 and / or competitively inhibits HJ5.1. In some embodiments, the epitope binding agent that specifically binds to an epitope within amino acids 1-103 of Tau-441, inclusive, is HJ8.5 or is an epitope binding agent that binds to the same epitope as HJ8.5 and / or competitively inhibits HJ8.5. In some embodiments, the epitope binding agent that specifically binds to an epitope within amino acids 104-221 of Tau-441, inclusive, is Tau1 or is an epitope binding agent that binds to the same epitope as Tau1 and / or competitively inhibits Tau1. Methods for identifying the epitope to which an antibody specifically binds and assays to assess competitive inhibition between two antibodies are known in the art.

[0081] Phosphorylation of specific amino acids (i.e., "sites") in tau results in phosphorylated tau (p-tau) isoform. The methods of the present disclosure provide a means to measure the stoichiometry of phosphorylation at one or more specific amino acids of tau. In some embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T111, S113, T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231. In some embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T111, T181, T153, and T217. In other embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T181, and T217. In other embodiments, the methods herein include measuring tau phosphorylation at one or more residues including T217. In other embodiments, the methods herein include measuring tau phosphorylation at one or more residues, including T181. In other embodiments, the methods herein include measuring tau phosphorylation at two or more residues, including T181 and T217. In other embodiments, the methods herein include measuring tau phosphorylation at three or more residues, including T181 and T217.

[0082] Applicants have developed a highly sensitive and specific mass spectrometry (MS) method using parallel reaction monitoring (PRM) to discover tau phosphorylation sites and initially quantify the abundance of phosphorylation sites in isolated tau protein. However, the present disclosure is not limited to any one particular method for quantitatively assessing site-specific phosphorylation of tau. A suitable method should distinguish tau isoforms that differ only in the phosphorylation state of a single amino acid, distinguish p-tau isoforms phosphorylated at different amino acids, and quantify changes in phosphorylation that occur at specific sites independent of the overall changes in total tau. Three approaches for quantifying changes in phosphorylation stoichiometry that occur at specific sites independent of the overall changes in total tau are detailed in the Examples. 1) relative comparison between phosphopeptide isomers, which can be used to estimate the relative abundance of each phosphopeptide sharing the same sequence; 2) standardization of phosphopeptides using any peptide from tau protein as a reference; and 3) absolute quantification for each phosphorylated and non-phosphorylated peptide using internal synthetic labeled standards, where the absolute quantification value for each phosphopeptide is standardized with any absolute quantification value obtained for any peptide from tau protein. All three approaches use internal standardization for comparison of relative phosphorylation changes for each site. Other methods known in the art may also be used.

[0083] In an exemplary embodiment, the site-specific phosphorylation of tau is measured by high-resolution mass spectrometry. Suitable types of mass spectrometry are known in the art. These include, but are not limited to, quadrupole, time-of-flight, ion trap and Orbitrap, as well as hybrid mass spectrometers that combine different types of mass spectrometers in one configuration (e.g., Orbitrap Fusion™ Tribrid™ mass spectrometer from ThermoFisher Scientific). Additional processing of the isolated tau sample may be performed prior to MS analysis. For example, tau may be proteolytically digested. Suitable proteases include, but are not limited to, trypsin, Lys-N, Lys-C and Arg-N. If affinity purification is used to generate the isolated tau sample, digestion may occur after elution of tau from the immobilized ligand or while tau is bound. Following one or more clean-up steps, the digested tau peptides may be separated by a liquid chromatography system that is compatible with a high-resolution mass spectrometer. The chromatography system may be optimized by routine experimentation to generate the desired LC-MS pattern. A variety of LC-MS techniques may be used to quantitatively analyze site-specific tau phosphorylation. Non-limiting examples include selected reaction monitoring, parallel reaction monitoring, selected ion monitoring, and data-independent acquisition. As mentioned above, any quantitative analysis of site-specific tau phosphorylation should consider the overall change in total tau. In an exemplary embodiment, the mass spectrometry protocol outlined in the examples is used.

[0084] "Total tau" as used herein refers to all tau isoforms in a given sample.Tau can be found in soluble and insoluble compartments, in monomer and aggregate form, in regular or irregular structures, inside or outside cells, and can be complexed with other proteins or molecules.Therefore, the source of biological sample (e.g. brain tissue, CSF, blood, etc.) and any downstream processing of biological sample will affect the overall amount of tau isoforms in a given sample.

[0085] Total tau can be measured by monitoring the abundance of unmodified tau peptides. For each phosphorylated tau site, tau peptides that share a common amino acid sequence with the phosphorylated peptide of interest can be used preferentially to measure total tau levels, although any peptide from the tau sequence can be used. Measurement of tau peptides can be performed by mass spectrometry, and the accuracy of the measurement can be improved by using a labeled internal standard as a reference. Alternatively, total tau can be measured by immunoassay or other methods to quantify tau concentration.

[0086] III. Use of p-tau and Aβ measurements The present disclosure also encompasses the use of measurements of p-tau and Aβ species (e.g., pT217 / T217, pT181 / T181, and Aβ42 / 40) in blood or CSF as biomarkers of pathological features and / or clinical symptoms of tauopathy to diagnose, stage, select appropriate treatment for a given disease stage, and modify a given treatment regimen (e.g., change dose, switch to another drug or treatment modality, etc.). The pathological features may be aspects of tau pathology (e.g., amount of tau deposits, presence / absence of post-translational modifications, amount of post-translational modifications, etc.). Alternatively or in addition to tau deposits, the pathological features may be tau-independent. For example, when the tauopathy is Alzheimer's disease, amyloid beta (Aβ) is deposited in the brain or in the arteries of the brain. The clinical symptoms may be dementia measured by a clinically validated instrument (e.g., MMSE, CDR-SB, etc.), or other clinical symptoms associated with tauopathy. It is also contemplated to use the measurements of p-tau and Aβ species in blood or CSF as biomarkers of other pathological features and clinical symptoms known in the art for 3R-tauopathies and 4R-tauopathies. Advantageously, p-tau and Aβ species, including but not limited to pT217 / T217, pT181 / T181, and Aβ42 / 40, distinguish not only between diseased and healthy conditions, but also between various tauopathies.

[0087] Thus, in one aspect, the present disclosure provides a method for measuring tauopathy-related pathology in a subject, comprising quantifying one or more p-tau species and Aβ species in a biological sample obtained from the subject, such as a blood sample or a CSF sample, wherein the amount of p-tau species and Aβ species quantified represents tauopathy-related pathology in the subject's brain. The tauopathy can be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. The disease-related pathology can be tau deposition, tau post-translational modifications, amyloid plaques in the brain and / or cerebral arteries, or other pathological features known in the art. The subject may or may not have clinical symptoms of tauopathy. In a preferred embodiment, at least one p-tau species quantified is pT217. In a further embodiment, two or more p-tau species quantified are pT217 and pT181. In still further embodiments, the Aβ species quantified are Aβ40 and Aβ42 species. In yet still further embodiments, pT217 / T217 and Aβ42 / 40, and optionally pT181 / T181, are quantified. In yet further embodiments, a composite pT217 / T217×Aβ42 / 40 value is quantified.

[0088] In another aspect, the present disclosure provides a method for diagnosing a subject with symptoms of tauopathy, comprising quantifying one or more p-tau species and Aβ species in a biological sample obtained from the subject, such as a blood sample or CSF sample, and diagnosing tauopathy if the quantified p-tau species and Aβ species deviate from a control population that does not have clinical signs or symptoms of tauopathy and is amyloid negative as measured by PET imaging and / or CSF Aβ42 / 40 measurement, or deviate from or resemble the same quantified p-tau and Ab species from a population diagnosed with 3R tauopathy, mixed 3R / 4R tauopathy, or 4R tauopathy. In a preferred embodiment, at least one p-tau species quantified is pT217. In a further embodiment, two or more p-tau species quantified are pT217 and pT181. In still further embodiments, the Aβ species quantified are Aβ40 and Aβ42 species. In yet still further embodiments, pT217 / T217 and Aβ42 / 40, and optionally pT181 / T181, are quantified. In yet further embodiments, a composite pT217 / T217×Aβ42 / 40 value is quantified.

[0089] In another aspect, the present disclosure provides a method for measuring the stability of tauopathy disease in a subject, comprising quantifying one or more p-tau species and Aβ species in a first biological sample obtained from the subject, and quantifying one or more p-tau species and Aβ species in a second biological sample obtained from the same subject at a later time (e.g., weeks, months, or years), and calculating the difference between the quantified p-tau species and Aβ species between the samples, wherein a statistically significant increase in the quantified p-tau species in the second sample indicates disease progression, a statistically significant decrease in the quantified p-tau species in the second sample indicates disease improvement, and no change indicates disease stability. The tauopathy can be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. The subject may or may not have clinical symptoms of the disease, and may or may not be receiving tau therapy. In some examples, tau therapy is administered to the subject one or more times during the period between the collection of the first biological sample and the collection of the second biological sample, and the measure of disease stability is an indication of the effectiveness or lack thereof of tau therapy. In a preferred embodiment, at least one p-tau species quantified is pT217. In a further embodiment, two or more p-tau species quantified are pT217 and pT181. In yet a further embodiment, the Aβ species quantified are Aβ40 and Aβ42 species. In yet a further embodiment, pT217 / T217 and Aβ42 / 40, and optionally pT181 / T181, are quantified. In yet a further embodiment, a composite pT217 / T217×Aβ42 / 40 value is quantified.

[0090] In another aspect, the present disclosure provides a method for treating a subject with a tauopathy, comprising quantifying one or more p-tau species and Aβ species in a biological sample obtained from the subject, such as a blood sample or CSF sample; and providing the subject with a tau therapy to improve the measurement of disease-related pathology or clinical symptoms, wherein the subject has a quantified p-tau species that is at least 1 standard deviation, preferably at least 1.3 standard deviation, more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ, or 1.5σ different, respectively, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy, and that is amyloid negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean can be used as a criterion for treating the subject. The tauopathy may be a 3R-tauopathy, a mixed 3R / 4R-tauopathy, or a 4R-tauopathy. The measure of disease-related pathology may be tau deposition measured by PET imaging, tau post-translational modifications measured by mass spectrometry or other suitable methods, amyloid plaques in the brain or cerebral arteries measured by PET imaging, amyloid plaques measured by Aβ42 / 40 in CSF, or other pathological features known in the art. The clinical symptoms may be dementia measured by clinically validated instruments (e.g., MMSE, CDR-SB, etc.), or other clinical symptoms known in the art for 3R- and 4R-tauopathy. In a preferred embodiment, at least one p-tau species quantified is pT217. In a further embodiment, the two or more p-tau species quantified are pT217 and pT181. In yet a further embodiment, the Aβ species quantified are Aβ40 and Aβ42 species. In still yet further embodiments, pT217 / T217 and Aβ42 / 40, and optionally pT181 / T181, are quantified.In yet further embodiments, the composite pT217 / T217×Aβ42 / 40 value is quantified. Many tau therapies target specific pathophysiological changes. For example, Aβ targeting therapies are generally designed to reduce Aβ production, antagonize Aβ aggregation, and increase brain Aβ clearance; tau targeting therapies are generally designed to change tau phosphorylation patterns, antagonize tau aggregation (general antagonism of tau or antagonism of specific tau isoforms), or increase NFT clearance; various therapies are designed to reduce CNS inflammation or brain insulin resistance, etc. However, not all tauopathies share the same pathophysiological changes. Thus, the efficacy of these various tau therapies can be improved by administering them to subjects who are correctly identified as having 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy.

[0091] Suitable biological samples and methods for measuring tau phosphorylation and Aβ species are described in Section II, the disclosure of which is incorporated herein by reference.

[0092] In certain embodiments, the present disclosure provides a method for distinguishing MAPT R406W tauopathy, comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species; and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and quantifying pT217 / T217 and Aβ42 / 40 values ​​to distinguish MAPT R406W tauopathy from Alzheimer's disease and healthy conditions.

[0093] In another specific embodiment, the present disclosure provides a method for distinguishing MAPT R406W tauopathy, comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species and Aβ species; and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample, wherein increased pT217 / T217 values ​​and normal Aβ42 / 40 values ​​distinguish MAPT R406W tauopathy from healthy conditions. In one aspect, the calculated change significantly deviates from the mean value in a control population that does not have brain amyloid plaques as measured by PET imaging. "Significantly deviate from the mean" includes values ​​at least 1 standard deviation, preferably at least 1.3 standard deviation, or more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ or 1.5σ, respectively, where σ is the standard deviation defined by normal distribution measured in a control population that does not have brain amyloid plaques as measured by PET imaging). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean may be used to diagnose the subject. The sample may be obtained from a subject who may or may not have a clinical diagnosis. In a further embodiment, the subject may carry one of the genetic mutations known to cause tauopathy. In an alternative embodiment, the subject may not have a genetic mutation known to cause tauopathy.

[0094] In another specific embodiment, the present disclosure provides a method for distinguishing MAPT R406W tauopathy, comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species and Aβ species; and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample, wherein a decrease in pT217 / T217 and an increase in Aβ42 / 40 values ​​distinguishes MAPT R406W tauopathy from AD. In one aspect, the calculated change significantly deviates from the mean value in a control population that does not have brain amyloid plaques as measured by PET imaging. "Significantly deviate from the mean" includes values ​​at least 1 standard deviation, preferably at least 1.3 standard deviation, or more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is the standard deviation defined by the normal distribution measured in AD populations with brain amyloid plaques as measured by PET imaging). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean can be used to diagnose the subject. The sample can be obtained from a subject who may or may not have a clinical diagnosis. In a further embodiment, the subject may carry one of the genetic mutations known to cause tauopathy. In an alternative embodiment, the subject may not have a genetic mutation known to cause tauopathy.

[0095] In another specific embodiment, the present disclosure provides a method for distinguishing MAPT R406W tauopathy, the method comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species; and quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample, the pT217 / T217×Aβ42 / 40 value distinguishing MAPT R406W tauopathy from Alzheimer's disease, 4R-tauopathy, and healthy conditions.

[0096] In another specific embodiment, the present disclosure provides a method for distinguishing MAPT R406W tauopathy, comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species and Aβ species; and quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample, wherein an increase in pT217 / T217×Aβ42 / 40 value distinguishes MAPT R406W tauopathy from a healthy state. In one aspect, the calculated change significantly deviates from the mean value in a control population without brain amyloid plaques measured by PET imaging. "Significantly deviate from the mean" includes values ​​that are at least 1 standard deviation, preferably at least 1.3 standard deviation, or more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ or 1.5σ, respectively, where σ is the standard deviation defined by normal distribution measured in a control population that does not have brain amyloid plaques as measured by PET imaging). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean may be used to diagnose the subject. Samples may be obtained from subjects that may or may not have a clinical diagnosis. In further embodiments, the subject may carry one of the genetic mutations known to cause tauopathy.

[0097] In another specific embodiment, the present disclosure provides a method for distinguishing MAPT R406W tauopathy, comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species and Aβ species; and quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample, wherein an increase in pT217 / T217×Aβ42 / 40 value distinguishes MAPT R406W tauopathy from AD. In one aspect, the calculated change significantly deviates from the mean value in an AD population with brain amyloid plaques measured by PET imaging. "Significantly deviate from the mean" includes values ​​that are at least 1 standard deviation, preferably at least 1.3 standard deviation, or more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is the standard deviation defined by the normal distribution measured in AD populations with brain amyloid plaques as measured by PET imaging). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean may be used to diagnose the subject. Samples may be obtained from subjects who may or may not have received a clinical diagnosis. In further embodiments, the subject may carry one of the genetic mutations known to cause tauopathy.

[0098] In another specific embodiment, the present disclosure provides a method for distinguishing MAPT R406W tauopathy, comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species and Aβ species; and quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample, wherein an increase in pT217 / T217×Aβ42 / 40 value distinguishes MAPT R406W tauopathy from 4R-tauopathy. In one aspect, the calculated change significantly deviates from the mean value in a 4R-tauopathy population without brain amyloid plaques as measured by PET imaging. "Significantly deviate from the mean" includes values ​​at least 1 standard deviation, preferably at least 1.3 standard deviation, or more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ or 1.5σ, respectively, where σ is the standard deviation defined by the normal distribution measured in a 4R-tauopathy population that does not have brain amyloid plaques as measured by PET imaging). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean may be used to diagnose the subject. Samples may be obtained from subjects who may or may not have a clinical diagnosis. In further embodiments, the subject may carry one of the genetic mutations known to cause tauopathy.

[0099] In another specific embodiment, the disclosure provides a method for distinguishing sporadic frontotemporal dementia (FTD), comprising: providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched in one or more p-tau species; and quantifying a pT181 / T181 value in the processed sample, where the pT181 / T181 value distinguishes sporadic FTD from Alzheimer's disease, other tauopathies, and healthy conditions.

[0100] In another specific embodiment, the present disclosure provides a method for distinguishing sporadic frontotemporal dementia (FTD), comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species; and quantifying the pT181 / T181 value in the processed sample, wherein the decrease in pT181 / T181 value distinguishes sporadic FTD from Alzheimer's disease. In one aspect, the calculated change significantly deviates from the mean value in a control population that does not have brain amyloid plaques as measured by PET imaging. "Significantly deviate from the mean" includes values ​​at least 1 standard deviation, preferably at least 1.3 standard deviation, or more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is the standard deviation defined by the normal distribution measured in AD populations with brain amyloid plaques as measured by PET imaging). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean may be used to diagnose the subject. Samples may be obtained from subjects who may or may not have a clinical diagnosis. In further embodiments, the subject may carry one of the genetic mutations known to cause tauopathy. In alternative embodiments, the subject may not have a genetic mutation known to cause tauopathy.

[0101] In another specific embodiment, the present disclosure provides a method for distinguishing sporadic frontotemporal dementia (FTD), comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species; and quantifying the pT181 / T181 value in the processed sample, wherein the decrease in pT181 / T181 value distinguishes sporadic FTD from healthy conditions.In one aspect, the calculated change significantly deviates from the mean value in a control population that does not have brain amyloid plaques as measured by PET imaging. "Significantly deviate from the mean" includes values ​​at least 1 standard deviation, preferably at least 1.3 standard deviation, or more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ or 1.5σ, respectively, where σ is the standard deviation defined by normal distribution measured in a control population that does not have brain amyloid plaques as measured by PET imaging). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean may be used to diagnose the subject. The sample may be obtained from a subject who may or may not have a clinical diagnosis. In a further embodiment, the subject may carry one of the genetic mutations known to cause tauopathy. In an alternative embodiment, the subject may not have a genetic mutation known to cause tauopathy.

[0102] In another specific embodiment, the present disclosure provides a method for distinguishing sporadic frontotemporal dementia (FTD), comprising: providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched with one or more p-tau species; and quantifying the pT181 / T181 value in the processed sample, wherein the decrease in pT181 / T181 value distinguishes sporadic FTD from healthy conditions.In one aspect, the calculated change significantly deviates from the mean value in a non-sporadic FTD tauopathy population that does not have brain amyloid plaques as measured by PET imaging. "Significantly deviate from the mean" includes values ​​that are at least 1 standard deviation, preferably at least 1.3 standard deviation, or more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is the standard deviation defined by the normal distribution measured in non-sporadic FTD tauopathy populations that do not have brain amyloid plaques as measured by PET imaging). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean can be used to diagnose the subject. Samples can be obtained from subjects that may or may not have a clinical diagnosis. In further embodiments, the subject may carry one of the genetic mutations known to cause tauopathy. In alternative embodiments, the subject may or may not have a genetic mutation known to cause tauopathy.

[0103] In another specific embodiment, the disclosure provides a method for measuring MAPT R406W tauopathy disease progression in a subject, comprising providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more p-tau and Aβ species; and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and providing a second processed CSF or blood sample obtained from the subject after (e.g., days, weeks, months) the first sample. providing a second CSF or blood sample, wherein the second CSF or blood sample is enriched in the same pTau and Aβ species, and quantifying the pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and calculating a difference between the quantified pT217 / T217 and Aβ42 / 40 values ​​of the second sample and the first sample, wherein a statistically significant difference in the quantified pT217 / T217 and Aβ42 / 40 values ​​in the second sample is indicative of disease progression in the subject.

[0104] In another specific embodiment, the disclosure provides a method for measuring MAPT R406W tauopathy disease progression in a subject, comprising providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more p-tau and Aβ species; and quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; providing a second processed CSF or blood sample obtained from the subject after (e.g., days, weeks, months) the first sample, providing two CSF ​​or blood samples, wherein the two CSF ​​or blood samples are enriched in the same p-tau and Aβ species; and quantifying the pT217 / T217 and Aβ42 / 40 values ​​in the processed samples; and calculating a difference between the quantified pT217 / T217 and Aβ42 / 40 values ​​of the second sample and the first sample, wherein there is no statistically significant difference in the quantified pT217 / T217 and Aβ42 / 40 values ​​in the second sample, indicating disease stability in the subject.

[0105] In another specific embodiment, the disclosure provides a method for measuring MAPT R406W tauopathy disease progression in a subject, comprising providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more p-tau and Aβ species; and quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample, wherein the second processed CSF or blood sample is obtained from the subject after (e.g., days, weeks, months) the first sample. providing a second CSF or blood sample, wherein the second CSF or blood sample is enriched in the same p-tau and Aβ species, and quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample; and calculating a difference between the quantified composite pT217 / T217×Aβ42 / 40 values ​​of the second sample and the first sample, wherein a statistically significant difference in the quantified composite pT217 / T217×Aβ42 / 40 value in the second sample is indicative of disease progression in the subject.

[0106] In another specific embodiment, the disclosure provides a method for measuring MAPT R406W tauopathy disease progression in a subject, comprising providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more pTau and Aβ species; and quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample; providing a second processed CSF or blood sample obtained from the subject after (e.g., days, weeks, months) the first sample, providing a second CSF or blood sample, wherein the second CSF or blood sample is enriched in the same p-tau and Aβ species; and quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample; and calculating a difference between the quantified composite pT217 / T217×Aβ42 / 40 values ​​of the second sample and the first sample, wherein there is no statistically significant difference in the quantified composite pT217 / T217×Aβ42 / 40 value in the second sample, indicating disease stability in the subject.

[0107] In another particular embodiment, the present disclosure provides a method for measuring sporadic frontotemporal dementia (FTD) disease progression in a subject, comprising: providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more p-tau species; and quantifying a pT181 / T181 value in the processed sample; providing a second processed CSF or blood sample obtained from the subject after (e.g., days, weeks, months) the first sample, the second CSF or blood sample being enriched in the same p-tau species, and quantifying the pT181 / T181 value in the processed sample; and calculating a difference between the quantified pT181 / T181 values ​​in the second sample and the first sample, wherein a statistically significant difference in the quantified combined pT181 / T181 value in the second sample indicates disease progression in the subject.

[0108] In another particular embodiment, the present disclosure provides a method for measuring sporadic frontotemporal dementia (FTD) disease progression in a subject, comprising: providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more p-tau species; and quantifying a composite pT181 / T181 value in the processed sample; providing a second processed CSF or blood sample obtained from the subject after (e.g., days, weeks, months) the first sample, the second CSF or blood sample being enriched in the same p-tau species, and quantifying the pT181 / T181 value in the processed sample; and calculating a difference between the quantified pT181 / T181 values ​​in the second sample and the first sample, where there is no statistically significant difference in the quantified pT181 / T181 values ​​in the second sample, indicating stability of the disease in the subject.

[0109] Instead of or in addition to using site-specific tau phosphorylation and Aβ species measurements, optionally with total tau measurements, in any of the above embodiments, ratios calculated from measured phosphorylation levels, or ratios calculated from measured phosphorylation levels and total tau, can be used. Both approaches are detailed in the examples. Mathematical operations other than ratios may also be used. For example, the examples use site-specific tau phosphorylation values ​​in various statistical models (e.g., linear regression, LME curve, LOESS curve, etc.) in conjunction with other known biomarkers (e.g., MAPT status, APOE ε4 status, age, sex, cognitive test scores, functional test scores, etc.). The selection of measurements and the selection of mathematical operations may be optimized to maximize the specificity of the method. For example, the accuracy of diagnosis may be evaluated by the area under the ROC curve, and in some embodiments, a ROC AUC value of 0.7 or more (e.g., 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, etc.) is set as the threshold.

[0110] Brain amyloid plaques in humans are routinely measured by amyloid positron emission tomography (PET). For example, 11 C-Pittsburgh Compound B (PiB) PET imaging is commonly used to detect Aβ plaque pathology. The standard uptake value ratio (SUVR) of cortical PiB-PET is used to reliably identify significant cortical Aβ plaques and classify subjects as PIB positive (SUVR≧1.25) or negative (SUVR<1.25). Thus, in the above embodiment, a control population without brain amyloid plaques as measured by PET imaging may refer to a population of subjects with cortical PiB-PET SUVR<1.25. Other values ​​of PiB binding (e.g., mean cortical binding potential) or analysis of regions of interest other than cortical regions may also be used to classify subjects as PIB positive or negative. Other PET imaging agents may also be used.

[0111] In another specific embodiment, the disclosure provides a method for treating a subject in need thereof, comprising: (a) providing a processed CSF or blood sample obtained from the subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species; (b) quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and (c) administering a treatment to the subject to alter tau pathology, wherein the subject's processed CSF or blood sample have quantified pT217 / T217 and Aβ42 / 40 values ​​that differ by about 1.5σ or more, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of a tauopathy and is amyloid negative as measured by PET imaging, and the amount of quantified pT217 / T217 values ​​is indicative of tau pathology in the brain of the subject. In some embodiments, administering a treatment to the subject to alter tau pathology alters or stabilizes the amount of quantified p-tau species.In some embodiments, the therapeutic agent is a cholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, an antidepressant (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), a gamma-secretase inhibitor, a beta-secretase inhibitor, an anti-Aβ antibody (including an antigen-binding fragment, variant or derivative thereof), an anti-tau antibody (including an antigen-binding fragment, variant or derivative thereof), an anti-T ... or derivatives), anti-TREM2 antibodies (including antigen-binding fragments, variants or derivatives thereof), TREM2 agonists, stem cells, dietary supplements (e.g., lithium water, omega-3 fatty acids with lipoic acid, long chain triglycerides, genistein, resveratrol, curcumin and grape seed extract, etc.), serotonin receptor 6 antagonists, p38 alpha MAPK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g. TRx0237, methylthionimium chloride, etc.), treatments to improve glycemic control (e.g. insulin, exenatide, liraglutide pioglitazone, 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 agonists, beta-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT1A and 1D receptor agonists, glutaminyl-peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensives, statins, and any combination thereof.In an exemplary embodiment, the pharmaceutical composition may include a kinase inhibitor. Suitable kinase inhibitors may inhibit thousand-and-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5, or Fyn. In another exemplary embodiment, the pharmaceutical composition may include a phosphatase activator. As a non-limiting example, the phosphatase activator may increase the activity of protein phosphatase 2A. In some embodiments, the treatment is a pharmaceutical composition comprising a tau targeted therapy, including, but not limited to, an active pharmaceutical ingredient that alters tau phosphorylation patterns, antagonizes tau aggregation, or increases the clearance of pathological tau isoforms and / or aggregates. In some embodiments, the treatment is an anti-Aβ antibody, an anti-tau antibody, an anti-TREM2 antibody, a TREM2 agonist, a gamma-secretase inhibitor, a beta-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau protein aggregation inhibitor. In one embodiment, an increase in pT217 / T217 values ​​and normal Aβ42 / 40 values ​​compared to healthy controls indicates that the subject is treated with MAPT R406W therapy. In another embodiment, a decrease in pT217 / T217 values ​​and an increase in Aβ42 / 40 values ​​compared to AD populations indicates that the subject is treated with MAPT R406W therapy or tau therapy. In yet another embodiment, an increase in pT217 / T217 values ​​and a decrease in Aβ42 / 40 values ​​compared to MAPT R406W populations indicates that the subject is treated with AD therapy. In yet another embodiment, an increase in the combined pT217 / T217×Aβ42 / 40 values ​​compared to control or AD populations indicates that the subject is treated with tau therapy.

[0112] In another specific embodiment, the present disclosure provides a method for treating a subject in need thereof, comprising: (a) providing a processed CSF or blood sample obtained from the subject, the CSF or blood sample being enriched in one or more p-tau species and Aβ species; (b) quantifying the pT181 / T181 value in the processed sample; and (c) administering a treatment to the subject to change tau pathology, wherein the subject's processed CSF or blood sample has a quantified MTBR tau species or a ratio of quantified MTBR tau species that differ by about 1.5σ or more, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid negative as measured by PET imaging, and the amount of quantified p-tau species represents the tau pathology in the subject's brain. In some embodiments, administering a treatment to the subject to change tau pathology changes or stabilizes the amount of p-tau species.In some embodiments, the treatment includes treatment with a cholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, an antidepressant (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), a gamma-secretase inhibitor, a beta-secretase inhibitor, an anti-Aβ antibody (including an antigen-binding fragment, variant or derivative thereof), an anti-tau antibody (including an antigen-binding fragment, variant or derivative thereof), an anti-T ... or derivatives), anti-TREM2 antibodies (including antigen-binding fragments, variants or derivatives thereof), TREM2 agonists, stem cells, dietary supplements (e.g., lithium water, omega-3 fatty acids with lipoic acid, long chain triglycerides, genistein, resveratrol, curcumin and grape seed extract, etc.), serotonin receptor 6 antagonists, p38 alpha MAPK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g. TRx0237, methylthionimium chloride, etc.), treatments to improve glycemic control (e.g. insulin, exenatide, liraglutide pioglitazone, 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 agonists, beta-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT1A and 1D receptor agonists, glutaminyl-peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensives, statins, and any combination thereof.In an exemplary embodiment, the pharmaceutical composition may include a kinase inhibitor. Suitable kinase inhibitors may inhibit thousand-and-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5 or Fyn. In another exemplary embodiment, the pharmaceutical composition may include a phosphatase activator. As a non-limiting example, the phosphatase activator may increase the activity of protein phosphatase 2A. In some embodiments, the treatment is a pharmaceutical composition that includes a tau targeted therapy, including, but not limited to, an active pharmaceutical ingredient that alters tau phosphorylation patterns, antagonizes tau aggregation, or increases the clearance of pathological tau isoforms and / or aggregates. In some embodiments, the treatment is an anti-Aβ antibody, an anti-tau antibody, an anti-TREM2 antibody, a TREM2 agonist, a gamma-secretase inhibitor, a beta-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau protein aggregation inhibitor. In one embodiment, a decrease in pT181 / T181 values ​​compared to healthy controls indicates that the subject is being treated with a sporadic FTD therapy or a tau therapy.

[0113] In each of the above embodiments, the pharmaceutical composition can include an imaging agent. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radionuclide-labeled antibodies, etc.).

[0114] In each of the above embodiments, the method may further include quantifying one or more of pT205 / T205, pT208 / T208, pT111 / T111, pT153 / T153, or any combination thereof.

[0115] IV. Clinical Trials Another aspect of the present disclosure is a method for selecting subjects for clinical trials, particularly clinical trials of Aβ or tau therapy, if all other criteria of clinical trials are met.In one embodiment, the method for selecting subjects for clinical trials may include: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched with one or more p-tau species and Aβ species; (b) quantifying the pT217 / T217 value and the Aβ42 / 40 value in the processed sample; and (c) selecting the subject for clinical trials of MAPT R406W tauopathy if the pT217 / T217 value is increased, the Aβ42 / 40 value is approximately the same as that of a healthy control population, and the subject does not have brain amyloid plaques as measured by PET imaging. In another embodiment, a method for selecting subjects in a clinical trial may include: (a) providing a processed CSF or blood sample obtained from the subject, where the CSF or blood sample is enriched in one or more p-tau and Aβ species; (b) quantifying pT217 / T217 and Aβ42 / 40 values ​​in the processed sample; and (c) excluding the subject from a clinical trial for AD (or Aβ therapy) if the pT217 / T217 value is increased and the Aβ42 / 40 value is approximately the same as a healthy control population, and the subject does not have brain amyloid plaques as measured by PET imaging.

[0116] In another embodiment, it is a method for selecting subjects for clinical trials, particularly clinical trials of Aβ or tau therapy, if all other criteria of clinical trials are met.In one embodiment, the method for selecting subjects for clinical trials can include: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched with one or more p-tau species and Aβ species; (b) quantifying the combined pT217 / T217×Aβ42 / 40 value in the processed sample; and (c) selecting subjects for clinical trials of MAPT R406W tauopathy if the combined pT217 / T217×Aβ42 / 40 value is increased compared to healthy control population. In another embodiment, a method for selecting subjects in a clinical trial may include: (a) providing a processed CSF or blood sample obtained from the subject, where the CSF or blood sample is enriched for one or more p-tau and Aβ species; (b) quantifying a composite pT217 / T217×Aβ42 / 40 value in the processed sample; and (c) excluding the subject from a clinical trial for AD if the composite pT217 / T217×Aβ42 / 40 value is increased compared to an AD population.

[0117] In another embodiment, the method is for selecting subjects for clinical trials, particularly clinical trials for Aβ or tau therapy, if all other criteria of the clinical trial are met.In one embodiment, the method for selecting subjects for clinical trials may include: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched with one or more p-tau species; (b) quantifying the pT181 / T181 value in the processed sample; and (c) selecting the subject for clinical trials for sporadic FTD therapy if the pT181 / T181 value is reduced compared to a healthy control population.In another embodiment, the method for selecting subjects for clinical trials may include: (a) providing a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is enriched with one or more p-tau species; (b) quantifying the pT181 / T181 value in the processed sample; and (c) excluding the subject from clinical trials for AD if the combined pT181 / T181 value is reduced compared to an AD population. The phrase "a control population free of brain amyloid plaques as measured by PET imaging" is defined in Section III.

[0118] Instead of or in addition to using measurements of site-specific tau phosphorylation, or optionally total tau, in any of the above embodiments, ratios calculated from the measured phosphorylation levels, or ratios calculated from the measured phosphorylation levels and total tau can be used. The ratios calculated from the measured phosphorylation levels can be ratios between pT181 and pT205, pT217 and pT205, or pT181 and pT217. The ratios calculated from the measured phosphorylation levels and total tau can be ratios between pT181 and total tau, p-T205 and total tau, or pT217 and total tau. Mathematical operations other than ratios may also be used. For example, in the examples, site-specific tau phosphorylation values ​​in various statistical models (e.g., linear regression, LME curves, LOESS curves, etc.) are used in conjunction with other known biomarkers (e.g., APOE ε4 status, age, sex, cognitive test scores, functional test scores, etc.).

[0119] The design of clinical trials for AD and FTD therapy can be greatly aided by the methods disclosed herein. Many clinical trials are designed to test the efficacy of imaging or therapeutic agents that target specific pathophysiological changes that occur before the onset of AD symptoms. As discussed above in Section III, the efficacy of these various agents can be improved by administering the agents to subjects with certain site-specific tau phosphorylation levels as measured by the methods disclosed and exemplified herein. Similarly, clinical trials that select subjects with symptoms of Aβ pathology or tau-only pathology can also benefit from being able to accurately distinguish between enrollee pathologies to determine whether efficacy is related to a specific pathology. Thus, in clinical trials, measuring tau phosphorylation levels as described herein, especially before selecting subjects for the treatment arm of the clinical trial, may result in reduced trials and / or improved outcomes. In some cases, the methods described herein can be developed and used as companion diagnostics for therapeutic agents.

[0120] In each of the above embodiments, the subject can be enrolled in a treatment arm of a clinical trial. "Treatment" is defined above. The subject enrolled in the treatment arm of the clinical trial can be administered a pharmaceutical composition. In some embodiments, the pharmaceutical composition can include an imaging agent. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radionuclide-labeled antibodies, etc.). Alternatively, the pharmaceutical composition can include an active pharmaceutical ingredient.Non-limiting examples of active pharmaceutical ingredients include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., 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 (including antigen-binding fragments, variants or derivatives thereof), stem cells, dietary supplements (e.g., lithium water, omega-3 fatty acids with lipoic acid, long chain triglycerides, genistein, resveratrol, curcumin and grape seed extract, etc.), antagonists of serotonin receptor 6, p38 alpha MAPK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors ( TRx0237, methylthionimium chloride, etc.), treatments to improve glycemic control (e.g. insulin, exenatide, liraglutide pioglitazone, 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 agonists, beta-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT1A and 1D receptor agonists, glutaminyl-peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents In an exemplary embodiment, the pharmaceutical composition may include a kinase inhibitor.Suitable kinase inhibitors may inhibit thousand-and-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5, or Fyn. In another exemplary embodiment, the pharmaceutical composition may include a phosphatase activator. As a non-limiting example, the phosphatase activator may increase the activity of protein phosphatase 2A.

[0121] In each of the above embodiments, the subject may or may not be asymptomatic. As used herein, an "asymptomatic subject" refers to a subject that does not show any signs or symptoms of tauopathy. Alternatively, the subject may exhibit signs or symptoms (e.g., memory loss, misplacing things, mood or behavior changes, etc.) but may not show sufficient cognitive or functional impairment for clinical diagnosis. A symptomatic or asymptomatic subject may have Aβ amyloidosis, but prior knowledge of Aβ amyloidosis is not a prerequisite for treatment. In yet 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 hereditary tauopathy. In an alternative embodiment, the subject may not have a genetic mutation known to cause hereditary tauopathy.

[0122] The following examples are included to demonstrate preferred embodiments of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention. However, those skilled in the art should understand in light of this disclosure that modifications can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. Accordingly, all matter described or shown in the accompanying drawings shall be interpreted as illustrative and not limiting in meaning. EXAMPLES

[0123] The following examples illustrate various iterations of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention. However, those skilled in the art should understand in light of this disclosure that modifications can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. Therefore, all matters described or shown in the accompanying drawings shall be interpreted as illustrative and not limiting in meaning. [Example 1]

[0124] MAPT R406W increases tau T217 phosphorylation in the absence of amyloid pathology Hyperphosphorylation of tau at threonine 217 (pT217) in cerebrospinal fluid (CSF) has recently been associated with early amyloidosis and may serve as a sensitive biomarker for Alzheimer's disease (AD). However, it remains unclear whether other tauopathies induce the pT217 modification. To determine whether the pT217 modification is specific to AD, we measured CSF pT217 in AD and other tauopathies.

[0125] Using immunoprecipitation and mass spectrometry, CSF T217 phosphorylation occupancy (pT217 / T217) and amyloid beta (Aβ)42 / 40 ratios were compared between cognitively normal individuals and those with symptomatic AD, progressive supranuclear palsy, corticobasal syndrome, and sporadic and familial frontotemporal dementia.

[0126] Individuals with AD had high CSF pT217 / T217 and low Aβ42 / 40. In contrast, cognitively normal individuals and the majority of individuals with 4R tauopathies had low CSF pT217 / T217 and normal Aβ42 / 40. A subgroup of individuals with increased CSF pT217 / T217 and normal Aβ42 / 40 ratios was identified, most of whom were MAPT R406W mutation carriers. The diagnostic accuracy of CSF Aβ42 / 40 and CSF pT217 / T217 alone and in combination was compared. We show that CSF pT217 / T217 × CSF Aβ42 / 40 is a sensitive composite biomarker that can separate MAPT R406W carriers from cognitively normal individuals and individuals with other tauopathies.

[0127] MAPT R406W is a tau mutation that causes a 3R+4R tauopathy similar to AD but without the amyloid neuropathology. Thus, this example shows that changes in the CSF pT217 / T217 ratio are not specific to AD but may reflect a common downstream tau pathophysiology shared by 3R+4R tauopathies.

[0128] Alzheimer's disease (AD) is characterized by plaque deposition of amyloid beta 42 peptide (Aβ42) and aggregation of hyperphosphorylated tau in neurofibrillary tangles, neuritic plaques, and neuropil threads in the brain. Concomitant decreases in the Aβ42 / 40 ratio and increases in phosphorylated tau (p-tau) species in cerebrospinal fluid (CSF) have been used as biomarkers of AD amyloidosis and as a surrogate for AD tau neuropathology, respectively. Increasing evidence suggests that tau phosphorylation at threonine 217 in CSF, measured as absolute concentration (pT217) or phosphorylation occupancy (pT217 / T217), is a specific and more sensitive biomarker of AD, surpassing the well-established measurement of CSF p-tau levels at threonine 181 (pT181). Increased CSF pT181 levels are strongly associated with increased CSF total tau concentrations and are assumed to reflect tau neuropathology. However, CSF pT217 and pT217 / T217 correlate more strongly with amyloid neuropathology measured by amyloid Pittsburgh compound B (PiB) positron emission tomography (PET) imaging than total CSF tau. Furthermore, pT217 can predict the onset of AD clinical symptoms in kindreds with AD mutations better than pT181. Thus, it remains unclear whether T217 hyperphosphorylation is an early and direct consequence of amyloidosis or a downstream marker of tau pathology. In this context, it is also unclear whether CSF pT217 / T217 is altered in primary tauopathy in the absence of amyloid pathology.

[0129] Other neurodegenerative dementia diseases associated with tau neuropathology, such as progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), and behavioral variant frontotemporal dementia (bvFTD), currently lack CSF or imaging biomarkers. Diagnosis relies primarily on clinical assessment and can be confirmed after brain autopsy. The lack of reliable in vivo biomarkers poses challenges to the accuracy of clinical diagnosis and impacts the design and implementation of clinical trials. Previous studies have primarily used immunoassays to measure absolute CSF pT181 concentrations, suggesting that increased pT181 is specific to AD. However, overall changes in CSF tau isoform concentrations may contribute to absolute pT181 concentrations without a relative change in pT181 phosphorylation. To fully interpret changes in CSF pT181, pT181 phosphorylation occupancy, measured as the pT181 / T181 ratio, is essential. Furthermore, several recent studies using immunoassays and mass spectrometry (MS) have shown that increased CSF pT217 concentrations are specific to AD and are not observed in other neurodegenerative diseases. However, previous studies have often not taken into account amyloid co-neuropathology, which is frequently increased in aging and many neurodegenerative diseases.

[0130] To evaluate the effect of tau phosphorylation in non-AD tauopathies, including PSP, CBS, and bvFTD, we measured CSF p-tau and CSF Aβ using sequential immunoprecipitation (IP) and MS. CSF pT217 / T217 and pT181 / T181 ratios were calculated to distinguish changes in tau phosphorylation from CSF total tau fluctuations. CSF Aβ42 / 40 ratios were calculated within the same participants and used as a surrogate for amyloid neuropathology. Cohorts of cognitively normal age-matched controls (AMC) and individuals with symptomatic AD, PSP, CBS, and sporadic and familial FTD were analyzed. Correlations between CSF p-tau and CSF Aβ42 / 40 ratios were assessed in each disease group, and the diagnostic relevance of CSF p-tau alone and in combination with CSF Aβ42 / 40 was evaluated to assess the ability to distinguish individuals with symptomatic AD from those with other neurodegenerative dementia diseases.

[0131] result Participants and study workflow: Participant demographics and clinical characteristics are summarized in Table 1. For analytical purposes, the study cohort was divided into groups. The "AD" group (n=80) included patients with clinically "typical" AD with predominant amnesia (n=66) and patients with focal mutations (n=14). bvFTD MAPT R406W mutation carriers (n=5) were grouped as "R406W". All neurodegenerative diseases other than AD and MAPT R406W mutation carriers were grouped as "4R tauopathy" (n=74). This group included individuals with PSP (n=16), CBS (n=15), CBS-PSP continuum (n=7), sporadic bvFTD (n=28), and bvFTD MAPT P301L mutation carriers (n=3), which were primarily 4R tauopathies with 4R tau as the predominant isoform in brain aggregates. It should be noted that although sporadic bvFTD are listed in the "4R tauopathy" group, they were not pathologically confirmed and may include a small number of 3R tauopathies such as FTLD-tau, FTLD-TDP43, FTLD-FUS, and Pick's disease. One of these participants was later found to have a C9orf72 mutation. Cognitively normal controls were named "controls" (n=98), which included cognitively normal AMC (n=64), YNC (n=26), and brain tumor patients (n=8).

[0132] [Table 1]

[0133] Individuals in the YNC (42.3 ± 2.4), brain tumor (50.2 ± 2.6), and MAPT P301L (37.2 ± 3.6) groups were younger than participants with AMC (73.0 ± 0.8), and neurodegenerative diseases including AD (73.3 ± 0.9), CBS (68.6 ± 2.6), CBS PSP continuum (70.7 ± 1.4), PSP (71.0 ± 2.6), and sporadic bvFTD (62.1 ± 1.3) (Table 1).

[0134] For all 252 CSF baseline samples and 8 CSF follow-up samples, CSF Aβ42, Aβ40, pT217, T217, pT181, and T181 concentrations were measured by sequential IP / MS methods. CSF Aβ42 / 40, pT217 / T217, and pT181 / T181 ratios were calculated. The workflow used to classify the different clinical groups is shown in Figure 1F.

[0135] Determination of cutoffs for IP / MS CSF Aβ42 / 40 and CSF pT217 / T217: To define an amyloid positivity cutoff for CSF Aβ42 / 40 measured by IP / MS, we used amyloid PiB-PET results from 48 participants in the WashU-A cohort (cutoff 0.18; Figure 1F, Table 1). CSF Aβ42 / 40 was significantly decreased in the amyloid-PiB+ cohort (Figure 3A). ROC curve analysis was performed and a Youden's index value of 0.086 was selected as the cutoff for CSF Aβ42 / 40 to maximize discrimination between cohorts (area under the curve [AUC] = 0.921, p < 0.0001; Figure 3B).

[0136] To determine the p-tau abnormality cutoff for CSF pT217 / T217, we used CSF Aβ42 / 40 values ​​in amyloid-PiB+ patients from the WashU-A cohort (Figure 1F). Another subset of 37 participants with MS only had additional CSF Aβ42 / 40 measurements. CSF pT217 / T217 was significantly increased in amyloid+ individuals based on PiB-PET and CSF Aβ42 / 40 measurements (Figure 3C). ROC curve analysis was performed and a Youden's index value of 4.76 was calculated as the cutoff for CSF pT217 / T217 (AUC=0.983, p<0.0001; Figure 3D).

[0137] The same ROC analysis was performed for CSF pT217, pT181, and total tau concentrations, and phosphorylation occupancy at T181 (pT181 / T181, Figures 3E-3L). The AUCs for each of these biomarkers were 0.949 (pT217), 0.816 (pT181), 0.698 (total tau), and 0.934 (pT181 / T181), respectively, supporting previous findings that CSF pT217 / T217 is an excellent discriminatory AD biomarker.

[0138] Association between IP / MS CSF Aβ42 / 40 and CSF pT217 / T217: To assess the relationship between IP / MS CSF Aβ42 / 40 and pT217 / T217, both ratios were plotted for each of the 255 participants. Based on the calculated 0.086 and 4.8 cutoffs for CSF Aβ42 / 40 and CSF pT217 / T217, respectively, quadrants were defined as follows: I (amyloid-, ptau+), II (amyloid+, ptau+), III (amyloid+, ptau-), and IV (amyloid-, ptau-) (Figures 1A-1E, Figure 3B).

[0139] In quadrant II (amyloid+, p-tau+), 88% (73 / 83) of individuals were clinically identified as having AD (Figure 1B). Overall, 91% (73 / 80) of AD patients were plotted in II (Figure 4A). The seven participants who were clinically identified as having AD were divided into quadrants I (n=3), III (n=1), and IV (n=3, Figures 1B-1D). A subset of controls (AMC [n=5], CBS [n=2], PSP [n=2], and bvFTD [n=1]) were also assigned to quadrant II (Figures 1A and 1B).

[0140] Eighty-four percent (82 / 98) of the controls plotted in the IV, and 55% (82 / 150) of the IV individuals were controls (Figure 1E, Figure 4B). Eighty percent (12 / 15) of the CBS and 81% (13 / 16) of the PSP were also in the IV (Figures 4C and 4D), as was 71% (5 / 7) of the CBS PSP continuum.

[0141] CSF Aβ42 / 40 and CSF pT217 / T217 were negatively associated, showing an L-shaped curve (Figure 1). To better understand the dynamic association between CSF Aβ42 / 40 and CSF pT217 / T217 profiles in relation to the AD continuum, we assessed and measured amyloid burden with PiB-PET and AV45-PET imaging, and tau aggregation with AV1451-PET imaging in a subset of participants (Figure 5). Aβ aggregation measured by both PET tracers gradually and significantly increased from quadrants IV, III to II (Figures 5A and 5B). All controls in quadrant III were AMC (Figure 1A), and none belonged to YNC (<64 years old). However, within AMC, age was not significantly different in quadrants III (74.8 ± 2.4) and IV (72.4 ± 0.6). These suggest that individuals in quadrant III with amyloid positivity may be defined as presymptomatic AD without abnormal tau phosphorylation. In contrast, tau aggregation measured by AV1451-PET increased only in quadrant II (Figure 5C), confirming that CSF Aβ42 / 40 and amyloid PET change before CSF pT217 / T217 and tau PET. Importantly, a significant negative correlation between CSF Aβ42 / 40 and CSF pT217 / T217 was observed in quadrants III and IV (pTau-), including most controls, PSP, CBS, and bvFTD (Figure 6).

[0142] MAPT R406W carriers had increased pT217 / T217 ratios without amyloid pathology: Quadrant I (amyloid-, pTau+) was populated with individuals with bvFTD, PSP, AD, or CBS (Figure 1C). Interestingly, 45% (5 / 11) had bvFTD, and all but one (80%, 4 / 5) were MAPT R406W mutation carriers (Figure 4F, Table 2). All (5 / 5) MAPT R406W mutation carriers analyzed in this study were amyloid negative (quadrants I and IV), supporting the absence of amyloid neuropathology in MAPT R406W carriers (Table 2). Only one of five MAPT R406W mutation carriers who was in their 40s and asymptomatic throughout the study was CSF pT217 / T217 negative (quadrant IV) at both baseline and follow-up visits; all other MAPT R406W mutation carriers were CSF pT217 / T217 positive (quadrant I) regardless of presymptomatic / onset status. One participant (#5) who was asymptomatic at baseline and developed dementia at the follow-up visit 4 years later had CSF pT217 / T217 just below the threshold at baseline (quadrant IV), but CSF pT217 / T217 increased at follow-up (quadrant I), suggesting that long-term changes in this biomarker may reflect disease progression. In comparison, the other five participants who had follow-up visits within 1 year (one with PSP in quadrant I, two with CBS and two with AMC in quadrant IV) remained in the same quadrant between baseline and follow-up. These results suggest that increasing age and onset of symptoms are associated with increased CSF pT217 / T217 in MAPT R406W mutation carriers.

[0143] [Table 2]

[0144] Diagnostic value of IP / MS CSF Aβ42 / 40, pT217 / T217, and composite biomarkers in AD and MAPTR406W mutation carriers: We next compared the diagnostic performance of IP / MS CSF Aβ42 / 40 and CSF pT217 / T217 using a composite biomarker consisting of pT217 / T217 multiplied by CSF Aβ42 / 40 and CSF pT217 / T217 divided by Aβ42 / 40 (Figure 2, Table 3, Figure 7). This analysis compared four previously defined clinical groups ("AD", "R406W", "4R tauopathy", and "controls"). When used alone, the CSF Aβ42 / 40 ratio and CSF pT217 / T217 only separate AD from the other three groups (Figure 2A and 2B). However, the R406W group had a significantly increased CSF Aβ42 / 40 × pT217 / T217 composite biomarker compared to that observed in the control and 4R tauopathy groups (Figure 2C). This composite biomarker showed excellent ability to separate the R406W group from the 4R tauopathy (AUC = 0.948) and control groups (AUC = 0.961). Using this composite biomarker, 100% of MAPT R406W mutation carriers scored above the cutoff of 0.50 for R406W vs. control (Table 3). CSF pT217 / T217 divided by CSF Aβ42 / 40 performed similarly to CSF ​​Aβ42 / 40 and CSF pT2117 / T217 alone and failed to discriminate R406W from the other groups (Figure 2D, Table 3).

[0145] [Table 3-1] [Table 3-2]

[0146] IP / MS CSF total tau and p-tau concentrations are not valid biomarkers for MAPT R406W carriers: Neither CSF pT217, pT181, total tau, nor phosphorylation occupancy at T181 (pT181 / T181) was as efficient as the composite biomarker CSF pT217 / T217×CSFAβ42 / 40 in separating MAPT R406W mutation carriers from individuals with other neurodegenerative dementia diseases (Figures 8D, 9D). CSF pT181 / T181 was significantly decreased in sporadic bvFTD (including FTLD-tau, FTLD-TDP43, and FTLD-FUS) compared to AD, AMC, and MAPT R406W mutation carriers (Figures 8D, 9D). When regrouped, CSF pT181 / T181 was able to separate the sporadic bvFTD group from AD (AUC=0.959), controls (AUC=0.752), and other tauopathies, such as CBS, PSP, and FTD-MAPT, including R406W and P301L (AUC=0.707; Figure 10). However, the specificity and sensitivity of CSF pT181 / T181 to separate sporadic bvFTD from other tauopathies and controls was not as high as that of CSF pT217 / T217 in identifying MAPT R406W mutation carriers.

[0147] Consideration MAPT R406W mutation carriers have increased pT217 / T217 ratios without amyloid pathology: CSF pT217 / T217 strongly correlates with amyloid pathology measured by amyloid PET in AD, but it was not proven whether pT217 / T217 is a readout for CSF amyloid pathology or tau pathology. This example showed a specific correlation between CSF Aβ42 / 40 and CSF pT217 / T217 in individuals with symptomatic AD. Even in presymptomatic AD, where subtle changes in phosphorylation are observed, CSF Aβ42 / 40 and CSF pT217 / T217 are correlated, consistent with previous reports showing a correlation between PiB-PET and CSF pT217 / T217. Neither CSF Aβ42 / 40 nor CSF pT217 / T217 was altered in other tauopathies, including PSP, CBS, and most sporadic and familial forms of FTD. However, MAPT R406W mutation carriers were found to have increased CSF pT217 / T217 independent of amyloid pathology, demonstrating that increased CSF pT217 / T217 is indeed a biomarker of pathological tau modification common to AD and MAPT R406W-associated dementias, and that amyloid pathology is not a prerequisite for this modification. This suggests the existence of common downstream tau pathology in AD and MAPT R406W mutation carriers, resulting in unique tau phosphorylation changes in the brain, leading to increased CSF pT217 / T217. Alternatively, two different upstream mechanisms, one involved in amyloid deposition and the second involved in the MAPT R406W mutation, could lead to activation of a similar pathway, ultimately causing tau hyperphosphorylation and aggregation.

[0148] Similarities of MAPT R406W mutations with AD: MAPT R406W mutation-associated lesions share multiple clinical and neuropathological similarities with AD. Unlike other MAPT mutation carriers, MAPT R406W mutation carriers have a later age of onset, with clinical symptoms such as memory loss appearing on average in the mid-50s, and progressing slowly. Most pathological MAPT mutations, such as P301L, are located in or around exon 10 and usually cause aggregation of 4R tau isoforms, resulting in 4R tauopathy. In contrast, MAPT mutations, such as R406W and V337M, are located at the C-terminus of tau protein within a domain common to both 3R and 4R tau isoforms, resulting in 3R+4R mixed brain lesions. Thus, MAPT R406W mutations can be classified as a 3R+4R tauopathy, similar to AD, and distinct from other 4R (CBS, PSP, bvFTD associated with MAPT mutations located in exon 10) or 3R (Pick's disease) tauopathies.

[0149] Filament structures in tau aggregates have recently been elucidated by cryo-electron microscopy in various tauopathies, such as AD and chronic traumatic encephalopathy (CTE) (3R+4R), CBS (4R), and Pick's disease (3R). Consistent with the neuropathological findings, tau domains shared by 3R and 4R isoforms are involved in AD and CTE tau aggregation, while 4R and 3R-specific domains are involved in corticobasal degeneration and Pick's disease aggregation, respectively. Although no such structural data are available for MAPT R406W, AD, MAPT R406W, and V337M, AD, MAPT R406W, and V337M have paired helical filament structures, and AD tau PET tracers such as AV1451 bind to some extent in presymptomatic MAPT R406W and V337M mutation carriers but not in other tauopathies, suggesting that tau aggregates in these 3R+4R tauopathies have similar characteristics. However, how hyperphosphorylation at T217 contributes to or is related to paired helical filament formation remains to be addressed. Previous studies have suggested that CSF T217 is hyperphosphorylated in early presymptomatic stages of AD and is detectable more than 20 years before clinical symptoms appear, while tau aggregates detected by PET imaging increase near the onset of symptoms. This example shows that in 3R+4R tauopathies, including MAPT R406W mutation carriers, (1) CSF pT217 / T217 becomes abnormal before the onset of symptoms when tau paired helical filament formation begins, but remains below the detection limit by tau PET imaging, and thereafter shows clear changes in tau PET imaging; or (2) CSF T217 hyperphosphorylation is not directly related to the formation of neurofibrillary tangles, but reflects abnormal cellular metabolism that affects tau and ultimately leads to tau aggregation.

[0150] A composite biomarker of CSF pT217 / T217 × CSF Aβ42 / 40 serves as a sensitive biomarker for MAPT R406W mutation carriers: We evaluated the diagnostic value of CSF pT217 / T217 and CSF Aβ42 / 40 alone and in combination. CSF pT217 / T217 levels were increased in MAPT R406W mutation carriers. However, the degree of increase was much smaller compared with AD and MAPT, and CSF pT217 / T217 alone could not separate R406W mutation carriers from the control group or the 4R tauopathy group, including PSP, CBS, sporadic bvFTD, and FTD-MAPT P301L (Figures 2A, 2B, and 6). Previous studies have shown that CSF and plasma pT181 concentrations are elevated in some cases of MAPT R406W mutation carriers, but the increase was moderate. These are consistent with the insufficient sensitivity obtained from this study using only CSF concentrations of pT181, pT217, or phosphorylation occupancy of T181 and T217 (pT181 / T181 and pT217 / T217) as R406W biomarkers. Through quadrant analysis, we demonstrated that both CSF pT217 / T217 and CSF Aβ42 / 40 are necessary to identify MAPT R406W mutation carriers with high accuracy. We also demonstrated that the combined biomarker, i.e., CSF pT217 / T217 × CSF Aβ42 / 40, contains sufficient sensitivity and specificity to distinguish MAPT R406W mutation carriers from controls and 4R tauopathies (Figure 2C, Figure 7C, AUC = 0.934, 0.960, respectively). This is comparable to the high specificity and sensitivity of CSF Aβ42 / 40 and CSF pT217 / T217 for distinguishing AD from controls (AUC=0.926, 0.952, respectively). The combination of CSF Aβ42 / 40 and pT217 / T217 ratios may be used in future studies to select presymptomatic MAPT R406W mutation carriers and possibly other 3R+4R tauopathies such as V337M.Furthermore, longitudinal measurements of CSF pT217 / T217 may reflect disease progression, suggesting that CSF Aβ42 / 40, CSF pT217 / T217, and combined biomarkers may serve as novel sensitive readouts in drug clinical trials for tauopathies that can assess target engagement in MAPT R406W mutation carriers.

[0151] Decreased CSF pT181 / T181 in sporadic bvFTD: Previous studies using immunoassays have shown mixed results with no or mild changes in CSF total tau or pT181 in bvFTD, PSP, and CBS patients. Consistent with multiple reports, this example showed no significant differences in CSF total tau or CSF pT181 concentrations alone between bvFTD, PSP, CBS, and control groups. However, calculation of phosphorylation occupancy within the same participants showed that CSF pT181 / T181 was significantly decreased in sporadic bvFTD. This can be achieved by normalizing the changes in pT181 per T181, taking into account the physiological increase in pT181 with increasing total tau, as well as individual differences such as age, sex, and genotype. The specificity and sensitivity (AUC<0.8) of the CSF pT181 / T181 biomarker in identifying sporadic bvFTD from controls or other tauopathies was not as high as the composite biomarker CSF pT217 / T217×CSF Aβ42 / 40 (AUC>0.9) in identifying MAPT mutation carriers, which may be due to the heterogeneity of the sporadic bvFTD cohort, including FTLD-tau, FTLD-TDP, and FTLD-FUS.

[0152] method Human studies: All studies were approved by the Institutional Review Board of Washington University in St. Louis, MO, USA and the Ethics Committee of Montpellier University Hospital (certified NFS96-900 CSF-NeuroBANK #DC-2008-417 [http: / / www.biobanques.eu] of the CHU Resource Center BB-0033-00031). All participants or their representatives consented to the collection and sharing of biofluid samples. Exclusion criteria included any conctradiction with lumbar puncture (LP) or lumbar catheter, including bleeding disorders, active anticoagulation, and active infection. Permission to handle personal data was granted by the French Data Protection Authority (CNIL) under the number 1709743v0.

[0153] Individuals with AMC and mild AD were recruited to the Knight Alzheimer Disease Research Center (ADRC) at Washington University School of Medicine as part of the Stable Isotope Labeling Kinetics (SILK) study. AMC were volunteers enrolled for research purposes and were cognitively normal. This included two distinct cohorts of symptomatic participants (WashU-A and WashU-B). Individuals from the WashU-A cohort participated in a 36-h lumbar catheter study. Individuals from the WashU-B cohort participated in the SILK study with five LPs over a 4-month period. Individuals were diagnosed by clinical assessment and classified according to the Clinical Dementia Rating (CDR). In addition to interviews with patients and collateral sources, brain PET imaging data and diagnostic CSF results were reviewed when available. This cohort includes clinical AD patients who did not have biomarkers consistent with AD and were classified as non-AD dementia. The WashU-A cohort was further classified by amyloid PET positivity based on PiB imaging. Young normal controls (YNC), aged 18–64 years, with no currently diagnosed neurological disease, were referred from Washington University Health Volunteer. Brain tumor patients were referred from Barnes Jewish Hospital. Patients with PSP, CBS, and sporadic bvFTD were referred from the affiliated Memory Diagnostic Center and Movement Disorders Clinic. MAPT P301L and R406W mutation carriers were clinically evaluated on-site at Washington University and referred from the Longitudinal Evaluation of Familial Frontotemporal Dementia Study (LEFFTDS; allftd.org / artfl-lefftds; Site PI NG). Eight participants (1 PSP, 3 MAPT R406W, 2 CBS, 2 AMC) underwent repeat LP and CSF collections.

[0154] Montpellier participants were referred from the Memory Resources and Research Center at Montpellier. They were classified into AD, CBS, PSP, bvFTD, and CBS-PSP continuum based on clinical, neuropsychological, brain imaging, and follow-up assessments. CSF biomarkers of Aβ42, tau, and pT181 concentrations were also measured by enzyme-linked immunosorbent assay (ELISA), and Aβ42 / 40 ratios were calculated. AD was diagnosed based on the ATN classification, according to accepted criteria; all AD participants had at least two abnormal CSF biomarkers. This includes the AD focal phenotype, which refers to the predominant language, behavioral, visuospatial, and apraxic phenotypes accompanied by CSF biomarkers of AD. Some PPA-endophenotype AD cases were included in the AD focal phenotype (n=5). CBS and PSP were diagnosed according to international criteria. bvFTD can result from frontotemporal lobar degeneration (FTLD)-tau, FTLD-TDP, and FTLD-FUS. Several language endophenotypes of FTLD were included in bvFTD (n=3). The CBS PSP continuum included patients with a CBS clinical phenotype who evolved to PSP during follow-up.

[0155] CSF collection: CSF from individuals with AD and AMC in the WashU-A cohort was collected via catheter as previously described. CSF from individuals with symptomatic AD, PSP, CBS, and bvFTD in the AMC and WashU-B cohorts was obtained by LP using gravity collection and centrifugation as previously described. CSF from MAPT mutant families was collected according to a standardized protocol at the Biomarker Core at the Washington University School of Medicine. CSF from individuals with brain tumors was obtained via a lumbar drain using a catheter either pre- or post-operatively.

[0156] CSF from the Montpellier cohort was collected using a standardized protocol for collection, centrifugation, and storage at the Memory Resources and Research Center at Montpellier. Briefly, an atraumatic needle was used for LP and CSF was collected into 10 mL polypropylene tubes and protein low-binding Eppendorf tubes. CSF was not centrifuged before aliquoting and storing at -80 °C. CSF tau and pT181 concentrations were measured using a standardized commercial INNOTEST sandwich ELISA X-MAP according to Fujirebio's instructions. CSF Aβ42 and Aβ40 were measured using Fujirebio's INNOTEST sandwich ELISA.

[0157] Sequential IP and MS methods for CSF Aβ and tau: CSF Aβ was analyzed as previously described with the following modifications. Detergent and chaotropic reagents (1% NP-40 final, 5 mmol / L guanidine, protease inhibitor cocktail) and tau ( 15 N-labeled 2N4R recombinant tau) and Aβ ( 15 A master mix containing N-labeled synthetic Aβ40, and 42) internal standards was prepared in low-binding Axygen tubes (Fisher Scientific, Pittsburgh, PA, USA, MCT-175). 500–1000 μL of CSF was added and immunoprecipitated with HJ5.1 mid-domain Aβ antibody. After washing, samples were digested with LysN protease, desalted, and analyzed on a XevoTQ-S mass spectrometer (Waters Corporation, Milford, MA, USA).

[0158] CSF tau and pTau were analyzed as previously described with the following modifications. HJ5.1 post-immunoprecipitation CSF samples containing tau internal standards were sequentially immunoprecipitated with tau1 mid-domain and HJ8.5 N-terminal tau antibodies. After washing, samples were trypsin digested, desalted, and analyzed on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The MS method for measuring pT217 and pT181 was described previously.

[0159] Amyloid and Tau PET Imaging: Amyloid PiB-PET, AV45-PET, and tau AV1451-PET imaging measurements were performed in a subset of AD and AMC participants from the Knight ADRC at the Washington University School of Medicine. Data were collected and processed as previously described. [Example 2]

[0160] Comparison of p-tau in non-AD tauopathies: LOAD vs. UCSF vs. NCRAD vs. CTRL vs. Tangle As previously reported, phosphorylation of T217 increases with amyloid beta. Abnormal tau phosphorylation was defined as values ​​above a cutoff defined by samples from participants in the following groups: amyloid beta negative (AB-), cognitively normal (CN), young normal control (YNC), brain metastasis (BM) or control (CTRL).

[0161] Considering this criterion, abnormal tau phosphorylation was observed in the majority of participants with corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) and Pick's disease (PiD). This trend is observed for CBD and PSP in three independent cohorts (UCSF, NCRAD, Tangled). The observed values ​​are in the same range as amyloid-positive participants (AB+CDR0) without clinical symptoms. Also, MAPT mutations N279K, S305S, V337M, 10+16 seem to induce CSF p-tau217 abnormalities. R406W, shown above, also has abnormal p-tau217. Thus, increased p-tau217 together with normal CSF Abeta42 / 40 seems useful to identify participants at risk for non-AD tauopathy (Figure 11).

[0162] Figure 12 shows that pTau181 is not sensitive enough to detect changes in tau phosphorylation in non-AD tauopathies. However, we observed that pTau181 was lower in TDP samples. Also, as shown in Figures 17-19, sporadic FTD (including FTD-tau, FTD-FUS, and FTD-TDP43) had reduced CSF pT181 / T181 ratios. Thus, pT181 / T181, when measured as a ratio, can separate sporadic FTD from other tauopathies.

[0163] As shown in Figure 13, pTau205 is mainly increased in amyloid-positive symptomatic (AB+CDR>0) and AD. N279K, S305S, V337M, and R406W are abnormally phosphorylated as seen for pTau217, but 10+16 is not abnormally phosphorylated. Some non-AD tauopathies (CBD, PSP, and Pick's disease) may be slightly higher than the control group, but the sensitivity of this site may be lower than pTau217.

[0164] pTau208 is associated with pTau217 in the LOAD cohort, but appears to be relatively low in AD compared to that observed for pTau217. N279K, S305S, V337M, and R406W are aberrantly phosphorylated, whereas 10+16 is not. Some non-AD tauopathies (CBD, PSP, and Pick's disease) may be slightly higher than controls, but the sensitivity of this site may be lower than pTau217 (Figure 14).

[0165] Figure 15 shows that pTau111 is associated with pTau217 in the LOAD cohort, but appears to be relatively low in AD compared to that observed for pTau217. Hyperphosphorylation of pTau217 is seen in CBD, PSP, or MAPT mutations, so no abnormal phosphorylation is observed in non-AD tauopathies. This may suggest that pTau111 is truly AD-specific and can be used to distinguish AD from non-AD tauopathies that are abnormally phosphorylated for pTau217.

[0166] As shown in Figure 16, similar to pTau111, pTau153 is associated with pTau217 in the LOAD cohort, but appears to be relatively low in AD compared to that observed for pTau217. As pTau217 hyperphosphorylation is seen in CBD, PSP, or MAPT mutations, no abnormal phosphorylation is observed in non-AD tauopathies. This suggests that pTau153 is truly AD-specific and can be used to distinguish AD from non-AD tauopathies that are abnormally phosphorylated for pTau217.

Claims

1. 1. A method for determining whether a subject has or is at increased risk for frontotemporal dementia (FTD) in a subject with symptoms of Alzheimer's disease, or a subject who is a carrier of a MAPT R406W mutation and has symptoms of Alzheimer's disease, comprising: (a) providing a processed CSF or blood sample obtained from the subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying pT217 / T217 and aβ42 / 40 values ​​in the processed sample; and (b)(i) an increased level of pT217 / T217 and a normal level of Aβ42 / 40 are detected compared to pT217 / T217 and Aβ42 / 40 levels in a healthy control population; or (ii) a decrease in the pT217 / T217 level and an increase in the Aβ42 / 40 level are detected compared to the pT217 / T217 level and the Aβ42 / 40 level in an Alzheimer's disease population; Determining that the subject has or is at increased risk for FTD. The method includes:

2. 2. The method of claim 1, wherein step (a) further comprises determining a composite pT217 / T217×Aβ42 / 40 value, and an increase in the composite value compared to a healthy control indicates that the subject has or is at increased risk of FTD.

3. 1. A method for determining whether a subject with symptoms of Alzheimer's disease has or is at increased risk for sporadic frontotemporal dementia (FTD), comprising: (a) providing a processed CSF or blood sample obtained from the subject, the CSF or blood sample being enriched in one or more p-tau species, and quantifying the pT181 / T181 value in the processed sample; and (b)(i) a decrease in the pT181 / T181 level is detected compared to the pT181 / T181 level in a healthy control population; or (ii) a decrease in the pT181 / T181 level is detected compared to the pT181 / T181 level in an Alzheimer's disease population; Determining that the subject has or is at increased risk for sporadic FTD. The method includes:

4. 1. A method for distinguishing MAPT R406W tauopathy, comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species; and (b) quantifying pT217 / T217 and Aβ42 / 40 values, and / or a combined pT217 / T217×Aβ42 / 40 value in said treated sample, (i) said increased pT217 / T217 levels and normal Aβ42 / 40 levels distinguish MAPT R406W tauopathy from healthy conditions; (ii) the decreased pT217 / T217 level and increased Aβ42 / 40 level distinguish MAPT R406W tauopathy from Alzheimer's disease; or (iii) the combined pT217 / T217×Aβ42 / 40 value distinguishes MAPT R406W tauopathy from Alzheimer's disease, 4R-tauopathy, and healthy conditions; Quantitative The method includes:

5. 1. A method for distinguishing sporadic frontotemporal dementia (FTD), comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau species; and (b) quantifying the pT181 / T181 value in the treated sample, the pT181 / T181 value distinguishing sporadic FTD from Alzheimer's disease, non-sporadic FTD tauopathies, and healthy conditions. The method includes:

6. 1. A method for measuring MAPT R406W tauopathy disease progression in a subject, the method comprising: (a) providing a first processed CSF or blood sample obtained from a subject, the first CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying pT217 / T217 and Aβ42 / 40 levels in the first processed sample; (b) providing a second processed CSF or blood sample obtained from the subject after the first sample, the second CSF or blood sample being enriched in the same p-tau and Aβ species as in step (a), and quantifying pT217 / T217 and Aβ42 / 40 levels in the second processed sample; and (c) calculating a difference between the quantified pT217 / T217 and Aβ42 / 40 values ​​of the second sample and the first sample, wherein a statistically significant difference in the quantified pT217 / T217 and Aβ42 / 40 values ​​in the second sample indicates disease progression in the subject. or the method comprises: (d) providing a first processed CSF or blood sample obtained from the subject, the first CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying a composite pT217 / T217×Aβ42 / 40 value in the first processed sample; (e) providing a second processed CSF or blood sample obtained from the subject after the first sample, the second CSF or blood sample being enriched in the same pTau and Aβ species as in step (d), and quantifying the composite pT217 / T217×Aβ42 / 40 value in the second processed sample; and (f) calculating a difference between the quantified composite pT217 / T217×Aβ42 / 40 value of the second sample and the first sample, wherein a statistically significant difference in the quantified composite pT217 / T217×Aβ42 / 40 value in the second sample indicates disease progression in the subject. Including, method.

7. 1. A method for measuring sporadic frontotemporal dementia (FTD) disease progression in a subject, comprising: (a) providing a first processed CSF or blood sample obtained from a subject, the first CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying the pT181 / T181 value in the first processed sample; (b) providing a second processed CSF or blood sample obtained from the subject after the first sample, the second CSF or blood sample being enriched in the same pTau and Aβ species as in step (a), and quantifying the pT181 / T181 value in the second processed sample; and (c) calculating a difference between the quantified pT181 / T181 values ​​in the second sample and the first sample, wherein a statistically significant difference in the quantified pT181 / T181 values ​​in the second sample indicates disease progression in the subject. The method includes:

8. 1. A pharmaceutical composition for use in a method of treating a subject having symptoms of Alzheimer's disease, said method comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species, and quantifying pT217 / T217 and Aβ42 / 40 values, and / or a combined pT217 / T217×Aβ42 / 40 value in the processed sample; and administering to said subject a pharmaceutical composition if increased pT217 / T217 levels and normal Aβ42 / 40 levels are detected compared to pT217 / T217 and Aβ42 / 40 levels in a healthy control population; administering a pharmaceutical composition to the subject if a decrease in pT217 / T217 levels and an increase in Aβ42 / 40 levels are detected compared to pT217 / T217 and Aβ42 / 40 levels in an Alzheimer's disease population; or administering the pharmaceutical composition to the subject if an increase in the combined pT217 / T217×Aβ42 / 40 level is detected compared to the combined pT217 / T217×Aβ42 / 40 level in a healthy control population, an Alzheimer's disease population, or a 4R-tauopathy population. Contains, or (b) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau and Aβ species; and quantifying the pT181 / T181 value in the processed sample; and administering the pharmaceutical composition to the subject if a decrease in the pT181 / T181 level is detected as compared to the pT181 / T181 level in a healthy control population, an Alzheimer's disease population, or a non-sporadic FTD tauopathy population. Including, Pharmaceutical compositions.

9. 1. A method for selecting subjects in a clinical trial, comprising: (a) providing a processed CSF or blood sample obtained from a subject, said CSF or blood sample being enriched in one or more p-tau and Aβ species; (b) quantifying pT217 / T217 and Aβ42 / 40 levels in the treated samples; and (c) selecting said subject for a clinical trial of FTD if the pT217 / T217 value is increased and the Aβ42 / 40 value is approximately the same as a healthy control population; or Excluding said subject from clinical trials of AD or Ab therapy if pT217 / T217 levels are increased and Aβ42 / 40 levels are similar to those of a healthy control population. The method includes:

10. 1. A method for selecting subjects in a clinical trial, comprising: (a) providing a processed CSF or blood sample obtained from a subject, said CSF or blood sample being enriched in one or more p-tau and Aβ species; (b) quantifying the combined pT217 / T217×Aβ42 / 40 value in the treated sample; and (c) selecting said subject for a clinical trial for FTD if said combined pT217 / T217×Aβ42 / 40 value is increased compared to a healthy control population; or and excluding a subject from a clinical trial for AD if said combined pT217 / T217×Aβ42 / 40 value is increased relative to an AD population. The method includes:

11. 1. A method for selecting subjects in a clinical trial, comprising: (a) providing a processed CSF or blood sample obtained from a subject, said CSF or blood sample being enriched in one or more p-tau species; (b) quantifying the pT181 / T181 level in the treated sample; and (c) selecting the subject for a clinical trial for sporadic FTD therapy if the pT181 / T181 value is decreased compared to a healthy control population; or and excluding the subject from a clinical trial for AD if the pT181 / T181 value is decreased relative to the AD population. The method includes:

12. 1. A method for distinguishing between Alzheimer's disease, comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau species; and (b) quantifying pT153 / T153 values ​​in the treated samples, wherein the pT153 / T153 values ​​distinguish Alzheimer's disease from non-AD tauopathies and healthy conditions, or increased pT153 / T153 values ​​compared to healthy control and non-Alzheimer's disease tauopathies populations distinguish Alzheimer's disease from non-AD tauopathies and healthy conditions. The method includes:

13. 1. A method for selecting subjects in a clinical trial, comprising: (a) providing a processed CSF or blood sample obtained from a subject, said CSF or blood sample being enriched in one or more p-tau species; (b) quantifying the pT153 / T153 level in the treated sample; and (c) selecting the subject for a clinical trial for an AD therapy if the pT153 / T153 value is increased compared to a healthy control population or a non-AD tauopathy population; or and excluding said subject from a clinical trial of a non-AD tauopathy therapy if said pT153 / T153 value is increased compared to a non-AD tauopathy population. The method includes:

14. 1. A method for distinguishing between Alzheimer's disease, comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau species; and (b) quantifying pT111 / T111 values ​​in the treated samples, wherein the pT111 / T111 values ​​distinguish Alzheimer's disease from non-AD tauopathy and healthy conditions, or increased pT111 / T111 values ​​compared to healthy control and non-Alzheimer's tauopathy populations distinguish Alzheimer's disease from non-AD tauopathy and healthy conditions. The method includes:

15. 1. A method for selecting subjects in a clinical trial, comprising: (a) providing a processed CSF or blood sample obtained from a subject, said CSF or blood sample being enriched in one or more p-tau species; (b) quantifying the pT111 / T111 level in the treated sample; and (c) selecting the subject for a clinical trial for an AD therapy if the pT111 / T111 value is increased compared to a healthy control population or a non-AD tauopathy population; or and excluding said subject from a clinical trial of a non-AD tauopathy therapy if said pT111 / T111 value is increased compared to a non-AD tauopathy population. The method includes:

16. 1. A method for distinguishing between tauopathies, comprising: (a) providing a processed CSF or blood sample obtained from a subject, the CSF or blood sample being enriched in one or more p-tau species; and (b) (i) quantifying the pT205 / T205 value in the treated sample, where the pT205 / T205 value distinguishes between a tauopathy and a healthy state, or an increased pT205 / T205 value distinguishes between a tauopathy and a healthy state; or (ii) quantifying the pT208 / T208 value in the treated sample, where the pT208 / T208 value distinguishes between a tauopathy and a healthy state, or an increase in the pT208 / T208 value distinguishes between a tauopathy and a healthy state. The method includes:

17. (a) a means for purifying one or more p-tau and / or Aβ species from a CSF or blood sample obtained from a subject; and (b) according to the method of any one of claims 1 to 7, 12, 14 and 16, (i) determining whether the subject has or is at increased risk of frontotemporal dementia (FTD); (ii) determining whether the subject has sporadic frontotemporal dementia (FTD) or is at increased risk for sporadic FTD; (iii) distinguish MAPT R406W tauopathy; (iv) measuring MAPT R406W tauopathy disease progression; (v) measuring sporadic frontotemporal dementia (FTD) disease progression; (vi) distinguishing Alzheimer's disease; or (vii) Differentiate tauopathies Instructions for Including the kit.

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