Methods for detecting MTBR tau isoforms and uses thereof

Abstract

The methods disclosed herein use a unique combination of processing steps to convert blood or CSF samples into samples suitable for quantification of MTBR tau species and other tau species. The invention also includes the use of MTBR tau species in blood or CSF to assess pathological features and / or clinical symptoms of 3R- and 4R-tauopathies for diagnosis, staging, and / or selection of appropriate treatments for a given disease stage and modification of a given treatment regimen.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 886,165, filed August 13, 2019, U.S. Provisional Application No. 62 / 970,950, filed February 6, 2020, and U.S. Provisional Application No. 63 / 044,836, filed June 26, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Government Rights This invention was made with government support under NS095773 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing Reference This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on date is named "665135_ST25.txt" and is 14 KB bytes in size.

[0004] Field The present invention encompasses methods for converting blood or cerebrospinal fluid (CSF) samples into samples suitable for quantifying microtubule binding region (MTBR) tau species by mass spectrometry, immunoassay, or other assays known in the art. The present invention also encompasses the use of MTBR tau species in blood or CSF to measure pathological features and / or clinical symptoms of 3R- and 4R-tauopathies for diagnosis, staging, and / or selection of appropriate treatments for a given disease stage. [Background technology]

[0005] background Accumulation of tau protein as insoluble aggregates in the brain is a hallmark of Alzheimer's disease and other neurodegenerative disorders termed tauopathies. Tau pathology is thought to propagate across brain regions and spread via the cell-to-cell transmission of specific pathological tau species in a prion-like manner, although the nature and dissemination process of these species (i.e., monomers, oligomers, and fibrils) are unknown (Frost et al., 2009; Goedert et al., 2010, 2017; Sanders et al., 2014; Wu et al., 2016; Mirbaha et al., 2018; Lasagna-Reeves et al., 2012). Tau is a full-length protein with six distinct isoforms. Furthermore, tau has over 100 post-translational modification sites, including phosphorylation, and multiple truncation sites (Meredith et al., 2013; Sato et al., 2018; Barthelemy et al., 2019; Cicognola et al., 2019; Blennow et al., 2020). Therefore, identifying the specific pathological tau species involved in tau spread is challenging. Several mass spectrometry (MS) studies have suggested that the microtubule-binding region (MTBR) of tau is enriched in aggregates in Alzheimer's disease brains (Taniguchi-Watanabe et al., 2016; Roberts et al., 2020). Furthermore, a series of cryo-electron microscopy (cryo-EM) studies have shown that the core structure of tau aggregates consists of subsegments of the MTBR domain, and the specific morphology depends on the tauopathy (Fitzpatrick et al., 2017; Falcon et al., 2018, 2019; Zhang et al., 2020). These findings strongly suggest that MTBR-tau is important for tau aggregation. However, these studies used postmortem brain tissue. The pathophysiology of corresponding extracellular MTBR-containing tau species in biological samples such as CSF and blood, which may serve as surrogate biomarkers of brain tau aggregates in living humans, remains largely unknown.

[0006] CSF samples are routinely obtained from study patients via lumbar puncture at their clinic visits. Previous CSF tau biomarker studies have shown that MTBR tau is absent from CSF and is concentrated in the N-terminal and mid-domain regions (Meredith et al., 2013; Sato et al., 2018). Species comprising the N-terminal to mid-domain appear to be actively secreted from neurons into the extracellular space after truncation between the mid-domain and the MTBR domain (Sato et al., 2018). Detection of MTBR tau species has been reported (Barthelemy et al., 2016b, a), but its correlation with disease has not yet been characterized. Recently, a tau species containing a truncation at residue 368 (tau368) within repeat region 4 (R4) was identified in CSF (Blennow et al., 2020). However, it is unclear whether tau368 reflects the entire pool of MTBR tau species, taking into account regions not captured by antibodies, truncations, and conformational variations.

[0007] Advances in high-resolution mass spectrometry technology have devised new methods for measuring the abundance of numerous proteins in biological samples. Despite advances in instrumentation and data analysis software, sample preparation remains an enormous challenge. The choice of sample preparation method can affect the observed metabolite profile and data quality, ultimately affecting the results of an experiment. This is especially true for proteins and peptides that are low in abundance in biological samples. Peptides fall under this umbrella and include many proteolytic fragments of full-length proteins that are variably produced in various disease processes. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there remains a need in the art for improved sample processing methods for quantifying low abundance MTBR tau species in biological fluids. [Means for solving the problem]

[0009] overview Among various aspects of the present invention, methods are provided for processing previously obtained biological samples to measure relative or absolute concentrations of tau by mass spectrometry.

[0010] One embodiment of the present invention includes a method for measuring tau in a biological sample, comprising: (a) preparing a biological sample selected from a blood sample or a CSF sample; (b) removing proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease, and then optionally desalting the resulting cleavage products by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect and measure the concentration of at least one of the tau proteolytic peptides.

[0011] Another aspect of the present invention includes a method for assessing tau in a biological sample, the method comprising: (a) depleting N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau in a biological sample, the biological sample being a blood sample or a CSF sample, by affinity depletion; (b) removing additional proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease, and then optionally desalting the resulting cleavage products by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau peptides to detect and measure the concentration of at least one of the tau proteolytic peptides.

[0012] Another aspect of the present invention includes a method for measuring tau in a biological sample, comprising: (a) depleting N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau in a biological sample that is a blood sample or a CSF sample by affinity depletion; (b) affinity-purifying MTBR tau; (c) cleaving the purified MTBR tau with a protease, and then optionally desalting the resulting cleavage products by solid-phase extraction to obtain a sample containing proteolytic peptides of MTBR tau; and (d) performing liquid chromatography-mass spectrometry on the sample containing proteolytic peptides of MTBR tau to detect and measure the concentration of at least one of the proteolytic peptides of MTBR tau.

[0013] Another aspect of the invention is a method for measuring tau in a biological sample, comprising: (a) depleting N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau in the biological sample, which is a blood sample or a CSF sample, by affinity depletion, wherein affinity depletion comprises contacting the biological sample with an epitope binding agent that specifically binds to an epitope within amino acids 1 to 221 (inclusive), preferably within amino acids 50 to 221 (inclusive), or more preferably within amino acids 104 to 221 (inclusive) of tau-441 (or within a similarly defined region of other full-length isoforms); and (b) depleting MTBR. The method includes affinity-purifying tau, wherein the affinity purification comprises contacting the product of step (a) with an epitope binding agent that binds to an epitope C-terminal to the epitope recognized by the epitope binding agent of step (a); (c) cleaving the purified MTBR tau with a protease, and then optionally desalting the resulting cleavage products by solid-phase extraction to obtain a sample containing proteolytic peptides of MTBR tau; and (d) performing liquid chromatography-mass spectrometry on the sample containing proteolytic peptides of MTBR tau to detect and measure the concentration of at least one of the proteolytic peptides of MTBR tau. In certain embodiments, the epitope binding agent of step (b) specifically binds to an epitope within amino acids 221 to 441 (inclusive) of tau-441 (or within a similarly defined region of other full-length isoforms). In certain embodiments, the epitope binding agent in step (b) specifically binds to an epitope within amino acids 235-441 (inclusive) of Tau-441 (or within a similarly defined region of other full-length isoforms). In certain embodiments, the epitope binding agent in step (b) specifically binds to an epitope within amino acids 235-368 (inclusive) of Tau-441 (or within a similarly defined region of other full-length isoforms). In certain embodiments, the epitope binding agent in step (b) specifically binds to an epitope within amino acids 244-368 (inclusive) of Tau-441 (or within a similarly defined region of other full-length isoforms).In certain embodiments, the epitope binding agent in step (b) specifically binds to an epitope within amino acids 244 to 299 (inclusive) of Tau-441 (or within a similarly defined region of other full-length isoforms).

[0014] Prior to use in the methods disclosed herein, biological samples may have been modified by removal of cellular debris, addition of components (e.g., protease inhibitors, isotope-labeled internal standards, detergents, chaotropic agents, etc.), and / or depletion of analytes (e.g., Aβ peptides, N-terminal tau, mid-domain tau, etc.).

[0015] The methods disclosed herein are particularly suitable for measuring MTBR tau. In the specific embodiments described above, the methods of the present invention may be used to measure the concentration of one or more tau tryptic peptides, including, but not limited to, IGST (SEQ ID NO: 2), VQII (SEQ ID NO: 4), LQTA (SEQ ID NO: 3), LDLS (SEQ ID NO: 5), HVPG (SEQ ID NO: 6), IGSL (SEQ ID NO: 7), and VQIV (SEQ ID NO: 9). In certain instances, it may be desirable to measure the concentration of two or more tau tryptic peptides and then calculate the ratio of the two values. As disclosed herein, ratios of HVPG (SEQ ID NO:6) to IGSL (SEQ ID NO:7), LQTA (SEQ ID NO:3) to IGSL (SEQ ID NO:7), IGST (SEQ ID NO:2) to IGSL (SEQ ID NO:7), VQII (SEQ ID NO:4) to IGSL (SEQ ID NO:7), LDLS (SEQ ID NO:5) to IGSL (SEQ ID NO:7), IGST (SEQ ID NO:2) to HVPG (SEQ ID NO:6), VQII (SEQ ID NO:4) to HVPG (SEQ ID NO:6), LDLS (SEQ ID NO:5) to HVPG (SEQ ID NO:6), and VQIV (SEQ ID NO:7) to LDLS (SEQ ID NO:5) can provide clinically meaningful information for tauopathy diagnosis and guidance in treatment decisions. In still further examples, it may be desirable to determine the presence / absence of and / or measure the concentration of one or more additional proteins in a biological sample.

[0016] Another aspect of the present invention provides a method for measuring tauopathy-related pathology in a subject, comprising quantifying one or more intermediate domain-independent MTRB tau species in a biological sample obtained from the subject, such as a blood sample or CSF sample, wherein the amount of the quantified intermediate domain-independent MTRB tau species or their ratio represents the tauopathy-related pathology in the subject's brain. The tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. The disease-related pathology may be tau deposition, tau post-translational modifications, amyloid plaques in the brain and / or cerebral arteries, or other pathological characteristics known in the art. The subject may or may not have clinical symptoms of tauopathy.

[0017] Another aspect of the present invention provides a method for diagnosing a tauopathy in a subject, comprising quantifying one or more intermediate domain-independent MTBR tau species in a biological sample obtained from the subject, such as a blood sample or CSF sample, and diagnosing the tauopathy when the quantified intermediate domain-independent MTBR tau species differ by about 1.5σ or more, where σ is the standard deviation defined by a normal distribution measured in a control population without clinical signs or symptoms of tauopathy and amyloid-negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF. The tauopathy may be a 3R-tauopathy, a mixed 3R / 4R-tauopathy, or a 4R-tauopathy. The subject may or may not have clinical symptoms of the disease.

[0018] Another aspect of the present invention provides a method for measuring disease stability in a subject, comprising quantifying one or more intermediate domain-independent MTBR tau species in a first biological sample obtained from the subject and then a second biological sample obtained from the same subject, where the second biological sample is obtained after the first biological sample (e.g., days, weeks, months, or years), and calculating the difference in the quantified MTBR tau species between the samples, where a statistically significant increase in the quantified MTBR tau species in the second sample indicates disease progression, a statistically significant decrease in the quantified MTBR tau species in the second sample indicates disease improvement, and no change indicates disease stability. The subject may or may not have clinical symptoms of the disease.

[0019] Another embodiment of the present invention provides a method of treating a subject with a tauopathy, comprising quantifying one or more intermediate domain-independent MTBR tau species in a biological sample obtained from the subject, such as a blood sample or CSF sample; and administering a treatment to the subject to improve a measure of disease-related pathology and / or clinical symptoms, wherein the quantified MTBR tau species in the subject 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 assessed by PET imaging and / or Aβ42 / 40 measurement in CSF. The tauopathy can be a 3R-tauopathy, a mixed 3R / 4R-tauopathy, or a 4R-tauopathy. The measure of disease-related pathology can be the amount of MTBR tau species and / or tau deposits assessed by PET imaging, tau post-translational modifications assessed by mass spectrometry or other suitable methods, amyloid plaques in the brain or cerebral arteries assessed by PET imaging, amyloid plaques measured by Aβ42 / 40 in CSF, or other pathological features known in the art. The clinical symptoms can be dementia assessed by a clinically validated instrument (e.g., MMSE, CDR-SB, etc.) or other clinical symptoms known in the art for 3R-, 3R / 4R-, and 4R-tauopathies.

[0020] These and other aspects and iterations of the invention are described in further detail below. [Brief explanation of the drawings]

[0021] This application contains at least one photograph submitted in color. Copies of this patent application publication with color photograph(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] [Figure 1] Figure 1 shows a schematic diagram of the longest human tau isoform (2N4R). The N-terminus (N-terminus), mid-domain, MTBR, and C-terminus (C-terminus) of this isoform are identified and vary in a predictable manner with respect to other tau isoforms (e.g., 2N3R, 1NR4, 1N3R, 0N4R, and 0N3R).

[0023] [Figure 2A] Figure 2A is a schematic diagram illustrating some methods of the present invention. The method detailed in the blue box (right) is one method. The combination of the red box (left) and the blue box (right) is another method.

[0024] [Figure 2B] Figure 2B is a schematic diagram illustrating some methods of the present invention. The method shown in the blue box (right) is one method. The combination of the red box (left) and the blue box (right) is another method.

[0025] [Figure 3A]Figure 3A is a graph comparing the effects of three sample processing methods on the ability to quantify tau peptides from a single test CSF sample. The CSF test samples were not from a single individual, and the disease state associated with the CSF was not available. Tau-441 peptides are shown on the x-axis, and the 14N / 15N ratio is shown on the y-axis. The relative positions of the epitopes recognized by antibodies HJ8.5 and Tau1 (each indicated as "Y") are indicated. In samples processed by the IP method (green triangles), tau tryptic peptides from the MTBR region, while detectable, were much lower in signal than tau tryptic peptides from the N-terminal to mid-domain, and were not quantifiable in human CSF from patients with chronic neurodegenerative diseases, including AD, and healthy volunteers. In contrast, these peptides were readily detectable in samples processed by the CX method (blue circles) or the PostIP-CX method (red squares).

[0026] [Figure 3B] Figure 3B shows how sample processing can affect the population of tau protein detected by downstream methods. In the IP method (encircled by a green dotted line), tau species with N-terminal and mid-domain epitopes recognized by antibodies (exemplified by HJ8.5 and Tau1, respectively) are immunoprecipitated. In the PostIP-CX method (encircled by a red dotted line), tau species present after immunoprecipitation and precipitation do not have epitopes recognized by the antibodies used in immunoprecipitation (exemplified by the "MTBR-C" diagram), or the epitopes are inaccessible (exemplified by the diagram of linearized tau). The CX method (encircled by a blue dotted line) performed without prior immunoprecipitation results in samples containing tau species derived from the IP and PostIP-CX methods.

[0027] [Figure 4]Figure 4 is a graph of pT217% (x-axis) versus Aβ42 / 40 concentration (y-axis) assessed in LOAD100 and LOAD60 CSF samples. The horizontal dotted line defines amyloid status as defined by CSF Aβ42 / 40 concentration (CSF Aβ42 / 40>0.1389=amyloid positive and CSF Aβ42 / 40<0.1389=amyloid negative). As shown, p217% correlates extremely well with amyloid status as defined by this cutoff.

[0028] [Figure 5] Figure 5 shows graphs of two tau tryptic peptides, TPPS and HVPG, quantified by mass spectrometry in CSF samples processed by the IP method described in Example 1 (IP_TPPS, left graph) or the PostIP-CX method described in Examples 1 and 2 (PostIP_HVPG, right graph). CSF samples were identified by CDR score and amyloid status. The graph between the two graphs shows the relative position of the tryptic peptides in tau-441. NS - not significant.

[0029] [Figure 6A] Figure 6A is a graph showing the amount of tau tryptic peptides, HVPG versus Aβ42 / 40, in CSF samples processed by the PostIP-CX method described in Examples 1 and 2. CSF samples were characterized by amyloid status - amyloid positive (red) or amyloid negative (blue). The data show that measurement of HVPG in CSF samples processed by the PostIP-CX method described in Examples 1 and 2 recapitulates the amyloid status in the brain, as evidenced by the strong correlation with amyloid status in terms of Aβ42 / 40.

[0030] [Figure 6B]Figure 6B is a graph showing the amount of tau tryptic peptide, HVPG vs. pT217% in CSF samples processed by the PostIP-CX method described in Examples 1 and 2. CSF samples were characterized by amyloid status - amyloid positive (red) or amyloid negative (blue). The data show that measurement of HVPG in CSF samples processed by the PostIP-CX method described in Examples 1 and 2 recapitulates the amyloid status in the brain, as evidenced by the strong correlation with amyloid status in terms of pT217%.

[0031] [Figure 7] Figures 7A, 7B, and 7C are graphs showing three tau tryptic peptides, LQTA (Figure 7A), HVPG (Figure 7B), and IGSL (Figure 7C), in CSF samples processed by the PostIP-CX method described in Examples 1 and 2. CSF samples are grouped by CDR score and amyloid status. The data show that LQTA is increased in amyloid-positive subjects compared to amyloid-negative subjects, even in the symptomatic stage; HVPG is increased in amyloid-positive subjects compared to amyloid-negative subjects, especially in the asymptomatic stage; and IGSL is increased in amyloid-positive subjects compared to amyloid-negative subjects and decreases after the symptomatic stage.

[0032] [Figure 8]Figures 8A, 8B, and 8C are graphs showing the amounts of three tau tryptic peptides, LQTA (Figure 8A), HVPG (Figure 8B), and IGSL (Figure 8C), respectively, in CSF samples processed by the PostIP-CX method described in Examples 1 and 2. Longitudinal samples from individual patients are shown with amyloid status indicated by symbols (CDR0 = black circle, CDR0.5 = blue triangle, CDR1 = red square, CDR2 = purple inverted triangle). Paired t-tests were used for the results of the first and second visits for individual participants. The amyloid-positive group showed significant directional changes for each patient. Also noteworthy was the change observed in participant A (indicated by the thick red line), who had a high tau-PET signal (>2 SUVR) and a change in CDR score from CDR1 to CDR2. For this patient, tau pathology progression led to an increase in LQTA (Fig. 8A), a decrease in HVPG (Fig. 8B), and a decrease in IGSL (Fig. 8C).

[0033] [Figure 9] Figures 9A, 9B, and 9C are graphs showing the amount of three tau tryptic peptides in CSF samples processed by the PostIP-CX method described in Examples 1 and 2 versus the CDR-SB score in the subjects from which the samples were obtained. The three tau tryptic peptides are LQTA, HVPG, and IGSL, respectively. Amyloid-positive subjects are blue circles, and amyloid-negative subjects are red squares. As shown by the accompanying statistical analysis, only LQTA significantly correlates with CDR-SB.

[0034] [Figure 10]10A, 10B, and 10C are graphs showing the amount of three tau tryptic peptides in CSF samples processed by the PostIP-CX method described in Examples 1 and 2 versus the Mini-Mental State Examination (MMSE) score in the subjects from whom the samples were obtained. The three tau tryptic peptides are LQTA, HVPG, and IGSL, respectively. Amyloid-positive subjects are blue circles, and amyloid-negative subjects are red squares. As shown by the accompanying statistical analysis, only LQTA significantly correlates with MMSE.

[0035] [Figure 11] 11A, 11B, and 11C are graphs showing the amount of three tau tryptic peptides in CSF samples processed by the PostIP-CX method described in Examples 1 and 2 versus the tau-PET score in the subject from whom the sample was obtained. The three tau tryptic peptides are LQTA, HVPG, and IGSL, respectively. Amyloid-positive subjects are blue circles, and amyloid-negative subjects are red squares. As shown by the accompanying statistical analysis, only LQTA significantly correlates with tau-PET. Other tryptic peptides of MTBR tau were not significantly correlated.

[0036] [Figure 12] Figures 12A and 12B are graphs showing the amount of tau tryptic peptide, LQTA, versus CDR-SB (Figure 12A) and MMSE (Figure 12B) in CSF samples processed by the PostIP-CX method described in Examples 1 and 2. The data showed that LQTA correlated significantly with cognitive function, as assessed by two different measures of cognitive impairment.

[0037] [Figure 13A]Figure 13A is a graph showing the amount of tau tryptic peptide, LQTA, in samples processed by the IP method (x-axis) and the PostIP-CX method (y-axis). Amyloid-positive subjects are identified with red symbols, and amyloid-negative subjects are identified with blue symbols. As shown by the accompanying statistical analysis, only "MTBR-associated LQTA" (measured in samples processed by the PostIP-CX method) was increased in the amyloid-positive group compared to the amyloid-negative group.

[0038] [Figure 13B-C] Figures 13B and 13C are graphs showing the amount of LQTA, a tryptic tau peptide, in CSF samples treated with the PostIP-CX or IP methods described in Examples 1 and 2, respectively. CSF samples are grouped by CDR score and amyloid status. The data show that the LQTA-specific feature (i.e., linear increase after the symptomatic stage) was observed only in "MTBR-associated LTQA." Data are presented as individual results (plots) and means (bars). Statistical significance: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. NS = non-significant. Statistical differences were assessed by one-way ANOVA with multiple comparison correction using the Benjamin-Hochberg false positive rate (FDR) method, with the FDR set at 5%.

[0039] [Figure 14] 14 is a graph showing receiver operating characteristic curves (ROC) comparing the sensitivity and specificity of LQTA, a tryptic peptide of tau measured by mass spectrometry following the IP method (blue (bottom) line) or the PostIP-CX method (red (top) line), for determining amyloid status. The curves show that PostIP-LQTA (MTBR-associated LQTA) better discriminates amyloid status than IP-LQTA.

[0040] [Figure 15A-B]Figures 15A and 15B show that the ratio of IGSL to HVPG enhances discrimination. Figure 15A is a graph showing the amount of tau tryptic peptides, IGSL and LQTA, expressed as a ratio (IGSL / LQTA), in CSF samples processed by the PostIP-CX method described in Examples 1 and 2. CSF samples are grouped by CDR score and amyloid status. Figure 15B is a graph showing the correlation between the IGSL / LQTA ratio and pT205%. pT205% was measured as previously described (Barthelemy, NR, Li, Y, Joseph-Mathurin, N. et al. Nat Med 26, 398-407 (2020)). IGSL / LQTA shows a very close correlation with pT205, which is regulated near the onset of onset AD.

[0041] [Figure 15C-D] Figures 15C and 15D show that the ratio of IGSL to HVPG enhances discrimination. Figure 15C is a graph showing the amounts of tau tryptic peptides, IGSL and HVPG, expressed as the ratio (IGSL / HVPG) in CSF samples processed by the PostIP-CX method described in Examples 1 and 2. CSF samples are grouped by CDR score and amyloid status. Figure 15D is a graph showing the correlation between the IGSL / LQTA ratio and pT217%. IGSL / HVPG correlates very closely with pT217, which recapitulates the amyloid status.

[0042] [Figure 16]Figure 16 shows the progression of tau pathology through different phases of Alzheimer's disease. Measurement of four different soluble tau species and insoluble tau in participants with definitive Alzheimer's disease mutations revealed tau-related changes (y-axis) over a course of approximately 40 years (x-axis), which differed based on disease stage and other measurable biomarkers. Starting with the development of fibrillar amyloid pathology, phosphorylation at positions 217 (purple) and 181 (blue) begins to increase. As neuronal dysfunction increases (based on metabolic changes), phosphorylation at position 205 (green) increases along with soluble tau (orange). Finally, with the onset of neurodegeneration (based on brain atrophy and cognitive decline), Tau-PET tangles (red) begin to develop, and phosphorylation at positions 217 and 181 begins to decrease. Taken together, this highlights the dynamic and branching patterns of soluble and aggregated tau in the disease course and their close correlation with amyloid pathology.

[0043] [Figure 17] Figure 17 is a diagram of a theoretical model showing how the accessibility of various regions of MTBR-tau to cleavage may change during the progression of Alzheimer's disease (AD) and why the MTBR-tau species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK) is a good surrogate for tau pathology across all AD stages. In presymptomatic AD stages, brain tau aggregates are immature, allowing broader access for proteases. MTBR-tau species, including MTBR-tau243, MTBR-tau299, and MTBR-tau354, are secreted into CSF. However, as the disease progresses, tau aggregates mature and form an increasingly rigid core, reducing protease access to MTBR-tau354 and then MTBR-tau299, while MTBR species, including MTBR-tau354 and then MTBR-tau299, are stabilized in CSF. However, MTBR-tau243 remains exposed throughout the disease and is available for protease digestion and release into CSF. An imbalance of these three species in the CSF is observed as a sign of brain tau aggregate formation. Note: In this figure, the size differences between MTBR tau species are not depicted.

[0044] [Figure 18A] FIG. 18A is a schematic representation of tryptic digest peptides from tau (gray bars) quantified in Example 3 and further described in FIGS. 18B and 18C.

[0045] [Figure 18B-C] Figures 18B and 18C are graphs showing that brain MTBR-tau species, including MTBR-tau 243, 299, and 354, are enriched in aggregated Alzheimer's disease brain insoluble extracts compared to control brain extracts, demonstrating that MTBR-tau is specifically deposited in Alzheimer's disease brains. The graphs show (Figure 18B) enrichment profiles of tau peptides from control and Alzheimer's disease brains (n ​​= 2, 6-8 brain region samples / group in the discovery cohort) and (Figure 18C) control (amyloid-negative, n = 8), very mild to moderate Alzheimer's disease (AD) (amyloid-positive, CDR = 0.5-2, n = 5), and severe AD brains (amyloid-positive, CDR = 3, n = 7) (total n = 20 in the validation cohort). The relative abundance of tau peptides was quantified against the mid-domain (residues 181-190) peptide for internal normalization. Species containing the upstream region of the microtubule-binding region (MTBR) domain (residues 243-254, MTBR-tau243) and repeat region 2 (R2) through R3 and R4 (residues 299-317, MTBR-tau299 and 354-369, MTBR-tau354, respectively) were highly enriched in the insoluble fraction of AD brains compared to controls, as assessed by CDRs, and were particularly enriched by the clinical stage of disease progression. MTBR-tau299 and MTBR-tau354 are located within the fibril core, while MTBR-tau243 is located outside the AD aggregate core (Fitzpatrick et al., 2017). Notably, residues 195-209 are decreased in AD brains, likely due to hyperphosphorylation. Data are presented as boxplots with Tukey's method, showing the median, interquartile interval, minimum, maximum, and individual outlier points. Statistical tests of significance: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05.

[0046] [Figure 19A] FIG. 19A is a schematic representation of tryptic digest peptides from tau (gray bars) quantified in Example 3 and further described in FIG. 19B and FIG. 19C, and the general binding sites of antibodies HJ8.5 and Tau1.

[0047] [Figure 19B] Figure 19B is a graph showing tau profiles in control human CSF. Tau peptides in control human CSF from a cross-sectional cohort of amyloid-negative and CDR=0 patients (n=30) were quantified by Tau1 / FIJ8.5 immunoprecipitation, focusing on the N-terminal to mid-domain tau. For quantification of species containing the microtubule-binding region (MTBR) and C-terminal regions, post-immunoprecipitation CSF samples were chemically extracted and analyzed sequentially. Using the Tau1 / FIJ8.5 immunoprecipitation method (blue circles), peptide recovery dramatically decreased after residue 222; therefore, only N-terminal to mid-domain tau peptides (residues 6–23 to 243–254) were quantified with this method (Sato et al., 2018). In contrast, the post-immunoprecipitation CSF chemical extraction method (red squares) allows quantification of the entire region encompassing the MTBR to C-terminal regions at concentrations of 0.4–7 ng / mL. Data are shown as averages.

[0048] [Figure 20]Figures 20A, 20B, and 20C are graphs showing the amounts of intermediate domain-independent MTBR tau 243 (Figure 20A), intermediate domain-independent MTBR tau 299 (Figure 20B), and intermediate domain-independent MTBR tau 354 (Figure 20C) in PostlP-CX CSF from a cross-sectional cohort. Intermediate domain-independent MTBR tau 243, intermediate domain-independent MTBR tau 299, and intermediate domain-independent MTBR tau 354 show distinct profiles with respect to amyloid plaques and clinical dementia stage. Amyloid-negative CDR = 0 (control, n = 30), amyloid-positive CDR = 0 (presymptomatic AD, n = 18), amyloid-positive CDR = 0.5 (very mild AD, n = 28), amyloid-positive CDR > 1 (mild-to-moderate AD, n = 12), and amyloid-negative CDR > 0.5 (non-AD cognitive impairment, n = 12). Intermediate domain-independent MTBR tau243 continues to increase with AD progression throughout all clinical stages. Intermediate domain-independent MTBR tau299 and MTBR tau354 concentrations continue to increase until the very mild AD stage (amyloid-positive and CDR = 0.5), after which they saturate (MTBR tau299) or decrease (MTBR tau354) at CDR > 1. P values ​​in red and blue indicate significant increases or decreases, respectively. Data are presented as individual results (plots) and means (bars). Statistical significance: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. NS=non-significant.

[0049] [Figure 21]Figures 21A, 21B, and 21C show graphs depicting the longitudinal rate of change (ng / mL / year) of (Figure 21A) mid-domain-independent MTBR tau 243, (Figure 21B) mid-domain-independent MTBR tau 299, and (Figure 21C) mid-domain-independent MTBR tau 354 in the CSF of amyloid-negative (-) or amyloid-positive (+) patients (total n=28 from the longitudinal cohort). The amyloid-positive group was further divided into various CDR alterations, including CDR=0-0 (n=7), CDR=0-0.5 (n=2), CDR=0-1 (n=1), CDR=0.5-0.5 (n=2), CDR=0.5-1 (n=1), and CDR=1-2 (n=1, participant A). While participants in the amyloid-negative group did not show significant longitudinal changes (mean values ​​close to 0), most participants in the amyloid-positive group showed longitudinal increases in MTBR tau concentrations. Notably, participant A, who showed the greatest cognitive change after clinical onset of Alzheimer's disease (CDR = 1-2), showed only an increase in CSF MTBR tau 243 as the disease progressed from mild (CDR = 1) to moderate (CDR = 2), while MTBR tau 299 and 354 decreased. These data indicate that CSF intermediate domain-independent MTBR tau species increase longitudinally with clinical progression of Alzheimer's disease.

[0050] [Figure 22] Figures 22A, 22B, and 22C are graphs showing (x-axis) Tau-PET (AV-1451) SUVR and (y-axis) intermediate domain-independent (Figure 22A) MTBR tau-243, (Figure 22B) MTBR tau-299, and (Figure 22C) MTBR tau-354 concentrations in PostIP-CX CSF (n=15 controls and n=20 from the Alzheimer's disease (AD) Tau-PET cohort). Open circles: controls, closed squares: AD. Intermediate domain-independent MTBR tau-243 showed the most significant correlation with Tau-PET SUVR (r=0.7588, p<0.0001). These data show that the CSF mid-domain-independent MTBR tau species containing sequence number 3 (LQTAPVPMPDLK) is highly correlated with Tau-PET SUVR measurements in tau-enriched individuals, while other mid-domain-independent MTBR tau regions are less correlated with tau-enriched individuals.

[0051] [Figure 23] Figures 23A and 23B are graphs showing that brain MTBR tau243, MTBR tau299, and MTBR tau354 are not enriched in Alzheimer's disease brain soluble extracts compared to control brain extracts. Figure 23A shows the enrichment profiles of tau peptides from control and Alzheimer's disease brains (n ​​= 2 with 8-10 brain region samples / group in the discovery cohort), and Figure 23B shows the enrichment profiles of tau peptides from control (amyloid-negative, n = 8), very mild to moderate Alzheimer's disease (AD) (amyloid-positive, CDR = 0.5-2, n = 5), and severe AD brains (amyloid-positive, CDR = 3, n = 7) in the validation cohort (total n = 20). Relative peptide abundance of tau peptides was quantified against the mid-domain (residues 181-190) peptide for internal normalization. No changes were observed in the microtubule-binding region (MTBR) domain-containing tau species in soluble tau species, in contrast to the increased insoluble MTBR tau species in Alzheimer's disease brains (Figure 18). Data are presented as Tukey boxplots showing the median, interquartile interval, minimum, maximum, and individual outlier points. Statistical significance: **p<0.01, *p<0.05.

[0052] [Figure 24] Figure 24 is a graph showing that MTBR tau354 correlates with tau368 in brain insoluble extracts, suggesting that the species do not differentiate with the progression of tau pathology. Mass spectrometry analysis of MTBR tau354 and tau368 species in brain insoluble extracts from control and Alzheimer's disease patients was performed using discovery cohort samples (23 brain samples total: 6 brain regions from Alzheimer's disease #1 participants, 8 brain regions from Alzheimer's disease #2 participants, 5 brain regions from control #1 participants, and 4 brain regions from control #2 participants). MTBR tau354 (residues 354-369) and its truncated form, tau368 (residues 354-368), showed a close correlation in brain insoluble extracts (Spearman r = 0.9783).

[0053] [Figure 25] Figures 25A, 25B, 25C, 25D, 25E, and 25F are graphs showing the quantification of tryptic peptides of MTBR tau in human CSF samples after processing with the PostIP-CX method followed by mass spectrometry (MS) analysis. Extracted MS chromatograms of mid-domain-independent MTBR tau 243 (Figures 25A, 25D), mid-domain-independent MTBR tau 299 (Figures 25B, 25E), and mid-domain-independent MTBR tau 354 (Figures 25C, 25F) are shown. Human CSF was obtained from amyloid-positive and CDR=0.5 very mild Alzheimer's disease participants from a cross-sectional cohort. Figures 25A, 25B, and 25C show peaks from endogenous tryptic peptides, while Figures 25D, 25E, and 25F show peaks from the internal standard (15N-labeled tau). The X- and Y-axes show the retention time and MS intensity of each peak, respectively.

[0054] [Figure 26]Figures 26A, 26B, 26C, 26D, 26E, 26F, 26G, 26H, 26I, 26J, and 26K are graphs showing concentrations of tryptic peptides of N-terminal tau and mid-domain tau in human CSF after sample processing by the IP method followed by MS analysis. N-terminal and mid-domain CSF tau species distinguish very early dementia from normal but do not correlate with dementia stage. Tau species in CSF from groups within the cross-sectional cohort, residues (Figure 26A) 6-23, (Figure 26B) 25-44, (Figure 26C) 45-67, (Figure 26D) 68-87, (Figure 26E) 88-126, (Figure 26F) 151-155, (Figure 26G) 181-190, (Figure 26H) 195-209, (Figure 26I) 212-221, (Figure 26J) 226-230, and (Figure 26K) 243-254 concentrations (residue numbering is based on tau 441). Amyloid-negative CDR = 0 (controls, n = 29), amyloid-positive CDR = 0 (presymptomatic AD, n = 18), amyloid-positive CDR = 0.5 (very mild AD, n = 27), amyloid-positive CDR > 1 (mild-to-moderate AD, n = 12), and amyloid-negative CDR > 0.5 (non-AD clinical impairment, n = 12). These tau species were isolated using the Tau1 / FIJ8.5 immunoprecipitation method (IP method). p-values ​​in red indicate significance. Data are shown as individual results (plots) and means (bars). Statistical significance: ****p < 0.001, ***p < 0.001, **p < 0.01, *p < 0.05. NS = non-significant.

[0055] [Figure 27]Figures 27A, 27B, 27C, 27D, 27E, 27F, 27G, and 27H are graphs showing the concentrations of tryptic peptides of MTBR tau in human CSF after sample processing by the PostIP-CX method followed by MS analysis. The distinctive Alzheimer's disease amyloid and clinical stage patterns are specific to the mid-domain-independent MTBR tau species in CSF. Only mid-domain-independent MTBR tau243 distinguishes more advanced clinical stages. Tau species in CSF obtained using chemical extraction from post-immunoprecipitation samples from participants within the cross-sectional cohort: residues (Figure 27A) 243-254 (MTBR tau 243), (Figure 27B) 260-267, (Figure 27C) 275-280, (Figure 27D) 282-290, (Figure 27E) 299-317 (MTBR tau 299), (Figure 27F) 354-369 (MTBR tau 354), (Figure 27G) 386-395, and (Figure 27H) 396-406 concentrations (residue numbering is based on tau 441). Amyloid-negative CDR = 0 (controls, n = 30), amyloid-positive CDR = 0 (pre-symptomatic AD, n = 18), amyloid-positive CDR = 0.5 (very mild AD, n = 28), amyloid-positive CDR > 1 (mild-to-moderate AD, n = 12), and amyloid-negative CDR > 0.5 (non-AD clinical impairment, n = 12). p-values ​​in red or blue indicate significant increases or decreases, respectively. Data are shown as individual results (plots) and means (bars). Statistical significance: ****p < 0.001, ***p < 0.001, **p < 0.01, *p < 0.05. NS = non-significant.

[0056] [Figure 28A-J]Figures 28A, 28B, 28C, 28D, 28E, 28F, 28G, 28H, 28I, and 28J are graphs showing concentrations of tryptic peptides of N-terminal tau and mid-domain tau in human CSF after sample processing by the PostIP-CX method followed by MS analysis. N-terminal and mid-domain CSF tau species do not correlate with dementia stage, regardless of purification method (see also Figure 26). Tau species in CSF from groups within the cross-sectional cohort, residues (Figure 28A) 6-23, (Figure 28B) 25-44, (Figure 28C) 45-67, (Figure 28D) 68-87, (Figure 28E) 88-126, (Figure 28F) 151-155, (Figure 28G) 181-190, (Figure 28H) 195-209, (Figure 28I) 212-221, and (Figure 28J) 226-230 concentrations. Amyloid-negative CDR = 0 (controls, n = 29), amyloid-positive CDR = 0 (pre-symptomatic AD, n = 18), amyloid-positive CDR = 0.5 (very mild AD, n = 27), amyloid-positive CDR > 1 (mild-to-moderate AD, n = 12), and amyloid-negative CDR > 0.5 (non-AD clinical impairment, n = 12). These tau species were isolated using the Tau1 / FIJ8.5 immunoprecipitation method followed by post-immunoprecipitation CSF chemical extraction (PostIP-CX). The sum of concentrations from the immunoprecipitation and post-immunoprecipitation CSF chemical extraction methods is shown for N-terminal to mid-domain tau species (as total concentrations). p values ​​in red indicate statistical significance. Data are shown as individual results (plots) and means (bars). Statistical significance: ****p < 0.001, ***p < 0.001, **p < 0.01, *p < 0.05. NS=not significant.

[0057] [Figure 28K] Figure 28K is a graph showing the total concentration of tau species containing residues 243-254 (summed concentrations from IP and PostIP-CX methods).

[0058] [Figure 29]Figure 29 is a graph showing that mid-domain-independent MTBR tau354 correlates with mid-domain-independent tau368 in CSF. CSF was processed by the PostIP-CX method as generally described in Example 3. Mid-domain-independent MTBR tau354 (residues 354-369) and its truncated form, tau368 (residues 354-368), show a close correlation (Spearman r = 0.8382). Results were obtained from a cross-sectional cohort sample (n = 100, including all clinical stages).

[0059] [Figure 30] Figures 30A, 30B, and 30C are graphs showing that CSF mid-domain-independent MTBR tau243 is highly correlated with clinical dementia grade-box sum (CDR-SB), whereas mid-domain-independent MTBR tau299 and mid-domain-independent MTBR tau354 are not. CDR-SB correlations with (Figure 30A) mid-domain-independent MTBR tau243, (Figure 30B) mid-domain-independent MTBR tau299, and (Figure 30C) mid-domain-independent MTBR tau354 concentrations in CSF. Mid-domain-independent MTBR tau243 in CSF from the amyloid-positive group showed a high correlation with CDR-SB (r=0.5562, p<0.0001). Results were obtained from a cross-sectional cohort sample (amyloid-negative n=42 and amyloid-positive n=58).

[0060] [Figure 31]Figures 31A, 31B, and 31C are graphs showing that CSF intermediate domain-independent MTBR tau243 is more highly correlated with Mini-Mental State Examination (MMSE) than intermediate domain-independent MTBR tau299 and intermediate domain-independent MTBR tau354. MMSE correlation with CSF concentrations of (Figure 31A) intermediate domain-independent MTBR tau243, (Figure 31B) intermediate domain-independent MTBR tau299, and (Figure 31C) intermediate domain-independent MTBR tau354. CSF intermediate domain-independent MTBR tau243 from the amyloid-positive group showed a high correlation with MMSE (r=0.5433, p<0.0001). Results were obtained from a cross-sectional cohort sample (amyloid-negative n=42 and amyloid-positive n=58).

[0061] [Figure 32] Figures 32A, 32B, and 32C are graphs showing the longitudinal increase of CSF mid-domain-independent MTBR tau243, mid-domain-independent MTBR tau299, and mid-domain-independent MTBR tau354 with progression of Alzheimer's disease clinical stages. Longitudinal changes in CSF mid-domain-independent (Figure 32A) MTBR tau243, (Figure 32B) MTBR tau299, and (Figure 32C) MTBR tau354 tau concentrations are shown in amyloid-negative (-) or amyloid-positive (+) patients. Black circles: CDR=0, blue triangles: CDR=0.5, red squares: CDR=1, purple inverted triangles: CDR=2. Participants whose CDR trajectories were stable (or decreasing) are indicated by dotted lines. Participants whose CDR increased between the first and second visits are indicated by solid lines. The bold red line in the amyloid-positive group indicates the longitudinal trajectory of a specific participant (Participant A) who experienced the greatest cognitive change since the onset of Alzheimer's disease (CDR = 1-2), suggesting that CSF MTBR tau243 also increases in mild (CDR = 1) to moderate (CDR = 2) AD. Statistical significance was assessed by paired t-test for visits 1 and 2 (amyloid-negative n = 14 and amyloid-positive n = 14). **p<0.01. NS = non-significant.

[0062] [Figure 33] FIG. 33 is a schematic diagram illustrating the method of the present invention.

[0063] [Figure 34] Figures 34A, 34B, and 34C are graphs showing the amounts of tryptic peptides LQTA (Figure 34A), HVPG (Figure 34B), and IGSL (Figure 34C) measured in CSF samples treated with the PostIP-CX method (top) versus the PostIP-CX method (bottom).

[0064] [Figure 35A] Figure 35A is a graph showing the amounts of tryptic peptides LQTA (left), IGST (center), and VQII (right) measured in samples processed by the PostIP-IP method (y-axis) versus the PostIP-CX method (x-axis). The upper part of the graph shows the relative position of each tryptic peptide in tau441. Both axes show absolute concentrations (ng / mL).

[0065] [Figure 35] Figure 35B is a graph showing the amounts of tryptic peptides LDLS (left), HVPG (center), and IGSL (right) measured in samples processed by the PostIP-IP method (y-axis) versus the PostIP-CX method (x-axis). The upper part of the graph shows the relative position of each tryptic peptide in tau441. Both axes show absolute concentrations (ng / mL).

[0066] [Figure 36A]Figure 36A is a graph showing the amounts of tryptic peptides LQTA (left), IGST (center), and VQII (right) measured in samples processed by the PostIP-IP method (y-axis) versus the PostIP-CX method (x-axis). The illustration above the graph shows the relative position of each tryptic peptide in tau441. Both axes show absolute concentrations (ng / mL). Samples from control subjects are blue circles; samples from amyloid-positive subjects without cognitive impairment (CDR<0.5) are red squares; and samples from amyloid-positive subjects with cognitive impairment (CDR>0.5) are black triangles.

[0067] [Figure 36B] Figure 36B is a graph showing the amounts of tryptic peptides LDLS (left), HVPG (center), and IGSL (right) measured in samples processed by the PostIP-IP method (y-axis) versus the PostIP-CX method (x-axis). The illustrations at the top of the graph show the relative position of each tryptic peptide in tau441. Both axes show absolute concentrations (ng / mL). Samples from control subjects are blue circles; samples from amyloid-positive subjects without cognitive impairment (CDR<0.5) are red squares; and samples from amyloid-positive subjects with cognitive impairment (CDR>0.5) are black triangles.

[0068] [Figure 37A] Figure 37A is a diagram of various full-length tau isoforms. The relative positions of tryptic peptides of several tau species are shown (e.g., LQTA, IGST, VQII, LDLS, HVPG, IGSL, VQIV). Each "Y" represents an antibody that specifically binds to the N-terminal (left), mid-domain (center), and MTBR (right) regions. The major cleavage site of tau, amino acid 224 of tau441, is shown as a dotted line.

[0069] [Figure 37B]Figure 37B is a graph showing the amounts of tryptic peptides VQIV and LDLS, expressed as ratios, in samples from control subjects (left, blue circles) and subjects with non-AD tauopathy (right, green circles), as determined by LC-MS after sample processing by the IP method described in Example 1. ns, not significant; Tukey's multiple comparison test.

[0070] [Figure 37C] Figure 37C is a graph showing the amounts of tryptic peptides VQIV and LDLS, expressed as a ratio, in samples from a control subject (left, blue circles) and a subject with non-AD tauopathy (right, green circles), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4.

[0071] [Figure 38] Figure 38 is a graph showing the abundance of tryptic peptides VQIV (x-axis) and LDLS (y-axis) in samples from control subjects (blue circles) and subjects with non-AD tauopathy (green triangles), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. ****p<0.0001; Tukey's multiple comparison test.

[0072] [Figure 39] Figure 39 is a graph showing the amounts of tryptic peptides VQIV and LDLS, expressed as ratios, in samples from a control subject (left, blue circles), a subject with AD (center, red circles), and a subject with a non-AD tauopathy (right, green circles), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. ns not significant; ****p<0.0001; Tukey's multiple comparison test.

[0073] [Figure 40]Figures 40A, 40B, 40C, 40D, 40E, and 40F are graphs showing a comparison of tau tryptic peptides in samples from a control subject (blue circles), a subject with AD (red squares), and a subject with a non-AD tauopathy (green triangles), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. The tau tryptic peptides are IGST (x-axis) and VQII (y-axis) in Figure 40A, LDLS (x-axis) and VQII (y-axis) in Figure 40B, IGSL (x-axis) and HVPG (y-axis) in Figure 40C, IGST (x-axis) and HPVG (y-axis) in Figure 40E, VQII (x-axis) and HPVG (y-axis) in Figure 40F, and IGST (x-axis) and HPVG (y-axis) in Figure 40F. Both axes show absolute concentrations (ng / mL).

[0074] [Figure 41] Figure 41 is a graph showing a comparison of the amounts of tau tryptic peptides IGST vs. HVPG (top left), VQII vs. HVPG (top right), and LDLS vs. HVPG (bottom) in samples from subjects with non-AD tauopathy, as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. The key legend on the right indicates each subject's non-AD tauopathy diagnosis. Both axes show absolute concentrations (ng / mL).

[0075] [Figure 42] Figures 42A, 42B, and 42C are graphs showing a comparison of the amounts of tau tryptic peptides IGST vs. IGSL (Figure 42A), VQII vs. IGSL (Figure 42B), and LDLS vs. IGSL (Figure 42C) in samples from control subjects (blue circles), subjects with AD (red squares), and subjects with non-AD tauopathy (green triangles), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. Both axes show absolute concentrations (ng / mL).

[0076] [Figure 43A]Figure 43A is a diagram of the MTBR region of tau, showing the relative positions of tryptic peptides IGST, VQII, LDLS, HVPG, and IGSL, as well as the relative position of the epitope specifically bound by antibody 77G7.

[0077] [Fig. 43B-E] Figures 43B, 43C, 43D, and 43E are graphs showing the ratios of tau tryptic peptides IGSL / IGST (Figure 43B), IGSL / VQII (Figure 43C), IGSL / LDLS (Figure 43D), and IGSL / HVPG (Figure 43E) in samples from non-AD subjects (left bar) and subjects with AD (right bar), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. For non-AD subjects, blue indicates subjects with PSP, green indicates subjects with FTD, red indicates subjects with CBD, and purple indicates subjects with sequential PSP-CBD. Statistical significance was determined by unpaired t-test with Welch's correction.

[0078] [Figure 44] Figures 44A, 44B, 44C, 44D, and 44E are graphs showing a comparison of the abundance of tau tryptic peptides IGSL vs. IGST (Figure 44A), IGSL vs. VQII (Figure 44B), IGSL vs. LDLS (Figure 44C), VQII vs. IGST (Figure 44D), VQII vs. LDLS (Figure 44E), and IGSL vs. HVPG (Figure 43E) in samples from non-AD subjects (blue) and subjects with AD (red), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. In Figures 44A, 44B, and 44C, non-AD subjects show scatter plots, while in Figures 44D, 44E, and 44F, AD and non-AD subjects show identical correlations.

[0079] [Figure 45]Figure 45 shows the correlation of various tau tryptic peptides measured in CSF from subjects with AD and non-AD tauopathies. Data highlighted in boxes suggest branching points that distinguish AD from non-AD tauopathies.

[0080] [Figure 46] Figure 46 shows a hypothesis of how CSF tau distinguishes non-AD tauopathies. As shown, non-AD tauopathies contain (1) less R1-R2 and (2) more R3-R4 in CSF than AD, as indicative of brain tau deposition.

[0081] [Figure 47] FIG. 47 is a graph showing the amounts of various tryptic peptides from brain-insoluble tau.

[0082] [Figure 48] Figures 48A, 48B, 48C, and 48D are graphs showing the ratios of tau tryptic peptides IGSL / IGST (Figure 48A), IGSL / VQII (Figure 48B), IGSL / LDLS (Figure 48C), and IGSL / HVPG (Figure 48D) in samples from control subjects (left bar), subjects with AD (center bar), and non-AD subjects (right bar) after sample processing by the PostIP-IP method described in Example 4. For non-AD subjects, black triangles indicate subjects with genetically confirmed P301L FTLD (4R-tauopathy), and black squares indicate subjects with genetically confirmed R406W FTLD (3R / 4R mix).

[0083] [Figure 49]Figures 49A, 49B, 49C, 49D, 49E, and 49F are graphs showing a comparison of the amounts of tau tryptic peptides IGSL vs. IGST (Figure 49A), IGSL vs. VQII (Figure 49B), IGSL vs. LDLS (Figure 49C), VQII vs. IGST (Figure 49D), VQII vs. LDLS (Figure 49E), and IGSL vs. HVPG (Figure 49F) in CSF samples from control subjects (blue), subjects with AD (red), and non-AD subjects (green), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. In Figures 49A, 49B, and 49C, non-AD subjects show scatter plots, while in Figures 49D, 49E, and 49F, controls, AD, and non-AD showed identical correlations. DETAILED DESCRIPTION OF THE INVENTION

[0084] Detailed Description MTBR tau exists as multiple peptides in blood and CSF. Detection and quantification of MTBR tau in these biological samples is hindered by the very low abundance of these polypeptides. The methods disclosed herein use a unique combination of processing steps to convert biological samples into samples suitable for quantification of MTBR tau and other tau species. For example, in some methods of the invention, processing steps deplete a protein while simultaneously enriching multiple tau proteins. In other methods of the invention, processing steps deplete a protein while simultaneously enriching multiple MTBR tau proteins. Some methods disclosed herein are particularly suitable for quantification of intermediate domain-independent MTBR tau species. The use of intermediate domain-independent MTBR tau species for evaluating clinical signs and symptoms of tauopathy, diagnosing tauopathy, and prescribing tauopathy treatment is also described. These and other aspects and iterations of the invention are described more thoroughly below.

[0085] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of this invention belong. Although many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of embodiments of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming embodiments of the present invention, the following terms are defined and used as follows:

[0086] As used herein, the term "about" refers to any quantitative variation, including, but not limited to, mass, volume, time, distance, and amount, and includes variations in quantity that may occur, for example, with common measuring techniques and equipment. Furthermore, given the nature of certain solid and liquid manipulation methods used, there is a certain degree of inadvertent error and variation due to differences in the manufacture, source, or purity of the components used to prepare the composition or carry out the method. The term "about" also includes these variations, which may be up to ±5%, and may also be ±4%, 3%, 2%, 1%, etc. Whether or not modified by the term "about," the claims include the equivalent value of the quantity.

[0087] As used herein, the term "antibody" refers to a complete antibody, i.e., an antibody consisting of two heavy chains and two light chains, as understood in the art, and also refers to any antibody-like molecule having an antigen-binding region, including, but not limited to, antibody fragments such as Fab', Fab, F(ab')2, single-domain antibodies, Fv, and single-chain Fv. The term "antibody" also refers to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and humanized antibodies. Techniques for producing and using various antibody-based constructs and fragments are well known in the art. Means for producing and characterizing antibodies are also well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference in its entirety).

[0088] 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. Typically, aptamers have molecular activity such as binding to a target molecule at a specific epitope (region). It is generally recognized that aptamers that are specific for binding to a polypeptide can be synthesized and / or identified by in vitro evolution methods. Means for producing and characterizing aptamers, including those by in vitro evolution methods, are well known in the art. See, for example, US Pat. No. 7,939,313, which is incorporated herein by reference in its entirety.

[0089] The term "Aβ" refers to a peptide derived from a region at the carboxy-terminus of a large protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are multiple Aβ forms that can have toxic effects: Aβ peptides are generally 37-43 amino acids in length but can have truncations and modifications that alter their overall size. They can be found in soluble and insoluble compartments, intracellularly or extracellularly, in monomeric, oligomeric, and aggregated forms, and can be complexed with other proteins or molecules. The harmful 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 fibrinogenic or insoluble and associated with disease states. The term "Aβ" generally refers to multiple Aβ species without distinguishing between them. Specific Aβ species are identified by peptide size, e.g., Aβ42, Aβ40, Aβ38, etc.

[0090] As used herein, the term "Aβ42 / Aβ40 value" refers to the ratio of the amount of Aβ42 in a sample obtained from a subject to the amount of Aβ40 in the same sample.

[0091] "Aβ amyloidosis" is defined as clinically abnormal Aβ deposition in the brain. Subjects determined to have Aβ amyloidosis are referred to herein as "amyloid positive," while subjects determined not to have Aβ amyloidosis are referred to herein as "amyloid negative." There are indicators of Aβ amyloidosis recognized in the art. At the time of the present invention, Aβ amyloidosis is measured directly by amyloid imaging (e.g., PiB PET, florbetapir, or other imaging methods known in the art) or indirectly by a decrease in cerebrospinal fluid (CSF) Aβ42 or a decrease in the CSF Aβ42 / 40 ratio. A mean cortical binding potential (MCBP) score >0.18 [ 11[C]PIB-PET imaging is indicative of Aβ amyloidosis, similar to cerebrospinal fluid (CSF) Aβ42 concentrations of approximately 1 ng / ml measured by immunoprecipitation and mass spectrometry (IP / MS). Alternatively, a cutoff ratio of CSF Aβ42 / 40 determined by PIB-PET that maximizes the accuracy of amyloid positivity prediction can be used. These or other values ​​known in the art and / or used in the Examples may be used alone or in combination for clinical confirmation of 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 have symptoms, 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 may include cognitive impairment, behavioral changes, abnormal language function, emotional dysregulation, seizures, dementia, and nervous system structural or functional impairment. 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 are at increased risk of developing diseases associated with Aβ amyloidosis.

[0092] "Clinical signs of Aβ amyloidosis" refers to the measurement of Aβ deposition, as known in the art. Clinical signs of Aβ amyloidosis can include, but are not limited to, Aβ deposits identified by amyloid imaging (e.g., PiB PET, florbetapir, or other imaging methods known in the art) or cerebrospinal fluid (CSF) Aβ42 or a decrease in the 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, which are incorporated herein by reference in their entirety. Clinical signs of Aβ amyloidosis may also include measurements of Aβ metabolism, particularly Aβ42 metabolism alone or compared to the metabolism of other Aβ variants (e.g., Aβ37, Aβ38, Aβ39, Aβ40 and / or total Aβ), as described in U.S. Patent Application Nos. 14 / 366,831, 14 / 523,148 and 14 / 747,453, each of which is incorporated herein by reference in its entirety. 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. Nevertheless, subjects with clinical signs of Aβ amyloidosis are at increased risk of developing diseases associated with Aβ amyloidosis.

[0093] "Candidate for amyloid imaging" refers to a subject who has been identified by a physician as an individual for whom amyloid imaging may be clinically justified. 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 Aβ plaque-related symptoms, one or more CAA-related symptoms, or a combination thereof. A physician may recommend amyloid imaging for such a subject and direct clinical care. As another non-limiting example, a candidate for amyloid imaging may be a potential clinical trial participant (control subject or test subject) for a disease associated with Aβ amyloidosis.

[0094] "Aβ plaque-related symptoms" or "CAA-related symptoms" refer to any symptoms caused by or associated with the formation of amyloid plaques or CAA, respectively, which are composed of regularly arranged fibrillar aggregates called amyloid fibrils. Examples of Aβ plaque-related symptoms may include, but are not limited to, neuronal degeneration, cognitive dysfunction, memory impairment, behavioral changes, emotional dysregulation, seizures, nervous system structure or function impairment, and an increased risk of developing or worsening Alzheimer's disease or CAA. Neuronal degeneration may include neuronal structural changes (including molecular changes such as intracellular accumulation of toxic proteins, protein aggregates, etc., and macro-level changes such as changes in the shape or length of axons or dendrites, changes in myelin sheath composition, myelin sheath loss, etc.), neuronal functional changes, neuronal function loss, neuronal death, or any combination thereof. Cognitive dysfunction may include, but is not limited to, difficulties with memory, attention, concentration, language, abstract thinking, creativity, executive function, planning, and organization. Behavioral changes may include, but are not limited to, aggressive behavior and language, impulsivity, decreased inhibitions, apathy, decreased initiation, 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 emotional incontinence. Seizures may include, but are not limited to, generalized tonic-clonic seizures, complex partial seizures, and non-epileptic, psychogenic seizures. Nervous system structural or functional disorders may include, but are not limited to, hydrocephalus, Parkinson's disease, sleep disorders, psychosis, balance and coordination disorders. This may include motor dysfunction such as monoparesis, hemiparesis, quadriplegia, ataxia, ballismus, and tremor. This may also include sensory loss or dysfunction, including olfactory, tactile, gustatory, visual, and auditory sensations. Additionally, this may include autonomic nervous system dysfunction such as bowel and bladder dysfunction, sexual dysfunction, and blood pressure and temperature regulation disorders. Finally, this can include hormonal dysfunction resulting from hypothalamic and pituitary dysfunction, such as deficiencies and dysregulation of growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, gonadotropin-releasing hormone, prolactin and numerous other hormones and modulators.

[0095] 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 awaiting medical care or treatment, undergoing medical care or treatment, or receiving medical care or treatment.

[0096] As used herein, the terms "control population," "normal population," or sample from a "healthy" subject refer to a subject or group of subjects who have been clinically determined to not have a tauopathy or Aβ amyloidosis or a disease associated with clinical Aβ amyloidosis (including, but not limited to, Alzheimer's disease) based on qualitative or quantitative test results.

[0097] As used herein, the term "blood sample" refers to a biological sample derived from blood, preferably peripheral (or circulating) blood. The blood sample may be whole blood, plasma, or serum, although plasma is generally preferred.

[0098] As used herein, the term "isoform" refers to any of several different forms of the same protein variant that result from 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.

[0099] The term "tau" refers to multiple isoforms encoded by the gene MAPT (or its homologs) and species thereof that have undergone in vivo C-terminal truncation, in vivo N-terminal truncation, in vivo post-translational modification, 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. For animals in which the gene has not been identified as MAPT, homologs can be identified by methods well known in the art.

[0100] In humans, there are six isoforms of tau, produced by alternative splicing of exons 2, 3, and 10 of MAPT. These isoforms range in length from 352 to 441 amino acids. Exons 2 and 3 each encode a 29-amino acid insert at the N-terminus (designated N), and full-length human tau isoforms may have both inserts (2N), one insert (1N), or no insert (0N). All full-length human tau isoforms also have three repeats of the microtubule-binding domain (designated R). Insertion of exon 10 at the C-terminus results in the insertion of a fourth microtubule-binding domain encoded by exon 10. Thus, full-length human tau isoforms may have four repeats of the microtubule-binding domain (exon 10 contains R1, R2, R3, and R4) or three repeats of the microtubule-binding domain (exon 10 contains 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 glycosylated. Human tau may or may not be proteolytically processed in vivo at the C-terminus, the N-terminus, or both the C-terminus and the N-terminus. Thus, the term "human tau" includes the 2N3R, 2N4R, 1N3R, 1N4R, 0N3R, and 0N4R isoforms, as well as species thereof that have undergone in vivo C-terminal truncation, in vivo N-terminal truncation, in vivo post-translational modification, or any combination thereof. Alternative splicing of the gene encoding tau similarly occurs in other animals.

[0101] As used herein, the term "tau-441" refers to the longest human tau isoform (2N4R), which is 441 amino acids in length. The amino acid sequence of tau441 is provided as SEQ ID NO: 1. The N-terminus (N term), mid-domain, MTBR, and C-terminus (C term) are shown for this isoform in FIG. 1. These regions vary in a predictable manner for other tau isoforms (e.g., 2N3R, 1NR4, 1N3R, 0N4R, and 0N3R). Thus, when an amino acid position is specified for tau441, one of skill in the art can determine the corresponding amino acid position for other isoforms.

[0102] As used herein, the term "N-terminal tau" refers to a tau protein or proteins that include two or more amino acids at the N-terminus of tau (such as, for example, amino acids 1-103 of tau441).

[0103] As used herein, the term "mid-domain tau" refers to a tau protein or proteins that include two or more amino acids of the mid-domain of tau (e.g., amino acids 104-243 of tau441).

[0104] As used herein, the term "MTBR tau" refers to a tau protein or proteins that contain two or more amino acids of the microtubule-binding region (MTBR) of tau (e.g., amino acids 244-368 of tau441).

[0105] As used herein, the term "C-terminal tau" refers to a tau protein or proteins that include two or more amino acids at the C-terminus of tau (e.g., amino acids 369-441 of tau441).

[0106] "Tau proteolytic peptide" refers to a peptide fragment of tau protein produced by in vitro proteolytic cleavage. "Tau tryptic peptide" refers to a peptide fragment of tau protein produced by in vitro cleavage with trypsin. Tau tryptic peptides may be referred to herein by their first four amino acids. For example, "LQTA" refers to the tryptic peptide LQTAPVPMPDLK (SEQ ID NO: 3). Non-limiting examples of other tryptic peptides identified by their first four amino acids include IGST (SEQ ID NO: 2), VQII (SEQ ID NO: 4), LDLS (SEQ ID NO: 5), HVPG (SEQ ID NO: 6), IGSL (SEQ ID NO: 7), VQIV (SEQ ID NO: 9), and TPPS (SEQ ID NO: 10).

[0107] Diseases associated with tau deposition in the brain are referred to herein as "tauopathies." The term "tau deposition" includes all forms of pathological tau deposits, including, but not limited to, neurofibrillary tangles, neuropil threads, and tau aggregates in degenerating neurites. Tauopathies known in the art include, but are not limited to, progressive supranuclear palsy (PSP), dementia pugilistica, chronic traumatic encephalopathy, chromosome 17-linked frontotemporal dementia and Parkinson's disease, Ritiko-Bodig disease, Parkinsonism-dementia complex of Guam, neurofibrillary tangle dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis complex, Hallervorden-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).

[0108] Tauopathies are classified according to the dominance of tau isoforms found in pathological tau deposits. Tauopathies in which tau deposits are primarily composed of tau with three MTBRs are referred to as "3R-tauopathies." Pick's disease is a non-limiting example of a 3R-tauopathies. For clarity, pathological tau deposits in some 3R-tauopathies may be predominantly 3R isoforms, with a mixture of 3R and 4R tau isoforms. Intracellular neurofibrillary-rich bodies (i.e., tau deposits) in the brains of subjects with Alzheimer's disease are generally thought to contain approximately equal amounts of both 3R and 4R isoforms. Tauopathies in which tau deposits are primarily 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 brains of some subjects with genetically confirmed FTLD cases, such as some V334M and R406W mutation carriers, display a mixture of 3R and 4R isoforms.

[0109] The clinical manifestation of tauopathy can be tau aggregates in brain, including but not limited to neurofibrillary tangles.Methods for detecting and quantifying tau aggregates in brain are known in the art (for example, Tau-PET using tau-specific ligands such as [18F]THK5317, [18F]THK5351, [18F]AV1451, [11C]PBB3, [18F]MK-6240, [18F]RO-948, [18F]PI-2620, [18F]GTP1, [18F]PM-PBB3 and [18F]JNJ64349311, [18F]JNJ-067).

[0110] As used herein, the term "treatment" or "treating" refers to the provision of medical care by a trained and licensed professional to a subject in need thereof. Medical care can be a diagnostic test, therapeutic treatment, and / or a prophylactic or preventative measure. The purpose of therapeutic and prophylactic treatment is to prevent or slow (reduce) an undesirable physiological change or disease / disorder. Beneficial or desired clinical results of therapeutic or prophylactic treatment include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), slowing or slowing of disease progression, improvement or palliation and remission (whether partial or total) of the disease state, whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to life expectancy without treatment. Those in need of treatment include those already with the disease, condition, or disorder, as well as those predisposed 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 any symptoms or clinical signs of a disease.

[0111] The term "tau therapy" collectively refers to any imaging agent, therapeutic treatment, and / or prophylactic or preventative measures intended for or used on subjects at risk of developing a tauopathy or clinically diagnosed with 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, nutritional supplements, and the like. Dietary supplements (e.g., lithium water, omega-3 fatty acids with lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin, and grapeseed extract), serotonin receptor 6 antagonists, p38 alpha MAPK inhibitors, recombinant granulocyte-macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513), tau protein aggregation inhibitors (e.g., TRx0237, methylthioninium chloride), treatments to improve glycemic control (e.g., insulin, exena) tide, 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, b-2 adrenoceptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenoceptor antagonists, 5-HT1A and 1D receptor agonists, These include, but are not limited to, glutaminyl-peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulators, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof.

[0112] II. Tau Measurement Methods The present invention provides a method for measuring tau in a biological sample by mass spectrometry. Generally speaking, the method for measuring tau in a biological sample of the present invention includes preparing a biological sample, treating the biological sample by depleting one or more proteins, then purifying tau, cleaving the purified tau with a protease, and optionally desalting the resulting cleavage products by solid-phase extraction to obtain a sample containing tau proteolytic peptides, and performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect at least one of the tau proteolytic peptides and measure its concentration (relative or absolute). Thus, in practice, the disclosed method uses at least one of the tau proteolytic peptides to detect and measure the amount of tau present in a biological sample.

[0113] In one example, the method of the present invention includes (a) preparing a biological sample selected from a blood sample or a CSF sample; (b) removing proteins from the biological sample by protein precipitation and separating the precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease and then optionally desalting the resulting cleavage products by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect and measure the concentration of at least one tau proteolytic peptide.

[0114] In other examples, the methods of the invention include (a) depleting N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau in a biological sample, the biological sample being a blood sample or a CSF sample, by affinity depletion; (b) (i) removing additional proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, followed by purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purifying MTBR tau, thereby producing (i) or (ii) enriched tau. (c) enriching the tau remaining after affinity depletion, which may be referred to as N-terminus-independent tau and / or mid-domain-independent tau; (c) cleaving the enriched tau with a protease and then optionally desalting the resulting cleavage products by solid phase extraction to obtain a sample containing tau proteolytic peptides; and (d) performing liquid chromatography-mass spectrometry (LC / MS) on the sample containing tau proteolytic peptides to detect and measure the concentration of at least one of the tau proteolytic peptides.

[0115] In another example, the method of the present disclosure includes (a) depleting N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau in a biological sample, which is a blood sample or a CSF sample, by affinity depletion; (b) removing additional proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease, and then optionally desalting the resulting cleavage products by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect and measure the concentration of at least one of the tau proteolytic peptides.

[0116] In another example, the method of the present invention includes (a) depleting N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau in a biological sample, which is a blood sample or a CSF sample, by affinity depletion; (b) affinity-purifying MTBR tau; (c) cleaving the purified MTBR tau with a protease, and then optionally desalting the resulting cleavage products by solid-phase extraction to obtain a sample containing proteolytic peptides of MTBR tau; and (d) performing liquid chromatography-mass spectrometry on the sample containing proteolytic peptides of MTBR tau to detect and measure the concentration of at least one of the proteolytic peptides of MTBR tau.

[0117] In another example, the method of the present invention includes (a) affinity purifying MTBR tau from a biological sample that is a blood sample or a CSF sample; (b) cleaving the purified MTBR tau with a protease, and then optionally desalting the resulting cleavage products by solid-phase extraction to obtain a sample containing proteolytic peptides of MTBR tau; and (c) performing liquid chromatography-mass spectrometry on the sample containing proteolytic peptides of MTBR tau to detect and measure the concentration of at least one of the proteolytic peptides of MTBR tau.

[0118] The present invention further contemplates determining the presence / absence of one or more proteins in a biological sample and / or measuring the concentration of one or more additional proteins in a biological sample in any of the above methods. In certain embodiments, the one or more proteins can be proteins depleted from the biological sample before tau purification. For example, in certain embodiments, N-terminal tau and / or mid-domain tau species can be identified and / or quantified separately from tau species (e.g., MTBR tau, C-terminal tau) by the quantification methods disclosed herein. Alternatively or additionally, Aβ, ApoE, or any other protein of interest can be identified and / or quantified by processing a portion of the biological sample in parallel, by depleting the protein of interest from the biological sample before use in the methods disclosed herein, or by depleting the protein of interest from the biological sample during the sample processing steps disclosed herein.

[0119] The steps of biological sample, appropriate internal standard and depletion of one or more proteins, tau purification, cleavage of purified tau with proteases and mass spectrometry are further detailed below.

[0120] Biological samples 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 care or treatment, may be undergoing medical care or treatment, or may be undergoing medical care 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. As specific examples, the tauopathy may be Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), or 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.).

[0121] CSF can be obtained by lumbar puncture with or without an indwelling CSF catheter. Multiple blood or CSF samples collected simultaneously from a subject can be pooled. Blood can be collected by venipuncture or fingertip sampling (or equivalent procedures) with or without a venous catheter. Once collected, blood or CSF samples can 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 can be used immediately or frozen and stored indefinitely. Prior to use in the methods disclosed herein, biological samples can be processed, if necessary or desired, to add protease inhibitors, isotope-labeled internal standards, detergents and chaotropic agents, and / or to deplete other components (e.g., proteins, peptides, metabolites).

[0122] The sample size used can vary depending on the sample type, the health status of the subject from whom the sample was obtained, and the analytes (in addition to tau) to be analyzed. CSF sample volumes can be about 0.01 mL to about 5 mL or about 0.05 mL to about 5 mL. In specific examples, sample sizes can be about 0.05 mL to about 1 mL CSF. Plasma sample volumes can be about 0.01 mL to about 20 mL.

[0123] (b) Isotopically labeled, internal tau standard Isotopically labeled tau can be used as an internal standard to account for the variation during sample processing and, if necessary, calculate absolute concentration.Generally, isotope-labeled, internal tau standard is added before significant sample processing, and if necessary, can be added one or more times.For example, see the method described in Figure 2 and Figure 33.

[0124] Described herein are multiple isotopically labeled internal tau standards. All incorporate a heavy isotope label into at least one amino acid residue. One or more full-length isoforms may be used. Alternatively or additionally, post-translationally modified tau isoforms and / or peptide fragments of tau may be used, as known in the art. Generally, the incorporated labeled amino acid residue increases the mass of the peptide without affecting its chemical properties, and the mass shift due to the presence of the isotopic label should be sufficient to allow for differentiation of the internal standard (IS) from the endogenous tau analyte signal in mass spectrometry methods. As shown herein, suitable heavy isotope labels include: 2 H, 13 C and 15 Examples of internal standard include, but are not limited to, N. Generally, about 1-10 ng of internal standard is sufficient.

[0125] (c) depletion of one or more proteins The methods of the invention include depleting one or more proteins from a sample. The term "depletion" refers to a quantitative or numerical reduction. Thus, a protein-depleted sample can have any amount of protein that is measurably less than the amount in the original sample.

[0126] Proteins can be depleted from a sample 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 multiple proteins can 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 an appropriate epitope-binding agent. As another non-limiting example, apolipoprotein E (ApoE) status can be determined by methods known in the art after 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. Targeted depletion of certain tau proteins is also used herein to enrich other tau proteins and / or remove proteins that interfere with mass spectrometry analysis. For example, in certain embodiments of the present invention, N-terminal tau protein and / or mid-domain tau protein are depleted from a sample before further sample processing for analysis by mass spectrometry. Downstream analysis of the depleted tau proteins may or may not be performed, and both options are contemplated in the methods of the present invention.

[0127] In some embodiments, target depletion can be performed by affinity depletion. Affinity depletion refers to a method of depleting a protein of interest from a sample by utilizing specific binding to a molecule. Typically, the molecule is a ligand (referred to as an immobilized ligand) attached to a solid support such as a bead, resin, or tissue culture plate. Ligand immobilization to the solid support can also occur after the ligand-protein interaction has occurred. Suitable ligands include antibodies, aptamers, and other epitope-binding agents. The molecule can also be a polymer or other material that selectively absorbs the protein of interest. As a non-limiting example, fatty oxyethylated alcohol-substituted polyhydroxymethylene (e.g., PHM-L LIPOSORB, Sigma-Aldrich) can selectively absorb lipoproteins (including ApoE) from serum. Two or more affinity depletion agents can be combined to sequentially or simultaneously deplete multiple proteins.

[0128] In certain embodiments, the methods of the invention involve affinity depletion of one or more proteins from a sample using at least one epitope binding agent that specifically binds to an epitope within amino acids 1-243 (inclusive) of Tau-441 (or within a similarly defined region of the 0N or 1N isoforms). In various embodiments, one, two, three, or more epitope binding agents may be used. When two or more epitope binding agents are used, they may be used sequentially or simultaneously.

[0129] In certain embodiments, the methods of the invention involve affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within the N-terminus of tau (e.g., amino acids 1-103, inclusive, of tau 441) and an epitope binding agent that specifically binds to an epitope within the mid-domain of tau (e.g., amino acids 104-243, inclusive, of tau 441). The epitope binding agents may be used sequentially or simultaneously.

[0130] In certain embodiments, the methods of the invention comprise affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds within epitope amino acids 1-35 of Tau 441 (inclusive) and an epitope binding agent that specifically binds within epitope amino acids 104-243 of Tau 441 (inclusive) (or within a similarly defined region of the 0N or 1N isoforms). The epitope binding agents may be used sequentially or simultaneously.

[0131] In certain embodiments, the methods of the invention comprise affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds to the epitope within amino acids 1-103 (inclusive) of Tau 441 (or within a similarly defined region of the 0N or 1N isoforms); an epitope binding agent that specifically binds to the epitope within amino acids 104-243 (inclusive) of Tau 441 (or within a similarly defined region of the 0N or 1N isoforms); and an epitope binding agent that specifically binds to the amyloid beta epitope. The epitope binding agents may be used sequentially or simultaneously.

[0132] In certain embodiments, the methods of the invention comprise affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds within the epitope amino acids 1-35 of Tau 441 (inclusive) (or within a similarly defined region of the 0N or 1N isoforms); an epitope binding agent that specifically binds within the epitope amino acids 104-243 of Tau 441 (inclusive) (or within a similarly defined region of the 0N or 1N isoforms); and an epitope binding agent that specifically binds to the amyloid beta epitope. The epitope binding agents may be used sequentially or simultaneously.

[0133] In certain embodiments, the methods of the invention comprise affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds to the epitope within amino acids 1-103 (inclusive) of Tau441 (or similarly defined regions of the 0N or 1N isoforms); and an epitope binding agent that specifically binds to the amyloid beta epitope. The epitope binding agents may be used sequentially or simultaneously.

[0134] In certain embodiments, the methods of the invention comprise affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds within the epitope amino acids 1-35 of tau441 (inclusive) (or within a similarly defined region of the 0N or 1N isoforms); and an epitope binding agent that specifically binds to the amyloid beta epitope. The epitope binding agents may be used sequentially or simultaneously.

[0135] In certain embodiments, the methods of the invention comprise affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds to the epitope within amino acids 104-243 (inclusive) of tau441 (or similarly defined regions of the 0N or 1N isoforms); and an epitope binding agent that specifically binds to the amyloid beta epitope. The epitope binding agents may be used sequentially or simultaneously.

[0136] In any of the above embodiments, the epitope binding agent may comprise an antibody or an aptamer. In certain embodiments, the epitope binding agent that specifically binds to amyloid beta is HJ5.1 or an epitope binding agent that binds to the same epitope as HJ5.1 and / or competitively inhibits HJ5.1. In certain embodiments, the epitope binding agent that specifically binds to the epitope within amino acids 1-103 (inclusive) of Tau441 is HJ8.5 or an epitope binding agent that binds to the same epitope as HJ8.5 and / or competitively inhibits HJ8.5. In certain embodiments, the epitope binding agent that specifically binds to the epitope within amino acids 104-221 (inclusive) of Tau441 is Tau1 or an epitope binding agent that binds to the same epitope as Tau1 and / or competitively inhibits Tau1. Methods for identifying epitopes specifically bound by antibodies and assays for assessing competitive inhibition between two antibodies are known in the art.

[0137] Alternatively, proteins can be depleted from samples by more conventional methods, such as ultrafiltration or protein precipitation with acids, organic solvents, or salts. Generally speaking, the use of these methods reliably reduces high abundance and high molecular weight proteins, which in turn enriches for low molecular weight and / or low abundance proteins and peptides (e.g., tau, Aβ, etc.).

[0138] In certain embodiments, proteins can be depleted from a sample by precipitation. That is, precipitation involves adding a precipitant to the sample, mixing thoroughly, incubating the sample with the precipitant to precipitate the protein, and separating the precipitated protein by centrifugation or filtration. The resulting supernatant can then be used in downstream applications. The amount of agent required can be determined experimentally by methods known in the art. Suitable precipitants 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.

[0139] As a non-limiting example, proteins can be depleted from a sample by acid precipitation using perchloric acid. As used herein, "perchloric acid" refers to 70% perchloric acid unless otherwise specified. In certain embodiments, perchloric acid is added to a final concentration of about 1% v / v to about 15% v / v. In other embodiments, perchloric acid is added to a final concentration of about 1% v / v to about 10% v / v. In other embodiments, perchloric acid is added to a final concentration of about 1% v / v to about 5% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3% v / v to about 15% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3% v / v to about 10% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3% v / v to about 5% v / v. In other embodiments, perchloric acid is added to a final concentration of 3.5% v / v to about 15% v / v, 3.5% v / v to about 10% v / v, or 3.5% v / v to about 5% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3.5% v / v. After the addition of perchloric acid, the sample is mixed well (e.g., using a vortex mixer) and maintained at a low temperature, typically for about 10 minutes or more, to promote precipitation. For example, the sample may be maintained for about 10 minutes to about 60 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to about 60 minutes. In other examples, the sample may be maintained for about 15 minutes to about 45 minutes, or about 30 minutes to about 45 minutes. In other examples, the sample may be maintained for about 15 minutes to about 30 minutes, or about 20 minutes to about 40 minutes. In other examples, the sample is maintained for about 30 minutes. The sample is then centrifuged at a low temperature to pellet precipitated proteins, and the supernatant containing soluble tau (i.e., the acid-soluble fraction) is transferred to a new container. As used above, "low temperature" refers to a temperature of 10°C or below. For example, low temperature can be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C. In certain embodiments, a narrow temperature range, e.g., about 3°C ​​to about 5°C or about 4°C, is preferred. In certain embodiments, low temperature can be achieved by placing the sample on ice.

[0140] Two or more methods from one 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 first depleted, followed by targeted depletion of one or more proteins. In yet another alternative, high abundance / molecular weight proteins can be first depleted, 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 proteins that are different from the first round of targeted depletion. Other iterations are readily apparent to those skilled in the art.

[0141] (d) Tau purification Another step of the method disclosed herein includes purifying tau, particularly MTBR tau. In some instances, the MTBR tau is N-terminal-independent and / or mid-domain-independent MTBR tau. The purification of tau can be partial or complete.

[0142] In certain embodiments, the methods of the present invention involve purifying tau by solid-phase extraction. Purifying tau by solid-phase extraction involves contacting a sample containing tau with a solid phase containing a tau-adsorbing sorbent, one or more wash steps, and eluting tau from the sorbent. Suitable sorbents include reversed-phase sorbents. Suitable reversed-phase sorbents are known in the art and include, but are not limited to, alkyl-bonded silica, aryl-bonded silica, styrene / divinylbenzene materials, and N-vinylpyrrolidone / divinylbenzene materials. In an exemplary embodiment, the reversed-phase material is a polymer containing N-vinylpyrrolidone and divinylbenzene or a polymer containing styrene and divinylbenzene. In an exemplary embodiment, the sorbent is Oasis HLB (Waters). Prior to contact with the tau-containing supernatant, the sorbent is generally preconditioned (e.g., with a water-miscible organic solvent, followed by a mobile phase-containing buffer) according to the manufacturer's instructions or as known in the art. Furthermore, the supernatant can be optionally acidified, as some reversed-phase materials retain ionized analytes more strongly than others. The use of volatile components in the mobile phase and for elution is preferred because it facilitates sample drying. In exemplary embodiments, the washing step can include using a liquid phase containing about 0.05% v / v trifluoroacetic acid (TFA) to about 1% v / v TFA, or an equivalent thereof. In certain examples, washing can use a liquid phase containing about 0.05% v / v to about 0.5% v / v TFA or about 0.05% v / v to about 0.1% v / v TFA. In certain examples, washing can use a liquid phase containing about 0.1% v / v to about 1.0% v / v TFA or about 0.1% v / v to about 0.5% v / v TFA. Bound tau is then eluted with a liquid phase containing about 20% v / v to about 50% v / v acetonitrile (ACN) or an equivalent thereof. In some instances, tau can be eluted with a liquid phase containing about 20% v / v to about 40% v / v ACN or about 20% v / v to about 30% v / v ACN. In some instances, tau can be eluted with a liquid phase containing about 30% v / v to about 50% v / v ACN or about 30% v / v to about 40% v / v ACN. The eluate can be dried by methods known in the art (e.g., vacuum drying (e.g., Speed-vac), lyophilization, evaporation under a nitrogen stream, etc.).

[0143] In one embodiment, the method of the present invention involves purifying MTBR tau by affinity purification. Affinity purification is a method for enriching a protein of interest by its specific binding to a molecule. Typically, the molecule is a ligand (referred to as an immobilized ligand) that is attached to a solid support, such as a bead, resin, or tissue culture plate. Immobilization of the ligand to the solid support can also occur after the ligand-protein interaction has occurred. Suitable ligands include antibodies, aptamers, and other epitope-binding agents. Purification of MTBR tau by affinity purification involves contacting a sample containing tau with a suitable immobilized ligand, one or more wash steps, and eluting MTBR tau from the immobilized ligand.

[0144] In certain embodiments, the methods of the invention involve purifying MTBR tau by affinity purification using at least one epitope binding agent that specifically binds to the epitope within amino acids 235-368 (inclusive) of tau 441 or within amino acids 244-368 (inclusive) of tau 441 (or within a similarly defined region of other full-length isoforms). In various embodiments, one, two, three, or more epitope binding agents can be used. When two or more epitope binding agents are used, they can be used sequentially or simultaneously. Non-limiting examples of suitable epitope-binding agents include antibodies 77G7, RD3, RD4, UCB1017, and PT76 described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, and antibodies E2814 and 7G6 described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, as well as other epitope-binding agents that specifically bind to the same epitope as these antibodies. In a further embodiment, the methods of the present invention include purifying MTBR tau by affinity purification using an epitope binding agent that specifically binds to an epitope within R1 of MTBR tau, an epitope binding agent that specifically binds to an epitope within R2 of MTBR tau, an epitope binding agent that specifically binds to an epitope within R3 of MTBR tau, an epitope binding agent that specifically binds to an epitope within R4 of MTBR tau, an epitope binding agent that specifically binds to an epitope unique to 3R tau, an epitope binding agent that specifically binds to an epitope unique to 4R tau, an epitope binding agent that specifically binds to an epitope spanning R1 and R2 of MTBR tau, an epitope binding agent that specifically binds to an epitope spanning R2 and R3 of MTBR tau, an epitope binding agent that specifically binds to an epitope spanning R3 and R4 of MTBR tau, or any combination thereof. In a specific example, the methods of the invention involve the purification of MTBR tau by affinity purification using an epitope binding agent that specifically binds to an epitope comprising amino acids 316-355 of tau 441 (or the same region of other full-length isoforms). In various embodiments, one, two, three, or more epitope binding agents can be used.When two or more epitope binding agents are used, they may be used sequentially or simultaneously.

[0145] In any of the above embodiments, the epitope binding agent can comprise an antibody or an aptamer. In certain embodiments, the epitope binding agent that specifically binds to an epitope within R3 and R4 of MTBR tau is 77G7 or an epitope binding agent that binds to the same epitope as 77G7 and / or competitively inhibits 77G7 (BioLegend). In certain embodiments, the epitope binding agent that specifically binds to an epitope unique to 3R tau is RD3 (de Silva et al., Neuropathology and Applied Neurobiology, 2003, 29: 288-302) or an epitope binding agent that binds to the same epitope as RD3 and / or competitively inhibits RD3. In certain embodiments, the epitope binding agent that specifically binds to an epitope unique to 4R tau is RD4 (de Silva et al., Neuropathology and Applied Neurobiology, 2003, 29: 288-302) or an epitope binding agent that binds to the same epitope as RD4 and / or competitively inhibits RD4.

[0146] (e) Cleavage of purified tau with proteases Another step of the method disclosed herein involves cleaving purified tau with a protease. Cleavage of purified tau with a protease involves contacting a sample containing purified tau with the protease under conditions suitable for digesting tau. When affinity purification is used, digestion may be performed after elution of tau from the immobilized ligand or while tau is bound. Suitable proteases include, but are not limited to, trypsin, Lys-N, Lys-C, and Arg-N. In a preferred embodiment, the protease is trypsin. The resulting cleavage products are compositions containing proteolytic peptides of tau. When the protease is trypsin, the resulting cleavage products contain tryptic digest peptides of tau. After proteolytic cleavage, the resulting cleavage products are typically desalted by solid-phase extraction.

[0147] (f) LC-MS Another step of the presently disclosed methods comprises performing liquid chromatography-mass spectrometry (LC-MS) on the proteolytic peptides of a sample comprising tau to detect and measure the concentration of at least one of the tau proteolytic peptides. Thus, in effect, the disclosed methods comprise the use of one or more tau proteolytic peptides for the detection and measurement of the amount of tau protein present in a biological sample.

[0148] In embodiments in which trypsin is the protease, tau proteolytic peptides indicative of the presence of MTBR tau include, but are not limited to, those listed in Table A. When different enzymes are used for digestion, the resulting proteolytic peptides may differ slightly, but this can be easily determined by one of ordinary skill in the art. Without wishing to be bound by theory, it is believed that variations in the amount of tryptic peptides between two biological samples of the same type reflect differences in the MTBR tau species that make up these biological samples. As disclosed herein, the amount of specific MTBR tau proteolytic peptides and the ratio of specific MTBR tau proteolytic peptides provide clinically meaningful information to guide treatment decisions. Therefore, methods that allow for the detection and quantification of MTBR tau tryptic peptides are useful in the diagnosis and treatment of many neurodegenerative diseases. [Table 1]

[0149] Tau proteolytic peptides can be separated using a liquid chromatography system compatible with a high-resolution mass spectrometer. Suitable LC-MS systems can include the use of a <1.0 mm ID column and a flow rate of less than about 100 μL / min. In a preferred embodiment, a nanoflow LC-MS system is used (e.g., an approximately 50-100 μm ID column and a flow rate of <1 μL / min, preferably about 100-800 nL / min, more preferably about 200-600 nL / min). In an exemplary embodiment, the LC-MS system can include the use of a 0.05 mm ID column and a flow rate of about 400 nL / min.

[0150] As is known in the art, tandem mass spectrometry can be used to improve resolution, or technology can be improved to achieve the resolution of tandem mass spectrometry using a single mass analyzer. Suitable types of mass spectrometers are known in the art. These include quadrupoles, time-of-flight, ion traps, and Orbitraps, as well as hybrid mass spectrometers that combine various types of mass analyzers in one structure (e.g., Orbitrap Fusion, each from ThermoFisher Scientific). TM Tribrid TM Mass spectrometer or Orbitrap Fusion TM Lumos TM Mass spectrometer, Orbitrap Tribrid TM Eclipse TM In an exemplary embodiment, the LC-MS system includes, but is not limited to, an Orbitrap Fusion LC-MS system. TM Tribrid TM Mass spectrometer, Orbitrap Fusion TM Lumos TM Mass spectrometer, Orbitrap Tribrid TM Eclipse TMThe mass spectrometer may be selected from mass spectrometers or quadrupoles with similar or improved ion focusing and ion transmission. A suitable mass spectrometry protocol can be developed by optimizing the number of ions collected before analysis (e.g., AGC settings using a byte trap) and / or injection time. In an exemplary embodiment, the mass spectrometry protocol outlined in the Examples is used.

[0151] III. Use of the MTBR Tau Assay The present invention also encompasses the use of measuring MTBR tau species, particularly mid-domain-independent MTBR tau species, in blood or CSF as a biomarker of pathological characteristics and / or clinical symptoms of tauopathy for diagnosis, staging, selection of treatment appropriate for a certain disease stage, and modification of certain treatment regimens (e.g., dose modification, switching to a different drug or treatment modality, etc.). Pathological characteristics can 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 characteristics can be tau-independent. For example, when the tauopathy is Alzheimer's disease, it can be amyloid beta (Aβ) deposits in the brain or cerebral arteries. The clinical symptoms can be dementia, as assessed by a clinically validated instrument (e.g., MMSE, CDR-SB, etc.) or any other clinical symptoms associated with tauopathy. Assessment of MTBR tau species, particularly mid-domain-independent MTBR tau species, in blood or CSF is also contemplated as a biomarker for other pathological features and clinical conditions known in the art as 3R- and 4R-tauopathies. Advantageously, MTBR tau species, including but not limited to mid-domain-independent MTBR tau species, not only distinguish between diseased and healthy states, but also between various tauopathies.

[0152] Thus, in one embodiment, the present invention provides a method for assessing tauopathy-related pathology in a subject, comprising quantifying one or more MTBR tau species in a biological sample obtained from the subject, such as a blood sample or CSF sample, wherein the amount of quantified MTBR tau species represents tauopathy-related pathology in the subject's brain. The tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. The disease-related pathology may be tau deposition, tau post-translational modifications, amyloid plaques in the brain and / or cerebral arteries, or other pathological characteristics known in the art. The subject may or may not have clinical symptoms of tauopathy. In a preferred embodiment, at least one MTBR tau species quantification is a middle domain-independent MTBR tau species. In a further embodiment, two or more MTBR tau species quantifications are middle domain-independent MTBR tau species. In still further embodiments, each MTBR tau species quantity is a middle domain-independent MTBR tau species.

[0153] In another embodiment, the present invention provides a method for diagnosing a tauopathy in a subject, comprising quantifying one or more MTBR tau species in a biological sample obtained from the subject, such as a blood sample or CSF sample, and diagnosing the tauopathy when the quantified MTBR tau species is greater than or equal to about 1.5σ, where σ is the standard deviation defined by a normal distribution measured in a control population without clinical signs or symptoms of tauopathy and amyloid-negative as assessed by PET imaging and / or Aβ42 / 40 measurement in CSF. The tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. The subject may or may not have clinical symptoms of the disease. In a preferred embodiment, at least one MTBR tau species quantification is a middle domain-independent MTBR tau species. In a further embodiment, two or more MTBR tau species quantifications are middle domain-independent MTBR tau species. In still further embodiments, each MTBR tau species quantity is a middle domain-independent MTBR tau species.

[0154] In another embodiment, the present invention provides a method for measuring the stability of tauopathy disease in a subject, comprising quantifying one or more MTBR tau species in a first biological sample obtained from the subject and then a second biological sample obtained from the same subject at a later time point (e.g., days, weeks, months, or years later), and calculating the difference in the quantified MTBR tau species between the samples, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates disease progression, a statistically significant decrease in the quantified MTBR tau species in the second sample indicates disease improvement, and no change indicates disease stability. The tauopathy may be a 3R-tauopathy, a mixed 3R / 4R-tauopathy, or a 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 instances, tau therapy is administered to the subject one or more times during the period between obtaining the first and second biological samples, and the disease stability assessment indicates the effectiveness or lack of effectiveness of the tau therapy. In a preferred embodiment, at least one MTBR tau species determination is an intermediate domain-independent MTBR tau species. In a further embodiment, two or more MTBR tau species determinations are intermediate domain-independent MTBR tau species. In yet a further embodiment, each MTBR tau species determination is an intermediate domain-independent MTBR tau species.

[0155] In another embodiment, the present invention provides a method for treating a subject with a tauopathy, comprising quantifying one or more MTBR tau species in a biological sample obtained from the subject, such as a blood sample or CSF sample; and providing the subject with a tau treatment to improve the assessment of disease-related pathology or clinical symptoms, wherein the subject's quantified MTBR tau species is at least 1 standard deviation above or below the mean, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or more preferably at least 2 standard deviations (i.e., 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is the standard deviation defined by a normal distribution evaluated in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative as assessed by PET imaging and / or Aβ42 / 40 measurement in CSF).In addition to using a threshold (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 disease-related pathology measurement 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 or other clinical symptoms known in the art for 3R- and 4R-tauopathies, assessed by a clinically validated device (e.g., MMSE, CDR-SB, etc.). In a preferred embodiment, at least one MTBR tau species quantification is a middle domain-independent MTBR tau species. In a further embodiment, two or more MTBR tau species quantifications are middle domain-independent MTBR tau species. In yet a further embodiment, each MTBR tau species quantification is a middle domain-independent MTBR tau species. Many tau treatments target specific pathophysiological changes.For example, Aβ-targeting therapies are generally designed to reduce Aβ production, antagonize Aβ aggregation, or increase brain Aβ clearance; tau-targeting therapies are generally designed to modify 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. Therefore, the efficacy of these various tau therapies can be improved by administering them to subjects accurately identified as having 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy.

[0156] The term "mid-domain-independent MTBR tau" refers to multiple MTBR tau species that lack all or substantially all of the mid-domain region of tau and thus also lack the N-terminal region. These mid-domain-independent MTBR tau species remain after tau species containing mid-domain tau have been partially or completely depleted from a biological sample, preferably a blood or CSF sample. Suitable biological samples are described in Section II(a), the disclosure of which is incorporated herein by reference. Depletion of mid-domain tau can be performed by targeted depletion of these tau species, for example, by affinity depletion using epitope binding agents that specifically bind to epitopes within the N-terminus or mid-domain of tau. Multiple epitope binding agents can also be used—for example, a first epitope binding agent that specifically binds to an epitope within the N-terminus of tau and a second epitope binding agent that specifically binds to an epitope within the mid-domain of tau. Further details can be found in Section II(c), the disclosure of which is incorporated herein by reference. Generally, at least 50% (e.g., 50%, 60%, 70%, 80%, 90% or more) of the target protein in the starting material is depleted. In certain embodiments, about 70% or more, about 80% or more, or about 90% or more of the target protein in the starting material is depleted. After depletion of mid-domain tau from the biological sample, the following steps may be performed: (1) enriching the remaining tau species, including mid-domain-independent MTBR tau, for example, by removing other proteins by precipitation and / or purifying tau protein by solid-phase extraction; or (2) selectively enriching the mid-domain-independent MTBR tau species, for example, by affinity purification using an epitope-binding agent that specifically binds to an epitope within MTBR. The term "enriched" means increasing the amount or number. Further details can be found in Section II, the disclosure of which is incorporated herein by reference. Preferably, the mid-domain-independent MTBR tau species are enriched at least 100-fold compared to the amount in CSF.In some instances, the intermediate domain-independent MTBR tau species can be enriched by about 100-fold to about 1000-fold, e.g., about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, or about 1000-fold. In some instances, the intermediate domain-independent MTBR tau species can be enriched by about 500-fold to about 1000-fold or more. MTBR tau can be quantified in processed CSF or blood samples obtained from a subject, where the CSF or blood sample is depleted of mid-domain tau as described in Section II or the Examples or by other methods known in the art (e.g., multiplexed assays (e.g., xMAP technology with Luminex, single molecule protein detection (e.g., Simoa® bead technology)), etc.), and then enriched for MTBR tau by LC-MS. In embodiments in which the biological sample is not depleted of mid-domain tau, tau is generally enriched in the manner and to the extent described above.

[0157] In each of the above embodiments, suitable MTBR tau species include the amino acid sequence of SEQ ID NO:2 (IGSTENLK), SEQ ID NO:3 (LQTAPVPMPDLK), SEQ ID NO:4 (VQIINK), SEQ ID NO:5 (LDLSNVQSK), SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), SEQ ID NO:8 (IGSLDNITHVPGGGN), SEQ ID NO:9 (VQIVYKPVDLSK), or combinations thereof. The MTBR tau species may include, but are not limited to, MTBR tau species comprising sequence and / or intermediate domain-independent MTBR species.The selection of the MTBR species to be evaluated may depend on the intended purpose of the method.For example, when tauopathy is 3R-tauopathy, the MTBR tau species comprising SEQ ID NO:9 (VQIVYKPVDLSK) may be decreased compared with mixed 3R / 4R-tauopathy or 4R-tauopathy, while the MTBR tau species comprising SEQ ID NO:2 (IGSTENLK), SEQ ID NO:4 (VQIINK), SEQ ID NO:5 (LDLSNVQSK), SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK) and / or SEQ ID NO:8 (IGSLDNITHVPGGGN) may be unchanged or increased compared with other tauopathy. Conversely, when a tauopathy is a 4R-tauopathy, MTBR species comprising SEQ ID NO:9 (VQIVYKPVDLSK) may be increased compared to mixed 3R / 4R-tauopathy or 3R-tauopathy, while MTBR tau species comprising SEQ ID NO:2 (IGSTENLK), SEQ ID NO:4 (VQIINK), SEQ ID NO:5 (LDLSNVQSK), SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), and / or SEQ ID NO:8 (IGSLDNITHVPGGGN) may be unchanged or decreased compared to other tauopathies. As a further example, 4R tauopathy may be distinguished from AD by quantitating MTBR tau species comprising SEQ ID NO:2 (IGSTENLK), SEQ ID NO:4 (VQIINK), SEQ ID NO:5 (LDLSNVQSK), SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), and / or SEQ ID NO:8 (IGSLDNITHVPGGGN). The use of intermediate domain-independent MTBR tau species can improve discrimination power, and this can be further improved by using the ratio of two different intermediate domain-independent MTBR tau species.For example, when tauopathy is 3R-tauopathy or mixed 3R / 4R-tauopathy, the ratio of SEQ ID NO:3 to SEQ ID NO:6, SEQ ID NO:3 to SEQ ID NO:8 or SEQ ID NO:6 to SEQ ID NO:8 can be used.When tauopathy is 4R-tauopathy, the ratio of SEQ ID NO:2, 4, 5 or 9 to SEQ ID NO:6, 7 or 8 can be used.Mathematical methods other than ratio can also be used.

[0158] While examples of the use of intermediate domain-independent MTBR tau243 may be helpful in illustrating the various aspects described above, such descriptions do not limit the scope of the present invention. "Intermediate domain-independent MTBR tau243" is described in detail in Example 3. It is a tryptic digest peptide of multiple intermediate domain-independent MTBR tau species, all of which have the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK). Measuring the amount of intermediate domain-independent MTBR tau243 is one way of measuring the amount of this particular group of intermediate domain-independent MTBR tau species in a given sample. As shown in Examples 2 and 3, increased amounts of CSF intermediate domain-independent MTBR tau243 mimic direct measurements of increased Aβ and tau deposition in the brain associated with Alzheimer's disease (AD). In other words, the amount of CSF intermediate domain-independent MTBR tau243 (and therefore the amount of CSF intermediate domain-independent MTBR tau comprising SEQ ID NO:3) represents AD-related pathology (e.g., tau deposition in the brain, Aβ deposition in the brain, etc.). These amounts can therefore be used to evaluate AD-related pathology, determine a subject's amyloid status, and diagnose AD in subjects without clinical symptoms of the disease. The amount of CSF intermediate domain-independent MTBR tau243 also reproduces changes measured during clinical disease duration of AD, as defined, for example, by the results of the MMSE or CDR-SB test. Thus, the amount of intermediate domain-independent MTBR tau243 (and therefore the amount of intermediate domain-independent MTBR tau comprising SEQ ID NO:3) can be used to diagnose and stage AD in subjects across the entire disease spectrum (e.g., preclinical to clinical). Utility for diagnosing and staging AD in subjects across the entire disease spectrum was observed with total tryptic digest peptides of intermediate domain-independent MTBR tau. See, for example, the mid-domain independent MTBR tau299 and mid-domain independent MTBR tau354 data in Example 3.This supports the use of SEQ ID NO:3 abundance (among other things) as a disease-specific biomarker for AD (and possibly other tauopathies), regardless of the measurement method (e.g., mass spectrometry, ELISA, etc.), where the set of peptides comprising "mid-domain-independent MTBR tau comprising SEQ ID NO:3" may be different (although possibly overlapping) from the set of peptides comprising "mid-domain-independent MTBR tau comprising SEQ ID NO:6." Following disease diagnosis and / or staging, subjects may be administered treatments to reduce or prevent further increases in the amount of mid-domain-independent MTBR tau243 in the CSF and / or reduce or prevent further increases in other clinical signs or symptoms of AD. Treatment selection is further guided by knowledge of the specific disease stage informed by the amount of mid-domain-independent MTBR tau243—for example, therapies designed to prevent Aβ deposition, reverse Aβ deposition, prevent tau deposition, reverse tau deposition, and improve clinical signs of disease, although likely overlapping, may be used in subjects with different amounts of mid-domain-independent MTBR tau243. The examples further demonstrate that CSF intermediate domain-independent MTBR tau243 is highly useful as a biomarker for AD, but not for non-AD tauopathies. Non-AD tauopathies are distinguished by quantifying and ratioing intermediate domain-independent MTBR tau species containing SEQ ID NO:2 (IGSTENLK), SEQ ID NO:4 (VQIINK), SEQ ID NO:5 (LDLSNVQSK), SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), or SEQ ID NO:8 (IGSLDNITHVPGGGN). While the examples demonstrate the above principle with CSF samples, blood samples are contemplated as a suitable alternative.

[0159] In certain embodiments, the present invention provides a method for assessing Alzheimer's disease (AD)-related pathology in a subject, comprising: preparing a processed CSF or blood sample from the subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the treated sample an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the amount of the quantified MTBR tau species or their ratio represents AD-related pathology in the subject's brain.

[0160] In another specific embodiment, the present invention provides a method for measuring Alzheimer's disease (AD)-associated tau precipitates in the brain of a subject, comprising: preparing a treated CSF or blood sample depleted of mid-domain tau and enriched for MTBR tau; and quantifying MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK) in the treated sample, wherein the amount of quantified MTBR tau species represents AD-associated tau precipitates in the brain of the subject.

[0161] In another specific embodiment, the present invention provides a method for measuring Alzheimer's disease (AD)-associated tau deposition in the brain of a subject, comprising: preparing a treated CSF or blood sample from the subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the treated sample an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the amount of the quantified MTBR tau species or their ratio represents AD-associated tau deposition in the brain of the subject.

[0162] In another specific embodiment, the present invention provides a method for determining amyloid status in a subject, the method comprising providing a processed CSF or blood sample from the subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK) in the processed sample, wherein the amount of quantified MTBR tau species represents AD-related amyloid beta deposits in the subject's brain and is predictive of amyloid positivity as determined by PIB-PET, e.g., PiB-PET SUVR as described in Ann Neurol 2016; 80:379-387.

[0163] In another specific embodiment, the present invention provides a method of diagnosing Alzheimer's disease, comprising: providing a processed CSF or blood sample from a subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the processed sample MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof; and diagnosing Alzheimer's disease when the quantified MTBR tau species differ by about 1.5 σ or more, where σ is greater than or equal to about 1.5 σ in a subject without clinical signs or symptoms of a tauopathy and assessed by PET imaging (e.g., PiB-PET as described in Ann Neurol 2016; 80:379-387). The method further provides a method for determining the standard deviation of Aβ42 / 40 in a control population that is amyloid-negative, as measured by PiB-PET SUVR (e.g., a standard deviation as defined by a normal distribution) and / or by Aβ42 / 40 measurement in CSF (e.g., a cutoff value for CSF Aβ42 / 40 calculated from PiB-PET SUVR (Ann Neurol 2016; 80:379-387) that maximizes sensitivity %+specificity %).

[0164] In another specific embodiment, the present invention provides a method for assessing the progression of Alzheimer's disease (AD) in a subject, comprising: providing a first processed CSF or blood sample and a second processed CSF or blood sample, wherein each processed sample is obtained from a single subject, and each processed sample is depleted of mid-domain tau and enriched for MTBR tau; and quantifying, for each processed sample, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof; and calculating the difference between the quantified MTBR tau species in the second sample and the first sample, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates progression of Alzheimer's disease in the subject.

[0165] In another specific embodiment, the present invention provides a method for distinguishing between 4R-tauopathies, comprising: preparing a processed CSF or blood sample from a subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the processed sample (i) an MTBR tau species having the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (ii) an MTBR tau species having the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK) or an MTBR tau species having the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN); wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0166] In another specific embodiment, the present invention provides a method for distinguishing between 4R-tauopathies, comprising: preparing a processed CSF or blood sample from a subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying (i) an MTBR tau species having the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (ii) an MTBR tau species having the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR tau species having the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), an MTBR tau species having the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), or any combination thereof, in the treated sample; wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0167] In another specific embodiment, the present invention provides a method for identifying a 4R-tauopathy, comprising providing a processed CSF or blood sample from a subject that is (a) depleted of N-terminal tau and mid-domain tau and (b) enriched for MTBR tau; and detecting in the processed sample: (i) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), or any of these. and (ii) quantifying an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0168] In another specific embodiment, the present invention provides a method for distinguishing between 4R-tauopathies, comprising: providing a processed CSF or blood sample from a subject that is (a) depleted of N-terminal tau and mid-domain tau and (b) enriched for MTBR tau; and quantifying in the processed sample (i) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), or a combination thereof, and (ii) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0169] In another specific embodiment, the present invention provides a method for distinguishing between 4R-tauopathies, comprising: providing a processed CSF or blood sample from a subject that is (a) depleted of N-terminal tau and mid-domain tau and (b) enriched for MTBR tau; and quantifying in the processed sample (a) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK) and (b) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (a) and (b) distinguishes between 4R-tauopathies and other tauopathies and healthy conditions.

[0170] In another specific embodiment, the present invention provides a method for distinguishing between 3R-tauopathies, comprising: providing a processed CSF or blood sample from a subject that is (a) depleted of N-terminal tau and mid-domain tau and (b) enriched for MTBR tau; and quantifying in the processed sample (a) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (b) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), SEQ ID NO: 4 (VQIINK), SEQ ID NO: 5 (LDLSNVQSK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), SEQ ID NO: 7 (IGSLDNITHVPGGGNK), SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (a) and (b) distinguishes between 3R-tauopathies, other tauopathies, and healthy conditions.

[0171] In another specific embodiment, the present invention provides a method for assessing tauopathy-associated pathology in a subject, comprising: preparing a processed CSF or blood sample from the subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the processed sample MTBR species comprising the amino acid sequence of SEQ ID NO:2 (IGSTENLK), MTBR species comprising the amino acid sequence of SEQ ID NO:3 (LQTAPVPMPDLK), MTBR species comprising the amino acid sequence of SEQ ID NO:4 (VQIINK), MTBR species comprising the amino acid sequence of SEQ ID NO:5 (LDLSNVQSK), MTBR species comprising the amino acid sequence of SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), MTBR species comprising the amino acid sequence of SEQ ID NO:8 (IGSLDNITHVPGGGN), MTBR species comprising the amino acid sequence of SEQ ID NO:9 (VQIVYKPVDLSK), or a combination thereof, wherein the amount of the quantified MTRB tau species or their ratio represents tauopathy-associated pathology in the subject's brain.

[0172] In another specific embodiment, the present invention provides a method for assessing tau deposition in a subject, comprising: preparing a processed CSF or blood sample from the subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the processed sample an MTBR species comprising the amino acid sequence of SEQ ID NO:2 (IGSTENLK), an MTBR species comprising the amino acid sequence of SEQ ID NO:3 (LQTAPVPMPDLK), an MTBR species comprising the amino acid sequence of SEQ ID NO:4 (VQIINK), an MTBR species comprising the amino acid sequence of SEQ ID NO:5 (LDLSNVQSK), an MTBR species comprising the amino acid sequence of SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), an MTBR species comprising the amino acid sequence of SEQ ID NO:8 (IGSLDNITHVPGGGN), an MTBR species comprising the amino acid sequence of SEQ ID NO:9 (VQIVYKPVDLSK), or a combination thereof, wherein the amount of the quantified MTBR tau species or their ratio represents tau deposition in the subject's brain.

[0173] In another specific embodiment, the present invention provides a method for assessing tau deposition in a subject, comprising: preparing a processed CSF or blood sample from the subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the processed sample MTBR species comprising the amino acid sequence of SEQ ID NO:2 (IGSTENLK), MTBR species comprising the amino acid sequence of SEQ ID NO:4 (VQIINK), MTBR species comprising the amino acid sequence of SEQ ID NO:5 (LDLSNVQSK), MTBR species comprising the amino acid sequence of SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), MTBR species comprising the amino acid sequence of SEQ ID NO:8 (IGSLDNITHVPGGGN), MTBR species comprising the amino acid sequence of SEQ ID NO:9 (VQIVYKPVDLSK), or a combination thereof, wherein the amount of the quantified MTBR tau species or their ratio represents tau deposition in the brain of a subject with a 3R-tauopathy or a 4R-tauopathy (i.e., a non-AD tauopathy).

[0174] The following specific embodiments relate to methods, including methods for assessing tau in a biological sample. In each of these embodiments, the method for measuring tau in a biological sample comprises: (a) depleting N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau in the biological sample by affinity depletion, and optionally depleting amyloid beta by affinity depletion, wherein the biological sample is a blood sample or a CSF sample, and the biological sample optionally contains an isotopically labeled, tau internal standard; (b) (i) removing additional proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant; and (ii) purifying tau from the supernatant by solid-phase extraction or (iii) affinity-purifying MTBR tau, thereby producing (i) or (ii) enriched tau; (c) cleaving the enriched tau with a protease, and then optionally desalting the cleavage products obtained by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (d) performing liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides to detect and quantify at least one of the tau proteolytic peptides. Further details of each of steps (a) through (d) can be found in Section II, which is incorporated herein by reference.

[0175] In another specific embodiment, the present invention provides a method for assessing Alzheimer's disease (AD)-related pathology in a subject, comprising measuring tau in a biological sample by the above-described method, wherein the tau measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the amount of MTBR tau species is indicative of AD-related pathology in the brain of the subject.

[0176] In another specific embodiment, the present invention provides a method for measuring Alzheimer's disease (AD)-associated tau deposits in the brain of a subject, comprising measuring tau in a biological sample by the above-described method, wherein the tau measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), and wherein the amount of the MTRB tau species is indicative of AD-associated pathology in the brain of the subject.

[0177] In another specific embodiment, the present invention provides a method for assessing Alzheimer's disease (AD)-associated tau deposition in the brain of a subject, comprising measuring tau in a biological sample by the above-described method, wherein the tau measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the amount of MTBR tau species is indicative of AD-related pathology in the brain of the subject.

[0178] In another specific embodiment, the present invention provides a method for determining the amyloid status of a subject, comprising measuring tau in a biological sample by the above method, wherein the tau measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), and wherein the amount of the MTRB tau species is indicative of AD-associated amyloid beta deposits in the subject's brain and is predictive of amyloid positivity as determined by PIB-PET.

[0179] In another specific embodiment, the present invention provides a method for diagnosing Alzheimer's disease, comprising measuring tau in a biological sample by the above method, wherein the measured tau is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof; and diagnosing Alzheimer's disease when the quantified MTBR tau species differ by about 1.5 σ or more, where σ is the standard deviation defined by a normal distribution measured in a control population without clinical signs or symptoms of tauopathy and who are amyloid-negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF.

[0180] In another specific embodiment, the present invention provides a method for assessing the progression of Alzheimer's disease (AD) in a subject, comprising measuring tau in a biological sample by the above method, wherein the measured tau is an MTBR tau species having the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species having the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species having the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species having the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof; and calculating the difference between the quantified MTBR tau species in the second sample and the first sample, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates progression of Alzheimer's disease in the subject.

[0181] In another specific embodiment, the present invention provides a method for distinguishing between 4R-tauopathy, comprising measuring tau in a biological sample by the above method, wherein the tau measured is (i) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (ii) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK) or an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN); and wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0182] In another specific embodiment, the present invention provides a method for distinguishing between 4R-tauopathy, comprising measuring tau in a biological sample by the above method, wherein the tau measured is (i) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (ii) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), or any combination thereof; wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0183] In another specific embodiment, the present invention provides a method for distinguishing between 4R-tauopathies, comprising measuring tau in a biological sample by the above method, wherein the tau measured is (i) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), or a combination thereof, and (ii) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0184] In another specific embodiment, the present invention provides a method for distinguishing between 4R-tauopathies, comprising measuring tau in a biological sample by the above method, wherein the tau measured is (i) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), or a combination thereof, and (ii) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0185] In another specific embodiment, the present invention provides a method for distinguishing between 4R-tauopathy, comprising measuring tau in a biological sample by the above method, wherein the tau measured is (i) an MTBR tau species having the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK) and (ii) an MTBR tau species having the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species having the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0186] In another specific embodiment, the present invention provides a method for assessing tauopathy-related pathology in a subject, comprising measuring tau in a biological sample that has been depleted of mid-domain tau and enriched for MTBR tau by the above-described method; and quantifying, in the treated sample, MTBR species comprising the amino acid sequence of SEQ ID NO:2 (IGSTENLK), MTBR species comprising the amino acid sequence of SEQ ID NO:3 (LQTAPVPMPDLK), MTBR species comprising the amino acid sequence of SEQ ID NO:4 (VQIINK), MTBR species comprising the amino acid sequence of SEQ ID NO:5 (LDLSNVQSK), MTBR species comprising the amino acid sequence of SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), MTBR species comprising the amino acid sequence of SEQ ID NO:8 (IGSLDNITHVPGGGN), MTBR species comprising the amino acid sequence of SEQ ID NO:9 (VQIVYKPVDLSK), or a combination thereof, wherein the amount of the quantified MTRB tau species or their ratio represents tauopathy-related pathology in the subject's brain.

[0187] In certain embodiments, the present invention provides a method for measuring tau deposition in a subject, comprising measuring tau in a biological sample that has been depleted of mid-domain tau and enriched for MTBR tau by the above method; and quantifying in the treated sample MTBR species comprising the amino acid sequence of SEQ ID NO:2 (IGSTENLK), MTBR species comprising the amino acid sequence of SEQ ID NO:3 (LQTAPVPMPDLK), MTBR species comprising the amino acid sequence of SEQ ID NO:4 (VQIINK), MTBR species comprising the amino acid sequence of SEQ ID NO:5 (LDLSNVQSK), MTBR species comprising the amino acid sequence of SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), MTBR species comprising the amino acid sequence of SEQ ID NO:8 (IGSLDNITHVPGGGN), MTBR species comprising the amino acid sequence of SEQ ID NO:9 (VQIVYKPVDLSK), or a combination thereof, wherein the amount of the quantified MTRB tau species or their ratio represents tau deposition in the brain of the subject.

[0188] In another specific embodiment, the present invention provides a method of treating a subject in need thereof, comprising: (a) providing a processed CSF or blood sample from the subject, wherein the processed CSF or blood sample is (i) depleted of mid-domain tau and (ii) enriched for MTBR tau; (b) detecting in the processed sample an MTBRMTBR tau species comprising the amino acid sequence of SEQ ID NO:2 (IGSTENLK), an MTBRMTBR tau species comprising the amino acid sequence of SEQ ID NO:3 (LQTAPVPMPDLK), an MTBRMTBR tau species comprising the amino acid sequence of SEQ ID NO:4 (VQIINK), an MTBRMTBR tau species comprising the amino acid sequence of SEQ ID NO:5 (LDLSNVQSK), an MTBRMTBR tau species comprising the amino acid sequence of SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), an MTBRMTBR tau species comprising the amino acid sequence of SEQ ID NO:7 (IGSLDNITHVPGGGNK), or an MTBRMTBR tau species comprising the amino acid sequence of SEQ ID NO:8 (IGSLDNITHVPGGGNK). and (c) quantifying the amount of MTBRMTBR tau species, the MTBRMTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), the MTBRMTBR tau species comprising the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK), or a combination thereof; and (c) administering a treatment to the subject to alter tau pathology, wherein the amount of the quantified MTBRMTBR tau species or the ratio of the quantified MTBRMTBR tau species in the treated CSF or blood samples of the subject differs by 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 assessed by PET imaging and / or Aβ42 / 40 measurement in CSF, and wherein the amount of the quantified MTRB tau species or the ratio thereof represents tau pathology in the subject's brain. In some embodiments, administering a treatment to the subject to alter tau pathology alters or stabilizes the amount of the quantified MTBR species.In certain embodiments, the treatment includes the use of a cholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, an antidepressant (e.g., selective serotonin reuptake inhibitor, an atypical antidepressant, an aminoketone, a selective serotonin and norepinephrine reuptake inhibitor, a tricyclic antidepressant, 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-TREM2 antibody (including an antigen-binding fragment, variant, or derivative thereof), an anti-T ... TREM2 agonists, including synthetic fragments, variants or derivatives, 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), serotonin receptor 6 antagonists, p38 alpha MAPK inhibitors, recombinant granulocyte macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513), tau protein aggregation inhibitors (e.g., TRx0237, methylthioninium chloride), blood Treatments to improve glucose 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, b-2 adrenoceptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenoceptor antagonists, 5-HT and pharmaceutical compositions comprising 1A 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 stimulators, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof.In exemplary embodiments, the pharmaceutical composition may contain a kinase inhibitor. Suitable kinase inhibitors may inhibit numerous amino acid kinases (TAOKs), CDKs, GSK-3p, MARKs, CDK5, or Fyn. In other exemplary embodiments, the pharmaceutical composition may contain a phosphatase activator. As a non-limiting example, a phosphatase activator may increase the activity of protein phosphatase 2A. In certain embodiments, the treatment is a pharmaceutical composition comprising a tau-targeting 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 certain 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.

[0189] In another specific embodiment, the present invention provides a method of treating a subject in need thereof, comprising: (a) providing a processed CSF or blood sample from the subject that is (i) depleted of mid-domain tau and (ii) enriched for MTBR tau; and (b) providing, in the processed sample, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:3 (LQTAPVPMPDLK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO:6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO:7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO:8 (IGSLDNITHVPGGGN). and (c) administering a treatment to the subject to change tau pathology, wherein the amount of quantified MTBR tau species or the ratio of quantified MTBR tau species in the treated CSF or blood samples of the subject differs by 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 assessed by PET imaging and / or Aβ42 / 40 measurement in CSF, and wherein the amount of quantified MTBR tau species or the ratio thereof represents the tau pathology in the brain of the subject.In some embodiments, administering a treatment to the subject to change tau pathology changes or stabilizes the amount of quantified MTBR species.In certain embodiments, the treatment includes the use of a cholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, an antidepressant (e.g., selective serotonin reuptake inhibitor, an atypical antidepressant, an aminoketone, a selective serotonin and norepinephrine reuptake inhibitor, a tricyclic antidepressant, 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-TREM2 antibody (including an antigen-binding fragment, variant, or derivative thereof), an anti-T ... TREM2 agonists, including synthetic fragments, variants or derivatives, 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), serotonin receptor 6 antagonists, p38 alpha MAPK inhibitors, recombinant granulocyte macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513), tau protein aggregation inhibitors (e.g., TRx0237, methylthioninium chloride), blood Treatments to improve glucose 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, b-2 adrenoceptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenoceptor antagonists, 5-HT and pharmaceutical compositions comprising 1A 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 stimulators, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof.In exemplary embodiments, the pharmaceutical composition may contain a kinase inhibitor. Suitable kinase inhibitors may inhibit numerous amino acid kinases (TAOKs), CDKs, GSK-3p, MARKs, CDK5, or Fyn. In other exemplary embodiments, the pharmaceutical composition may contain a phosphatase activator. As a non-limiting example, a phosphatase activator may increase the activity of protein phosphatase 2A. In certain embodiments, the treatment is a pharmaceutical composition comprising a tau-targeting 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 certain 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. For example, the present disclosure provides the following embodiments. [1] 1. A method for measuring tau in a biological sample, comprising: (a) providing a biological sample selected from a blood sample or a CSF sample, (i) optionally containing an isotopically labeled internal standard of tau, and (ii) optionally depleted of amyloid beta, N-terminal tau, mid-domain tau, or any combination thereof; (b) removing proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant; (c) tau was purified from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease, and then optionally desalting the resulting cleavage products by solid phase extraction to obtain a sample containing proteolytic peptides of tau; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect and quantify at least one tau proteolytic peptide; method. [2] 10. The method of claim 1, wherein the biological sample is depleted of amyloid beta, N-terminal tau, mid-domain tau, or any combination thereof. [3] The method of any one of claims 2 to 11, wherein (i) the biological sample is depleted of amyloid beta, N-terminal tau, and mid-domain tau, (ii) the biological sample is depleted of N-terminal tau and mid-domain tau, or (iii) the biological sample is depleted of mid-domain tau. [4] 2. The method of claim 1, wherein the solid phase in step (c) comprises a reversed-phase adsorbent that adsorbs tau. [5] step (b) comprises mixing an acid to precipitate proteins in the biological sample, and optionally the acid is perchloric acid; and / or In step (e), liquid chromatography-mass spectrometry is carried out by a nano-LC / MS system; Any of the methods 1 to 4 above. [6] 1. A method for measuring tau in a biological sample, comprising: (a) depleting N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau in a biological sample by affinity depletion, and optionally depleting amyloid beta by affinity depletion, wherein the biological sample is a blood sample or a CSF sample, and the biological sample optionally contains an isotopically labeled, tau internal standard; (b) enriching MTBR tau by a method comprising (i) removing additional proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, and then purifying tau from the supernatant by solid phase extraction, or (ii) affinity purifying MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) cleaving the enriched MTBR tau with a protease, and then optionally desalting the resulting cleavage products by solid phase extraction to obtain a sample containing proteolytic peptides of tau; and (d) performing liquid chromatography-mass spectrometry (LC / MS) on the sample containing tau proteolytic peptides to detect and quantify at least one tau proteolytic peptide; method. [7] 7. The method of claim 6, wherein step (a) comprises reducing (i) N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau, and (ii) amyloid beta. [8] the biological sample comprises human tau, and wherein step (a) further comprises: contacting the biological sample with at least one epitope binding agent that specifically binds to an epitope within amino acids 1 to 243 (inclusive) of Tau441; or contacting the biological sample with a first epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive) of Tau 441 and a second epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of Tau 441; or contacting the biological sample with a first epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive) of tau 441, a second epitope that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of tau 441, and a third epitope binding agent that specifically binds to an epitope of amyloid beta; or contacting the biological sample with a first epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive) of tau 441 and a second epitope binding agent that specifically binds to an epitope of amyloid beta; or contacting the biological sample with a first epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of Tau 441 and a third epitope binding agent that specifically binds to an epitope of amyloid beta; Method 6 above. [9] 9. The method of claim 8, wherein the epitope binding agent that specifically binds to amyloid beta is HJ5.1.

[10] 9. The method of claim 8, wherein the epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive) of Tau441 is HJ8.5.

[11] 9. The method of claim 8, wherein the epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of Tau441 is Tau1.

[12] 12. The method of any one of 6 to 11, wherein the solid phase extraction comprises a reverse phase adsorbent that adsorbs tau.

[13] the method for enriching MTBR tau comprises step (b)(i), wherein protein precipitation comprises mixing the biological sample with an acid to precipitate proteins, optionally the acid being perchloric acid; and / or In step (d), liquid chromatography-mass spectrometry is carried out by a nano-LC / MS system; Any of the methods 6 to 11 above.

[14] 14. The method of claim 13, wherein the solid phase extraction performed in steps (b) and (c) comprises a reversed phase adsorbent that adsorbs tau.

[15] a method for enriching MTBR tau comprising step (b)(ii), wherein affinity purification of MTBR tau comprises contacting the product of step (a) with an epitope binding agent that specifically binds to an epitope that is C-terminal to the epitope of step (a); and / or In step (d), liquid chromatography-mass spectrometry is carried out by a nano-LC / MS system; Any of the methods 8 to 11 above.

[16] The epitope binding agent of step (b) Within amino acids 221 to 441 (inclusive) of Tau441 or Within amino acids 235 to 441 (inclusive) of Tau441 or Within amino acids 235 to 368 (inclusive) of tau441 or Within amino acids 244 to 368 (inclusive) of Tau441 or Within amino acids 244 to 299 (inclusive) of Tau441 15. The method according to claim 15, wherein the antibody specifically binds to an epitope of

[17] 17. The method of claim 16, wherein the epitope binding agent is an antibody selected from the group consisting of 77G7, RD3, RD4, UCB0107, PT76, E2814, and 7G6.

[18] 18. The method of claim 15, 16 or 17, wherein the solid phase extraction performed in step (c) comprises a reversed phase adsorbent that adsorbs tau.

[19] 6. The method according to any one of 1 to 5 above, wherein the protease is trypsin.

[20] 19. The method of claim 19, wherein step (d) comprises detecting and quantifying at least one proteolytic peptide of tau, wherein the proteolytic peptide of tau has an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 9. [twenty one] 19. The method of claim 19, wherein step (d) comprises detecting and measuring the concentration of at least two tau proteolytic peptides, wherein the two tau proteolytic peptides have amino acid sequences selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. [twenty two] 21. The method of claim 21, wherein at least two tau proteolytic peptides have the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:6, SEQ ID NO:3 and SEQ ID NO:7, SEQ ID NO:3 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:8, SEQ ID NO:4 and SEQ ID NO:7, SEQ ID NO:4 and SEQ ID NO:8, SEQ ID NO:5 and SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:6, SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:9 and SEQ ID NO:5. [twenty three] 12. The method according to any one of 6 to 11 above, wherein the protease is trypsin. [twenty four] 23. The method of claim 23, wherein step (d) comprises detecting and measuring the concentration of at least one proteolytic peptide of MTBR tau, wherein the proteolytic peptide of tau has an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. [twenty five] 23. The method of claim 23, wherein step (d) comprises detecting and measuring the concentration of at least two proteolytic peptides of MTBR tau, wherein the two tau proteolytic peptides have amino acid sequences selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[26] 25. The method of claim 25, wherein at least two proteolytic peptides of MTBR tau have the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:6, SEQ ID NO:3 and SEQ ID NO:7, SEQ ID NO:3 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:8, SEQ ID NO:4 and SEQ ID NO:7, SEQ ID NO:4 and SEQ ID NO:8, SEQ ID NO:5 and SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:6, SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:9 and SEQ ID NO:5.

[27] 13. The method of claim 12, wherein the protease is trypsin.

[28] 14. The method of claim 13, wherein the protease is trypsin.

[29] 16. The method of claim 15, wherein the protease is trypsin.

[30] 30. Any of the methods 27, 28 or 29, wherein step (d) comprises detecting and measuring the concentration of at least one proteolytic peptide of MTBR tau, wherein the proteolytic peptide of tau has an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[31] 30. Any of the methods 27, 28 or 29, wherein step (d) comprises detecting and measuring the concentration of at least two proteolytic peptides of MTBR tau, wherein the proteolytic peptides of tau have amino acid sequences selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[32] 31. The method of claim 31, wherein at least two proteolytic peptides of MTBR tau have the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:7, SEQ ID NO:3 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:8, SEQ ID NO:4 and SEQ ID NO:7, SEQ ID NO:4 and SEQ ID NO:8, SEQ ID NO:5 and SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:6, SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:9 and SEQ ID NO:5.

[33] 33. Any of the methods 6 to 32, wherein the method further comprises detecting and measuring the concentration of N-terminal tau, mid-domain tau or amyloid beta removed from the biological sample in step (a).

[34] 1. A method for measuring Alzheimer's disease (AD)-related pathology in a subject, comprising: providing a processed CSF or blood sample from the subject that is depleted of mid-domain tau and enriched for MTBR tau; and and quantifying in the treated sample an MTBR tau species comprising the amino acid sequence of SEQ ID NO:3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:8, or a combination thereof, wherein the amount of the quantified MTBR tau species or a ratio thereof represents AD-related pathology in the subject's brain. method.

[35] A method for measuring Alzheimer's disease (AD)-related pathology in a subject, comprising measuring tau in a CSF or blood sample by any of the methods of 6 to 18; wherein the tau to be measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof, and wherein the amount of the measured MTBR tau species or the ratio thereof represents AD-related pathology in the subject's brain. method.

[36] 36. The method of claim 34 or 35, wherein the AD-related pathology is tau deposition in the brain of the subject.

[37] 36. The method of claim 34 or 35, wherein the AD-related pathological condition is amyloid beta deposition in the subject's brain or cerebral arteries.

[38] 1. A method for measuring Alzheimer's disease (AD)-associated tau deposition in the brain of a subject, comprising: providing a processed CSF or blood sample from the subject that is depleted of mid-domain tau and enriched for MTBR tau; and and quantifying in the treated sample MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK) in the treated CSF or blood sample, wherein the amount of quantified MTRB tau species represents AD-associated tau deposits in the brain of the subject. method.

[39] A method for measuring Alzheimer's disease (AD)-associated tau deposition in the brain of a subject, comprising measuring tau in a CSF or blood sample by any of the methods of 6 to 18; wherein the tau to be measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), and wherein the amount of the measured MTRB tau species represents AD-associated tau deposition in the subject's brain. method.

[40] 1. A method for measuring Alzheimer's disease (AD)-associated tau deposition in the brain of a subject, comprising: providing a treated CSF or blood sample from the subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the treated sample an MTBR tau species comprising the amino acid sequence of SEQ ID NO:3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:8, or a combination thereof, wherein the amount of the quantified MTBR tau species or a ratio thereof represents AD-associated tau deposits in the brain of the subject. method.

[41] A method for measuring Alzheimer's disease (AD)-associated tau deposition in the brain of a subject, comprising measuring tau in a CSF or blood sample by any of the methods of 6 to 18; wherein the tau to be measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof, and wherein the amount of the measured MTBR tau species or the ratio thereof represents AD-related tau deposition in the brain of the subject. method.

[42] A method of diagnosing Alzheimer's disease, comprising: providing a treated CSF or blood sample from a subject that has been depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the treated sample an MTBR tau species comprising the amino acid sequence of SEQ ID NO:3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:8, or a combination thereof; and Alzheimer's disease is diagnosed when the quantified MTBR tau species differ by more than about 1.5 σ, where σ is the standard deviation defined by a normal distribution measured in a control population that is amyloid-negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF. method.

[43] A method for diagnosing Alzheimer's disease, comprising measuring tau in a CSF or blood sample by any of the methods of 6 to 18, wherein the tau measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof; and Alzheimer's disease is diagnosed when the quantified MTBR tau species differ by more than about 1.5 σ, where σ is the standard deviation defined by a normal distribution measured in a control population that is amyloid-negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF. method.

[44] 1. A method for measuring Alzheimer's disease (AD) progression in a subject, comprising: providing a first processed CSF or blood sample and a second processed CSF or blood sample, wherein each processed sample is obtained from one subject, and each processed sample is depleted of mid-domain tau and enriched for MTBR tau; and quantifying, for each processed sample, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:8, or a combination thereof; and calculating a difference between the quantified MTBR tau species in the second sample and the first sample, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates progression of Alzheimer's disease in the subject; method.

[45] 1. A method for measuring Alzheimer's disease (AD) progression in a subject, comprising: measuring tau in the first and second CSF or blood samples by any of the methods 6 to 18 above, wherein the tau to be measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof; and calculating a difference between the quantified MTBR tau species in the second sample and the first sample, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates progression of Alzheimer's disease in the subject; method.

[46] 46. ​​Any of the methods 38 to 45, wherein the subject is amyloid-negative.

[47] 46. ​​The method of claim 46, wherein the subject does not have dementia.

[48] 46. ​​The method of claim 46, wherein the subject has dementia.

[49] 46. ​​Any of the methods 38 to 45, wherein the subject is amyloid-positive.

[50] 49. The method of claim 49, wherein the subject does not have dementia.

[51] 49. The method of claim 49, wherein the subject has dementia.

[52] 49. The method of claim 46 or 49, wherein the subject has a CDR score of 0.5 to 1.0.

[53] 49. The method of claim 46 or 49, wherein the subject has a CDR score of >1.0 to 2.0 (moderate AD).

[54] 49. The method of claim 46 or 49, wherein the subject has a CDR score of >2.0.

[55] 55. The method of any one of 38 to 54, further comprising amyloid beta quantification, N-terminal tau quantification, mid-domain tau quantification, tau post-translational modification quantification, or ApoE isoform identification in a biological or CSF sample.

[56] 1. A method for measuring tau pathology in the brain of a subject, comprising: providing a processed CSF or blood sample from the subject that is depleted of mid-domain tau and enriched for MTBR tau; and quantitating in the treated sample MTBR tau species comprising the amino acid sequence of SEQ ID NO:2, MTBR tau species comprising the amino acid sequence of SEQ ID NO:3, MTBR tau species comprising the amino acid sequence of SEQ ID NO:4, MTBR tau species comprising the amino acid sequence of SEQ ID NO:5, MTBR tau species comprising the amino acid sequence of SEQ ID NO:6, MTBR tau species comprising the amino acid sequence of SEQ ID NO:7, MTBR tau species comprising the amino acid sequence of SEQ ID NO:8, MTBR tau species comprising the amino acid sequence of SEQ ID NO:9, or a combination thereof, wherein the amount of quantified MTBR tau species or a ratio thereof is indicative of tau pathology in the brain of the subject. method.

[57] 19. A method for measuring tau pathology in the brain of a subject, comprising measuring tau in a CSF or blood sample by any of the methods set forth in 6 to 18, wherein the tau measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 9, or a combination thereof, wherein the amount of the quantified MTBR tau species or the ratio thereof represents tau pathology in the brain of the subject. method.

[58] 1. A method for identifying a 4R-tauopathy, comprising: providing a processed CSF or blood sample from the subject that is depleted of mid-domain tau and enriched for MTBR tau; and quantifying in the treated sample (a) an MTBR tau species comprising the amino acid sequence of SEQ ID NO:9 and (b) an MTBR tau species comprising the amino acid sequence of SEQ ID NO:4, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:5, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:7, or an MTBR tau species comprising the amino acid sequence of SEQ ID NO:8; wherein the ratio of the quantified MTBR species in (a) to the quantified MTBR species in (b) distinguishes between 4R-tauopathies. method.

[59] A method for identifying a 4R-tauopathy, comprising measuring tau in a biological sample by any of the methods set forth in 6 to 17, wherein the tau measured is (a) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 9 and (b) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, or an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8; wherein the ratio of the quantified MTBR species in (a) to the quantified MTBR species in (b) is discriminative of a 4R-tauopathy. method.

[60] 1. A method for distinguishing between a 3R-tauopathy or a 4R-tauopathy and Alzheimer's disease, comprising: providing a processed CSF or blood sample from a subject that is (a) depleted of mid-domain tau and (b) enriched for MTBR tau; and In the treated sample, (a) MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2, MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4, MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5, or a combination thereof, and (b) MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof, are quantified; wherein the ratio of the quantified MTBR species in (a) to the quantified MTBR species in (b) distinguishes between a 3R-tauopathy or a 4R-tauopathy and Alzheimer's disease. method.

[61] 19. A method for distinguishing between 3R-tauopathy or 4R-tauopathy and Alzheimer's disease, comprising measuring tau in a biological sample by any of the methods set forth in 6 to 18, wherein the tau measured is (a) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5, or a combination thereof, and (b) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof; wherein the ratio of the quantified MTBR species in (a) to the quantified MTBR species in (b) distinguishes between a 3R-tauopathy or a 4R-tauopathy and Alzheimer's disease. method.

[62] A method for diagnosing a 4R-tauopathy, comprising: measuring tau in a CSF or blood sample by any of the methods 6 to 18, wherein the tau to be measured is (a) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5, or a combination thereof; and (b) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof; and and diagnosing 4R-tauopathy when the quantified MTBR tau species differ by about 1.5 σ 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 / or Aβ42 / 40 measurement in CSF. method.

[63] 63. Any of the methods of 56 to 62, wherein the subject does not have dementia.

[64] 63. The method of claim 63, wherein the 4R-tauopathy is corticobasal degeneration, frontotemporal lobar degeneration, frontotemporal dementia, or progressive supranuclear palsy.

[65] 63. Any of the methods 56 to 62, wherein the subject has dementia.

[66] 65. The method of claim 65, wherein the 4R-tauopathy is corticobasal degeneration, frontotemporal lobar degeneration, frontotemporal dementia, or progressive supranuclear palsy.

[67] 1. A method of treating a subject in need thereof, comprising: providing a processed CSF or blood sample from a subject that is (a) depleted of mid-domain tau and (b) enriched for MTBR tau; quantitating in the treated sample MTBR tau species comprising the amino acid sequence of SEQ ID NO:2, MTBR tau species comprising the amino acid sequence of SEQ ID NO:3, MTBR tau species comprising the amino acid sequence of SEQ ID NO:4, MTBR tau species comprising the amino acid sequence of SEQ ID NO:5, MTBR tau species comprising the amino acid sequence of SEQ ID NO:6, MTBR tau species comprising the amino acid sequence of SEQ ID NO:7, MTBR tau species comprising the amino acid sequence of SEQ ID NO:8, MTBR tau species comprising the amino acid sequence of SEQ ID NO:9, or combinations thereof; and treating a subject to modify tau pathology, wherein treated CSF or blood samples from the subject have quantified MTRB tau species or a ratio of quantified MTRB tau species that differ by about 1.5 σ 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 / or Aβ42 / 40 measurement in CSF, and wherein the amount of quantified MTRB tau species or their ratio is representative of tau pathology in the subject's brain. method.

[68] 68. The method of claim 67, wherein the treatment alters or stabilizes the amount of the quantified MTBR species.

[69] Treatment may include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants, gamma-secretase inhibitors, beta-secretase inhibitors, anti-Aβ antibodies, anti-tau antibodies, anti-TREM2 antibodies, TREM2 agonists, stem cells, dietary supplements, serotonin receptor 6 antagonists, p38 alpha MAPK inhibitors, recombinant granulocyte-macrophage colony-stimulating factor, passive immunotherapy, active vaccines, tau protein aggregation inhibitors, treatments to improve glycemic control, 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 moieties 69. The method of claim 67 or 68, wherein the therapeutic agent is selected from the group consisting of an anti-inflammatory drug, ...

[70] 69. The method of claim 69, wherein the treatment is selected from the group consisting of 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, and a tau protein aggregation inhibitor.

[71] 71. The method of claim 70, wherein the kinase inhibitor is an inhibitor of a large number of amino acid kinases (TAOK), CDK, GSK-3p, MARK, CDK5 or Fyn.

[72] 71. The method of claim 70, wherein the phosphatase activator increases the activity of protein phosphatase 2A.

[73] 71. The method of claim 70, wherein the vaccine is CAD106 or AF20513.

[74] 71. The method of claim 70, wherein the tau protein aggregation inhibitor is TRx0237 or methylthioninium chloride.

[75] 70. The method of claim 70, wherein the anti-Aβ antibody is aducanumab. [Example]

[0190] The following examples illustrate various iterations of the present invention. It should be recognized by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present invention. However, those skilled in the art will recognize, based on the description herein, that changes can be made to the specific embodiments disclosed herein and still obtain like or similar results without departing from the spirit and scope of the present invention. Accordingly, all matter described or shown in the accompanying drawings should be interpreted in an illustrative and not a limiting sense.

[0191] Example 1 Several sample processing methods have been developed to enrich MTBR tau: immunoprecipitation of N-terminal tau and intermediate domain tau (IP) as described by Sato et al., 2018; chemical extraction (CX); and a combination of IP and CX methods (PostIP-CX). The CX and PostIP-CX methods were specifically developed for the detection and quantification of MTBR tau. An overview of these methods is shown in Figure 2.

[0192] Briefly, CSF (approximately 475 μL) was added as an internal standard. 15The mixture was mixed with a solution containing uniformly labeled N-tau441 (2N4R) (approximately 10 μL of a 100 pg / μL solution or approximately 5 μL of a 200 pg / μL solution). N-terminal tau and mid-domain tau species were immunoprecipitated with Tau1 and HJ8.5 antibodies, then processed and trypsin-digested as previously described (Sato et al., 2018).

[0193] For the CX method, CSF (approximately 475 μL) was used as an internal standard. 15 The mixture was mixed with a solution containing uniformly labeled N-tau441 (2N4R) (approximately 10 μL of a 100 pg / μL solution or approximately 5 μL of a 200 pg / μL solution). Tau was then chemically extracted. High-abundance CSF proteins were precipitated using 25 μL of perchloric acid. After mixing and incubation on ice for 15 minutes, the mixture was centrifuged at 20,000 g for 15 minutes at 4°C. The supernatant was further purified using an Oasis HLB 96 μElution Plate (Waters) according to the following steps: The plate was washed once with 300 μL of methanol and equilibrated once with 500 μL of 0.1% FA in water. The supernatant was added to an Oasis HLB 96 μElution Plate and allowed to adsorb to the solid phase. The solid phase was then washed once with 500 μL of 0.1% FA in water. Elution buffer (100 μL; 35% acetonitrile and 0.1% FA in water) was added, and the eluate was dried in a Speed-vac. The dried sample was dissolved in 50 μL of trypsin solution (10 ng / μL) in 50 mM TEABC and incubated at 37°C for 20 h.

[0194] For the PostIP-CX method, post-immunoprecipitation CSF (i.e., the supernatant remaining after the IP method above) was treated as described for the CX method.

[0195] After trypsin digestion, all samples were purified by solid-phase extraction using a C18 TopTip. During this purification process, 5 fmol each of the AQUA internal standard peptides, residues 354-369 (MTBR tau354) and 354-368 (tau368), was added for differential quantification. Before sample elution, 3% hydrogen peroxide and 3% FA in water were added to the beads, followed by overnight incubation at 4°C to oxidize methionine-containing peptides. The eluate was lyophilized and resuspended in 27.5 μL of 2% acetonitrile and 0.1% FA in water, followed by MS analysis on a nanoAcquity UPLC system coupled to an Orbitrap Fusion Lumos Tribrid or Orbitrap Tribrid Eclipse mass spectrometer (Thermo Scientific) operated in PRM mode.

[0196] As shown in Figure 3A, the CX and PostIP-CX methods resulted in samples containing MTBR tau that were detectable and quantifiable by mass spectrometry. Quantifiable signals for MTBR tau were not obtained with the IP method. Although unproven, it is believed that methods with similar sensitivity for detecting and quantifying MTBR tau may also be used.

[0197] Example 2 In this example, CSF samples from two clinical cohorts (LOAD100 and LOAD60) of subjects with late-onset Alzheimer's disease were analyzed. The Clinical Dementia Rating (CDR) scores and amyloid status of the samples used for this analysis are shown in Tables 1 and 2. CSF samples (approximately 500 μl each) were processed using the PostIP-CX method as described in Example 1 and evaluated by mass spectrometry. CSF Aβ42 and Aβ40 immunoprecipitation from CSF was measured by mass spectrometry as previously described (Patterson BW, et al., Ann Neurol 2015, 78: 439-453). pT217% was measured by mass spectrometry as previously described (Barthelemy, NR, et al., Alz Res Therapy, 2020, 12: 26).

[0198] The cutoff value for CSF Aβ42 / 40 was calculated from the PiB-PET SUVR results to determine amyloid status. Based on the established cutoff of >1.42 for PiB-PET SUVR (Ann Neurol 2016; 80:379-387), the (sensitivity% + specificity%) was maximized at 0.1389 for CSF Aβ42 / 40. Notably, pT217% showed excellent correlation with amyloid status as defined by the established cutoff, although with one outlier (Figure 4).

[0199] As shown in Figures 5–7, tau tryptic peptides associated with MTBR, measured in PostIP-CX samples, were significantly increased in amyloid-positive subjects. HVPG was most significantly increased, even in clinically asymptomatic subjects (Figures 5–6). Increases in HVPG and IGSL saturated after symptomatic onset, whereas LQTA continued to increase even after clinical onset (Figures 7–8). LQTA concentrations showed the highest correlation with tau pathology assessed by positron emission tomography (PET) (Pearson r = 0.84, n = 35) and cognitive test assessments (Figures 9–12). In some of the above analyses, data from amyloid-positive CDR1 and CDR2 samples were combined as CDRs > 1, and data from amyloid-negative CDR0.5 and CDR1 samples were combined as CDRs > 0.5. Statistical analysis was performed by one-way ANOVA adjusted for multiple comparisons using the Benjamin-Hochberg FDR method with the FDR set at 5%.

[0200] In particular, sample processing has been shown to affect the diagnostic utility of tau. As an example, the MTBR tau tryptic peptide HVPG distinguishes between amyloid-positive and amyloid-negative subjects in the presymptomatic stage in PostIP-CX samples, whereas this discriminatory power was not observed with the mid-domain tau tryptic peptide TPPS in IP samples (Figure 5). The amino acid sequence of the TPPS tryptic peptide is TPPSSGEPPK (SEQ ID NO: 10). Sample processing also significantly affected the ability to distinguish changes in MTBR tau abundance between various CSF samples. For example, the tryptic peptide LQTA showed a linear increase in post-symptomatic CSF samples in PostIP-CX samples but not in IP samples (Figure 13), indicating that PostIP-LQTA (mid-domain-independent MTBR tau243) distinguishes amyloid status better than IP-LQTA (Figure 14).

[0201] These data suggest that MTBR tau, which is enriched in AD brain aggregates, is also increased in AD CSF. It is hypothesized that the tryptic peptides LQTA and HVPG are part of the fuzzy coat, which are the starting point for tau fibril aggregation, respectively, while IGSL is located inside the core. The location of the LQTA peptide on the surface of the fibrils as the fuzzy coat increases its exposure, increasing the likelihood of its release into CSF. Given the role of HVPG in aggregation, immature fibrils still expose HPVG on their surface, while this peptide may be recruited to the core of mature fibrils. The location of IGSL in the fibril core may predict early AD stages.

[0202] Regardless of the underlying mechanism, the data suggest that the tryptic peptides HVPG and LQTA in CSF may be used as biomarkers to recapitulate amyloid status and tau pathology in AD, respectively. Notably, only LQTA showed a continuous increase throughout disease progression in terms of Tau-PET and amyloid status and cognitive decline, suggesting that this region is important for distinguishing tau pathology in AD. The use of these peptides in combination with the tryptic peptide IGSL and / or other biomarkers enhances the discriminatory power when staging a subject's disease trajectory (Figures 15-17). Furthermore, LQTA and other MTBR tau peptides may be used as biomarkers to distinguish between various tauopathies. [Table 2] [Table 3]

[0203] Example 3 This example details the existence and potential utility of MTBR tau species as Alzheimer's disease biomarkers. The results demonstrate that significant amounts of mid-domain-independent MTBR tau are present in CSF—i.e., tau species that are cleaved near the center of the polypeptide sequence (e.g., around amino acid 224 of tau441) resulting in a C-terminal fragment lacking the N-terminal and mid-domain regions ("C-terminal truncations"). Furthermore, various regions of CSF MTBR tau differentiate disease progression stages and correlate with tau aggregation in Alzheimer's disease brains. These findings provide new insights into the correlation between MTBR tau and CSF in the brain and support CSF tau as a biological biomarker for Alzheimer's disease.

[0204] Materials: Two distinct cohorts of human brain samples were used in the experiments in this example—a discovery cohort and a validation cohort. The discovery cohort included postmortem frozen brain tissue samples from two participants with Alzheimer's disease pathology and two control participants without pathology, provided by the Knight ADRC Pathology Core at Washington University School of Medicine. Each sample was classified by the National Institute on Aging and Alzheimer's Association as amyloid stage A3 (Thai phase) for amyloid deposition and Tau Braak stage VI, B3 for tau aggregation. Samples from each participant were collected from 6–10 brain regions, including the cerebellum, superior frontal gyrus, frontal pole, temporal lobe, occipital region, thalamus, amygdala, pons, parietal lobe, and striatum. As a validation cohort, additional postmortem frozen brain tissue samples from the parietal lobe of 20 participants (8 amyloid-negative and 12 amyloid-positive by CSF Aβ 42 / 40 ratio) were analyzed. The 12 amyloid-positive samples were further divided into clinical groups by Clinical Dementia Rating (CDR) score and classified as very mild to moderate Alzheimer's disease (amyloid-positive, CDR = 0.5-2, n = 5) or severe Alzheimer's disease (amyloid-positive, CDR = 3, n = 7). These human studies were approved by the Washington University Institutional Review Board.

[0205] Three different cohorts of human CSF samples were also used in the experiments in this example—a cross-sectional cohort, a longitudinal cohort, and a Tau-PET cohort (Tables 3A and 3B). CSF samples from 100 participants were collected from the Amyloid Beta (Aβ) Stable Isotope Labeling Kinetics (SILK) study (Patterson et al., 2015) for analysis as a cross-sectional cohort. This cohort is also referred to as the LOAD100 cohort in Example 2. CSF collection was performed as previously described (Patterson et al., 2015). Briefly, CSF was collected at baseline. Participants then received a leucine bolus infusion over 10 minutes. 6 mL of CSF was obtained every hour for 36 hours. The CSF aliquot collected at 30 hours was used for MS measurements of tau species in this study. Amyloid status was defined using the CSF Aβ 42 / 40 ratio, as previously reported (Patterson et al., 2015). The corresponding cutoff ratio (0.1389) maximized the predictive accuracy of amyloid positivity determined by Pittsburgh Compound B (PiB) PET. The amyloid group was further divided into clinical groups by CDR score, as shown in Table 3A. From the cross-sectional cohort, 28 participants (14 amyloid-positive and 14 amyloid-negative) were followed for 2–9 years to assess the longitudinal trajectory of tau species in CSF. CSF samples were collected and analyzed in the same manner as the cross-sectional cohort. The Tau-PET cohort consisted of 35 participants (20 amyloid-positive and 15 amyloid-negative, including 16 participants from the longitudinal cohort) who had Tau-PET AV-1451 normalized uptake value ratio (SUVR) measurements within 3 years of CSF collection. PET scans were performed as previously described ( Sato et al., 2018 ) and were performed for SUVR using partial volume effect-corrected regional diffuse function techniques ( Su et al., 2015 ). CSF samples were collected and analyzed in the same manner as the other cohorts. [Table 4] [Table 5]

[0206] Brain tau analysis by MS: Frozen brain tissue samples were sliced ​​using a cryostat at -20°C and collected into tubes. Tissue (300–400 mg) was sonicated at a concentration of 0.3 mg / μL in ice-cold buffer containing 25 mM tris-hydrochloride (pH 7.4), 150 mM sodium chloride, 10 mM ethylenediaminetetraacetic acid, 10 mM ethylene glycol tetraacetic acid, phosphatase inhibitor cocktail, and protease inhibitor cocktail. The homogenate was clarified by centrifugation at 11,000 g for 20 min at 4°C. The supernatant (whole brain extract) was aliquoted into new tubes and kept at -80°C until use. The whole brain extract was incubated with 1% sarkosyl for 60 min on ice, followed by ultracentrifugation at 100,000 g for 60 min at 4°C to obtain an insoluble pellet. The insoluble pellet was resuspended in 200 μL of PBS followed by sonication, and the insoluble suspension was kept at −80°C until use.

[0207] For soluble tau analysis, tau species in whole brain extracts were immunoprecipitated with Tau1 and HJ8.5 antibodies. Immunoprecipitated soluble tau species were treated and digested as previously described (Sato et al., 2018).

[0208] For insoluble tau analysis, the insoluble suspension (10–20 μL containing 2.5 μg of total protein) was mixed with 200 μL of lysis buffer (7 M urea, 2 M thio-urea, 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1.5% n-octylglucoside, 100 mM triethylammonium bicarbonate (TEABC)) followed by 100 μL of lysing buffer (7 M urea, 2 M thio-urea, 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1.5% n-octylglucoside, 100 mM triethylammonium bicarbonate (TEABC)) as an internal standard. 15Five microliters of a solution containing uniformly labeled N-tau441 (2N4R) (2 ng / μL, kindly provided by Dr. Guy Lippens, University of Lille, France) was added. Five microliters of 500 mM dithiothreitol was added to the suspension, followed by sonication. The resulting solution was mixed with 15 μL of 500 mM iodoacetamide and incubated for 30 minutes at room temperature in the dark. Protein digestion was performed using a previously reported filter-aided sample preparation method (Roberts et al., 2020). Briefly, each prepared solution was loaded onto a Nanosep 10K filter unit (PALL) and centrifuged. After washing the sample on the filter unit with 8 M urea in 100 mM TEABC solution, the immobilized proteins were digested on the filter using 0.25 μg of endoproteinase Lys-C for 60 minutes at 37°C. The samples were then further digested using 0.4 μg trypsin overnight at 37°C.

[0209] Digested samples (soluble and insoluble tau species) were collected by centrifugation and then desalted using a C18 TopTip (Glygen). During this purification process, 50 fmol each of AQUA internal standard peptides of residues 354-369 (MTBR tau354) and 354-368 (tau368) were added for differential quantification. Before sample elution, 3% hydrogen peroxide and 3% formic acid (FA) in water were added to the beads, followed by overnight incubation at 4°C to oxidize methionine-containing peptides. The eluate was lyophilized and resuspended in 27.5 μL of 2% acetonitrile and 0.1% FA in water, followed by MS analysis on a nanoAcquity UPLC system (Waters) coupled to an Orbitrap Fusion Tribrid or Orbitrap Tribrid Eclipse (Thermo Scientific) operated in parallel reaction monitoring (PRM) mode.

[0210] 16 brain tau peptides from both soluble and insoluble tau species were 15Quantification was performed by comparison of the corresponding isotopomer signals from N or AQUA internal standards (Table 4). Peptide profile comparison across brain samples was performed by normalizing each peptide amount by the mid-domain tau peptide (residues 181-190). [Table 6]

[0211] CSF tau analysis by MS: CSF (455 μL) was diluted with 10 μL of tau solution as an internal standard. 15 The mixture was mixed with a solution containing uniformly labeled N-tau 441 (2N4R) (100 pg / μL). Tau species consisting mainly of the N-terminal to mid-domain regions were immunoprecipitated with Tau1 and HJ8.5 antibodies. The immunoprecipitated tau species were treated and digested as previously described (Sato et al., 2018). Subsequently, 20 μL of 2N4R was added to the 2N4R solution. 15 N-tau internal standard (100 pg / μL) was added to the CSF after immunoprecipitation. Tau was then chemically extracted as previously reported (Barthelemy et al., 2016b), with some modifications. High-abundance CSF proteins were precipitated using 25 μL of perchloric acid. After mixing and incubation on ice for 15 minutes, the mixture was centrifuged at 20,000 g for 15 minutes at 4°C. The supernatant was further purified using an Oasis HLB 96 μElution Plate (Waters) according to the following steps: The plate was washed once with 300 μL of methanol and equilibrated once with 500 μL of 0.1% FA solution in water. The supernatant was added to the Oasis HLB 96 μElution Plate and allowed to adsorb to the solid phase. The solid phase was then washed once with 500 μL of 0.1% FA solution in water. Elution buffer (100 μL; 35% acetonitrile and 0.1% FA in water) was added, and the eluate was dried in a Speed-vac. The dried sample was dissolved in 50 μL of trypsin solution (10 ng / μL) in 50 mM TEABC and incubated at 37°C for 20 h.

[0212] After incubation of both the immunoprecipitated and chemically extracted samples, each tryptic digest was purified by solid-phase extraction using a C18 TopTip. During this purification process, 5 fmol of each of the AQUA internal standard peptides, residues 354-369 (MTBR tau 354) and 354-368 (tau 368), was added for differential quantification. Before sample elution, 3% hydrogen peroxide and 3% FA solution were added to the beads, followed by overnight incubation at 4°C to oxidize methionine-containing peptides. The eluate was lyophilized and resuspended in 27.5 μL of 2% acetonitrile and 0.1% FA solution, followed by MS analysis on a nanoAcquity UPLC system coupled to an Orbitrap Fusion Lumos Tribrid or Orbitrap Tribrid Eclipse mass spectrometer (Thermo Scientific) operated in PRM mode. Nineteen CSF tau peptides were quantified (Table 4). A schematic diagram of the CSF tau analysis procedure is shown in Figure 2.

[0213] Statistical Analysis: Differences in biomarker values ​​were assessed by one-way ANOVA unless otherwise noted. A two-sided p<0.05 was considered statistically significant and corrected for multiple comparisons using the Benjamin-Hochberg false positive rate (FDR) method with a 5% FDR setting (Benjamini and Hochberg, 1995). Spearman correlation was used to assess correlations between tau biomarkers and cognitive test measures and Tau-PET SUVR.

[0214] Results—Enrichment profiling of tau species in Alzheimer's disease brains: We hypothesized that tau aggregation in Alzheimer's disease brains would be reflected in the tau profile in CSF. Therefore, we first analyzed tau profiles in insoluble extracts from Alzheimer's disease and control brains (Figure 18B: discovery cohort) and then compared them with CSF tau profiles. Species containing residues 299–317 (MTBR tau 299) and 354–369 (MTBR tau 354), located between the R2 and R3 domains and within the R4 domain, respectively, were enriched 3–4-fold in insoluble extracts from Alzheimer's disease brains compared with controls. The upstream region of MTBR, encompassing residues 243–254 (MTBR tau 243), was also approximately threefold more abundant in Alzheimer's disease brains compared to controls, whereas species encompassing residues 260–267 and 275–280, located within the R1 and R2 domains, respectively, did not differ between Alzheimer's disease and control tissues. Other regions of tau were not enriched in Alzheimer's disease brains compared to controls. Notably, species encompassing residues 195–209 within the mid-domain were particularly low in Alzheimer's disease brains compared to controls, potentially resulting from extensive hyperphosphorylation occurring at residues 199, 202, 205, and 208 in insoluble tau aggregates (Malia et al., 2016). Notably, no changes were observed in the identified MTBR tau species in soluble tau (whole brain extracts) between control and Alzheimer's disease brains (Figure 23A). These results were replicated in brain samples from control (amyloid negative, n=8), very mild to moderate Alzheimer's disease (amyloid positive, CDR=0.5-2, n=5), and severe Alzheimer's disease (amyloid positive, CDR=3, n=7) participants (Figure 18C and Figure 23B: validation cohort), suggesting that MTBR tau 243, 299, and 354 species were specifically enriched in insoluble tau aggregates throughout disease progression.

[0215] Next, we tested the recently reported truncated tau368 (residues 354–368) species produced by asparagine endopeptidase (Zhang et al., 2014; Blennow et al., 2020) against its counterpart, the uncleaved MTBR tau354, and quantified both species in insoluble brain extracts (Figure 24). We found a high correlation between tau368 and MTBR tau354 (r = 0.9783), suggesting that truncation at residue 368 occurs at similar rates at different stages of brain pathology.

[0216] Results—Quantification of MTBR tau in CSF: To determine whether MTBR tau enrichment in Alzheimer's disease brain aggregates correlates with the levels of soluble tau species in CSF, we developed a method to analyze MTBR tau in CSF. The method involves chemical extraction of tau in CSF after immunoprecipitation (Tau1 / FIJ8.5), followed by MS analysis (Figure 2A). This method yielded sufficient recovery for MTBR peptide quantification (Figure 25). Tau peptide abundance recovered by the Tau1 / FIJ8.5 immunoprecipitation method prior to chemical extraction dramatically decreased after residue 222 (Sato et al., 2018). In contrast, the MTBR tau species concentration quantified by the PostIP-CX method was relatively low compared to the N-terminal to mid-domain region, but was still comparable to other regions of tau immunoprecipitated (Figure 19). CSF concentrations (calculated as the sum of values ​​from immunoprecipitation and chemical extraction methods) in normal control participants ranged from 8.2 to 32.0 ng / mL for mid-domain species (residues 151–155, 181–190, 195–209, and 212–221), 0.4 to 3.7 ng / mL for MTBR tau species (residues 243–254, 260–267, 275–280, 282–290, 299–317, and 354–369), and 6.5 to 5.1 ng / mL for non-MTBR C-terminal tau species (residues 386–395 and 396–406). The CSF concentrations of C-terminally-containing truncated tau species are in a range similar to those containing the mid-domain (residues 195–209 and 212–221), suggesting that the C-terminal side of tau is also cleaved in neuronal cells and secreted extracellularly in the same manner as N-terminal to mid-domain tau (Sato et al., 2018).

[0217] Results - CSF MTBR tau in an Alzheimer's disease cross-sectional cohort: To determine whether MTBR-containing species present in the extracellular space reflect Alzheimer's disease-related changes, CSF was analyzed in a cross-sectional cohort of amyloid-negative and amyloid-positive participants at various clinical stages: amyloid-negative CDR=0 (controls, n=30), amyloid-positive CDR=0 (pre-symptomatic AD, n=18), amyloid-positive CDR=0.5 (very mild AD, n=28), amyloid-positive CDR>1 (mild-moderate AD, n=12), and amyloid-negative CDR>0.5 (non-AD cognitive impairment, n=12).

[0218] First, we examined the CSF levels of three MTBR tau species (MTBR tau243, MTBR tau299, and MTBR tau354) that are specifically enriched in Alzheimer's disease brains (Figure 20). All three species were present in both Alzheimer's disease and control CSF, and levels were higher in the amyloid-positive group compared to the control group, even at the asymptomatic stage (CDR=0) (MTBR tau243 p=0.0170, MTBR tau299 p=0.0002, and MTBR tau354 p=0.0076). Notably, these species had distinct CSF characteristics after clinical disease onset. MTBR tau299 levels were 204% higher in presymptomatic AD compared with controls but saturated during very mild AD (CDR = 0.5) and mild-to-moderate AD (CDR > 1) (p = 0.2541), whereas MTBR tau354 levels were significantly lower in samples collected after symptom onset (p = 0.0345). In contrast, MTBR tau243 levels progressively increased across all disease stages, including after symptom onset (p = 0.0025). These results suggest that regional specificity within MTBR tau species can be discerned across various AD stages and that MTBR tau243 is a good AD stage-specific marker.

[0219] Next, we tested whether CSF MTBR tau species offer increased sensitivity and specificity for Alzheimer's disease staging compared with tau species containing other regions. Multiple species containing the N-terminal, mid-domain, MTBR, and C-terminal domains were quantified using region-specific methods (Figures 26, 27, and 28). N-terminal and mid-domain species were quantified by immunoprecipitation (IP method) and post-IP-CSF chemical extraction (PostIP-CX method), whereas the C-terminal species MTBR was quantified only by post-IP-CSF chemical extraction (PostIP-CX method) because immunoprecipitation did not yield a quantifiable signal. Levels of species containing the N-terminal domain quantified by the immunoprecipitation method did not differ between controls and asymptomatic patients (except for residues 6–23, p = 0.0362) or other nearby disease stages. Intermediate domain species levels by immunoprecipitation methods were significantly higher in the asymptomatic amyloid stage than in controls (except for residues 212-221, p = 0.0762), but the effective sizes were relatively modest (123%-168% vs. controls) compared with MTBR tau species (e.g., MTBR tau 299 levels were >200% greater than controls in the presymptomatic AD stage) and did not differ from later disease stages. Regardless of extraction method, MTBR tau 243, 299, and 354 species showed significant differences between controls and disease stages compared with N-terminal to intermediate domain species (residues 6-23 to 226-230, Figure 28). Profiles from other species containing the C-terminal domain to MTBR (residues 260–267, 275–280, 282–290, 386–395, and 396–406) were similar to the intermediate domain species and were not specific to the stage of Alzheimer's disease clinical dementia.

[0220] In summary, three representative species containing MTBR (MTBR tau243, MTBR tau299, and MTBR tau354) enriched in Alzheimer's disease brains (Figure 18) shared similar characteristics in CSF, with MTBR tau243 showing the highest specificity for Alzheimer's disease dementia stages. Notably, a high correlation was observed between the cleaved form of tau368 and the uncleaved form of MTBR tau354 in CSF (r = 0.8382) (Figure 29). MTBR tau243 was the only species that reliably distinguished the clinical stages of Alzheimer's disease.

[0221] Results—Mid-domain-independent MTBR tau243 as a specific biomarker for Alzheimer's disease staging: The gradual increase in levels of MTBR tau-243 species across the stages of Alzheimer's disease clinical dementia suggests that it may be a reliable predictor of disease progression. Next, we examined which MTBR tau species (MTBR tau-243, MTBR tau-299, and MTBR tau-354) had the highest correlation with cognitive test results, such as CDR-Box Sum (CDR-SB) and the Mini-Mental State Examination (MMSE). The mid-domain-independent MTBR tau-243 species in the amyloid-positive group was found to be highly correlated with both CDR-SB and MMSE (r=0.5562, p<0.0001 and r=-0.5433, p<0.0001, respectively) (Figures 30 and 31). Other species levels correlated much less or not significantly with cognitive tests (Table 5), suggesting that CSF MTBR tau243 specifically discriminates clinical stage and global disease progression through the progression from asymptomatic stages to clinical stages of Alzheimer's disease. [Table 7]

[0222] Results—CSF MTBR tau in the Alzheimer's Disease Longitudinal Cohort: A subset of participants (n=28) from the cross-sectional cohort was followed for 2–9 years to measure the longitudinal trajectory of MTBR tau in CSF (Table 6). MTBR tau species enriched in Alzheimer's disease brains (MTBR tau243, MTBR tau299, and MTBR tau354) significantly increased over time in the amyloid-positive group (p<0.01 by two-tailed paired t-test between visits 1 and 2), but not in the amyloid-negative group (Figure 32). The amyloid-negative group also showed a slight longitudinal increase in MTBR tau243, although it was lower than that observed in the amyloid-positive group (mean difference = 0.4926 and 2.208 for the amyloid-negative and amyloid-positive groups, respectively).

[0223] Figure 21 shows the longitudinal rate of change in MTBR tau species concentrations in individual participants. Notably, one participant (Participant A) with the highest CDR after disease onset (CDR = 1 to 2 over 7 years) showed specific trajectory profiles for each MTBR tau species. MTBR tau 243 continued to increase even in mild (CDR = 1) to moderate (CDR = 2), whereas MTBR tau 299 and MTBR tau 354 decreased in this participant's CSF after mild AD. Other participants in the amyloid-positive group were diagnosed with preclinical or very mild AD (CDR = 0 or 0.5, respectively) at the first visit, and the increasing trends in the various levels were observed in the majority of participants, supporting findings from the cross-sectional cohort. [Table 8]

[0224] Results - Correlation with Tau-PET Imaging: Tau pathology measured by Tau-PET scanning strongly correlated with cognitive decline and clinical stage of Alzheimer's disease (Arriagada et al., 1992; Johnson et al., 2016; Ossenkoppele et al., 2016; Bejanin et al., 2017; Jack et al., 2018; Gordon et al., 2019). We next tested whether MTBR tau in CSF correlated with brain tau pathology assessed by Tau-PET (Figure 22). The middomain-independent MTBR tau243 significantly correlated with Tau-PET SUVR (r = 0.7588, p < 0.0001), whereas the correlations for MTBR tau299 and MTBR tau354 were much lower (r = 0.4584, p = 0.0056 and r = 0.4375, p = 0.0086, respectively). Tau species containing residues 226–230 also showed a high correlation with Tau-PET SUVR (r = 0.6248, p < 0.0001, Table 7), but this was lower than that observed for MTBR tau243. This indicates that CSF MTBR tau243 and surrounding regions may be surrogate biomarkers for tau aggregation in the brain. The ability to specifically and quantitatively track tau pathology in the brain is a highly sought-after biomarker for Alzheimer's disease clinical trials. [Table 9]

[0225] Discussion: The MTBR region of tau has been primarily studied in brain aggregates but not extensively in CSF. In this study, we demonstrated the presence and quantification of the MTBR region of tau in CSF samples from human participants using a sensitive and antibody-independent method for analyzing CSF tau. Previous studies utilizing antibody-dependent assays (Meredith et al., 2013; Sato et al., 2018) may have failed to detect MTBR-containing tau species in CSF due to assay limitations, including antibody specificity or sensitivity or the ability to recover forms that may be selected by MTBR species in CSF. Alternatively, MTBR-tau can be cleaved by various proteases to produce fragments that are not detected by conventional immunoassays or immunoprecipitation followed by MS assays (Gamblin et al., 2003; Cotman et al., 2005; Zhang et al., 2014; Zhao et al., 2016; Chen et al., 2018; Quinn et al., 2018). Surprisingly, in this study, using the PostIP-CX method followed by mass spectrometry, robust concentrations of MTBR-tau species were measured, approximately 1%-10% compared to mid-domain tau species (Figure 19 and Figure 2A).

[0226] To date, it was unclear whether MTBR-tau contributes to extracellular tau proliferation, as extracellular levels were thought to be too low for pathology seeding and spread. These new findings on the stoichiometry of MTBR in CSF support the hypothesis that MTBR-containing species diffuse extracellularly, similar to pathological species. These measurements provide information on potential targets for anti-tau drug development for AD and provide a quantitative measure of targeting, as demonstrated by a 2- to 3-fold increase in MTBR-tau species in CSF from AD patients. However, a limitation is that pathological species may reside in interstitial fluid (ISF) rather than CSF (Colin et al., 2020). Although several reports have shown that CSF tau is primarily derived from ISF ( Reiber, 2001 ) and that human CSF from Alzheimer's disease patients can induce tau seeding in transgenic mouse models ( Skachkova et al., 2019 ), further studies are needed to clarify whether tau species detected in CSF reflect pathological tau that can propagate in the human brain.

[0227] Previous studies have shown that inoculation of Alzheimer's disease brain tau aggregates into mouse brains induced severe tau pathology (Guo et al., 2016; Narasimhan et al., 2017); however, no reports have identified pathological tau species in the extracellular space that are also linked to disease progression in humans. This led to the examination of CSF MTBR tau species changes in Alzheimer's disease and the exploration of their suitability as novel Alzheimer's disease biomarkers. CSF MTBR tau levels were found to be elevated in Alzheimer's disease and correspond to species enriched in the insoluble fraction of Alzheimer's disease brain. The finding that CSF MTBR tau correlates with Alzheimer's disease clinical stage and tau pathology suggests that MTBR tau is associated with tau proliferation in Alzheimer's disease; however, the nature (i.e., monomeric, oligomeric, or fibrillar species) and origin of extracellular CSF MTBR tau remain unknown. It is possible that CSF MTBR tau may originate from brain aggregates or neurons actively secreting monomeric species, and further studies should be designed to address this issue.

[0228] Interestingly, the trajectories of changes in CSF MTBR tau species were found to differ across different regions of the MTBR and across clinical stages of Alzheimer's disease. We hypothesize that this finding is due to structural changes in tau determined by recent cryo-EM findings. Cryo-EM analysis suggests an ordered β-sheet core of tau aggregates beginning at residue 306 (Fitzpatrick et al., 2017). Thus, MTBR tau 354 (including residues 354–369), MTBR tau 299 (including residues 299–317), and MTBR tau 243 (including residues 243–254) represent the interior, border, and exterior of the fibril core, respectively. In contrast to both MTBR tau 354 and MTBR tau 299, MTBR tau 243 levels gradually increased across all disease stages. MTBR tau243 and neighboring regions (i.e., residues 226–230) levels in CSF also highly correlated with Tau-PET SUVR performance (Figure 22 and Table 7), supporting the hypothesis that MTBR tau243 and possibly neighboring regions are deposited in brain tau aggregates and secreted extracellularly (Figure 17).

[0229] The discovery that MTBR tau is highly correlated with Alzheimer's disease pathology and clinical progression provides important insights into promising targets for therapeutic anti-tau drugs to treat tauopathies. For example, a novel tau antibody recognizing an epitope in the upstream region of MTBR (residues 235–250) exhibits significant and selective ability to attenuate tau dissemination from Alzheimer's disease and progressive supranuclear palsy brains in cell-based assays (Courade et al., 2018). These findings indicate that the upstream region of MTBR associates with extracellular, pathological tau. This is supported by the antibody's ability to attenuate tau pathological propagation to distal brain regions in transgenic mice injected with human Alzheimer's disease brain extracts (Albert et al., 2019). Other novel tau antibodies that recognize epitopes in the upstream region of MTBR (residues 249–258) show reduced induction of tau pathology in cells and in in vivo transgenic mouse models seeded with human Alzheimer's disease brain extracts (Vandermeeren et al., 2018). Antibodies targeting MTBR tau 299 and MTBR tau 354 species also attenuated tau pathology induced by seeding with P301L tau or Alzheimer's disease brain extracts (Weisova et al., 2019; Roberts et al., 2020), supporting the hypothesis that species containing specific regions of MTBR are responsible for spreading tau pathology in tauopathies.

[0230] Taken together, our findings demonstrate that MTBR-tau species in CSF exist as C-terminal fragments, are specifically increased in Alzheimer's disease, and mirror the enrichment seen in Alzheimer's disease brain aggregates. These findings suggest that specific MTBR-containing species (MTBR-tau299 and MTBR-tau243) are promising CSF biomarkers for assessing amyloid and tau pathology in Alzheimer's disease. In particular, the middomain-independent MTBR-tau243 parallels disease progression and tau pathology in Alzheimer's disease, potentially serving as a biomarker for tau pathology and a target for novel anti-tau antibody therapeutics.

[0231] Example 4 An additional sample processing method, termed "PostIP-IP," was developed and compared to the PostIP-CX method described in Examples 1 and 2. An exemplary workflow of the PostIP-IP method is shown in FIG.

[0232] CSF samples from the LOAD100 cohort described in Example 2 were processed by the PostIP-CX method (Example 1) or the PostIP-IP method (this example) and then analyzed by LC-MS as generally described in Example 2.

[0233] As shown in Figure 34A, the tryptic peptide LQTA exhibited different profiles between the two samples. For example, the amount of LQTA, which continuously increased even with clinical onset, measured in samples treated with the PostIP-CX method was not observed in samples treated with the PostIP-IP method. In contrast, the tryptic peptides HVPG and IGSL exhibited similar profiles between samples treated with the PostIP-CX and PostIP-IP methods (Figures 34B and 34C). Further analysis of additional tryptic peptides suggests that there may be a significant cleavage event occurring at R1 within the amino acid sequence between the LQTA and IGST peptides (Figure 35A).

[0234] The abundance of all tryptic peptides downstream of LQTA (i.e., C-terminus) showed good correlation between sample processing methods (R 2 Based on the R values ​​(see Figure 35), the R 2There was a significant increase in values. To explore this further, samples were grouped by CDR score—more specifically, cognitively impaired subjects (C1, CDR>0.5) and cognitively non-impaired subjects (CDR<0.5). As shown in Figure 36B, only cognitively impaired subjects showed a low correlation between samples processed with the PostIP-CX versus PostIP-IP method for HVPG and IGSL tryptic peptides. This may reflect the development of tau aggregates in the brain, which are recruited to tau regions containing HVPG and IGSL tryptic peptides in aggregates, thereby leading to changes in the amounts of tau species containing these peptides in CSF and other body fluids.

[0235] Overall, these data indicate that the choice of sample processing method influences the ability to detect MTBR tau species in CSF and other body fluids that recapitulate tau pathology in the CNS.

[0236] Example 5 In this example, CSF samples from subjects from three clinical cohorts were processed by the IP method (Example 1) and the PostIP-IP method (Example 4) and evaluated by mass spectrometry as generally described in Example 3. Samples were obtained from control subjects (n=93), subjects with amyloid-positive AD (n=41), and subjects with non-AD tauopathies (n=87). Subjects with non-AD tauopathies were clinically diagnosed with CBD or CBD / PSP (n=20), FTD (n=29), FTLD (R406W n=7, P301L n=3), PSP (n=18), and undetermined non-AD dementia (n=3). Tryptic peptides specific for the 3R and 4R isoforms were of particular interest. CSF Aβ42 / 40 was measured by mass spectrometry as generally described in Ovod et al., Alzheimers Dement J. Alzheimers Assoc., 2017, 13:841-849. Amyloid status was defined using a cutoff value of 0.085 (i.e., amyloid positive >0.085, amyloid negative <0.085). pT217% was measured by mass spectrometry as previously described.

[0237] As shown in Figure 37, the tryptic peptide VQIV / LDLS ratio in samples distinguishes between non-AD tauopathy and controls in samples treated with the PostIP-IP method (Figure 37C), but not the IP method (Figure 37B). Further analysis of samples treated with the PostIP-IP method showed that LDLS was less abundant in CSF from subjects with non-AD tauopathy compared to control subjects (Figure 38).

[0238] Increased VQIV / LDLS ratios were measured in PostIP-IP-treated samples from subjects with non-AD tauopathies, but not in samples from subjects with AD or control subjects (Figure 39). Further analysis of tryptic peptides after PostIP-IP sample processing identified a lower correlation between R1-R2 tryptic peptides (e.g., IGST, VQII, LDLS, etc.) and late R2-R3 tryptic peptides (e.g., HVPG, etc.) in subjects with non-AD tauopathies compared with subjects with AD or control subjects (Figure 40, Table 8). When comparing between non-AD tauopathies, certain subjects with PSP, CBD, and FTD were outliers (Figure 41). Similar results were obtained when comparing the tryptic peptide IGSL, but not HPVG (Figure 42).

[0239] Overall, these data suggest that the CSF tau profile measured after PostIP-IP sample processing reflects the brain tau aggregation state. For example, 4R-tauopathy contains brain insoluble tau enriched in the R2 region, including the VQII tryptic peptide, and some CSF samples from subjects with 4R-tauopathy showed reduced amounts of the tryptic peptides IGST, VQII, and LDLS relative to HVPG or IGSL, which was not observed in subjects with AD. Based on these data, methods for identifying 4R-tauopathy should focus on enrichment of the R2 region of MTBR tau. [Table 10]

[0240] Example 6 In this example, further analysis of CSF and brain samples from a single clinical cohort of subjects with non-AD tauopathy provided further evidence of the usefulness of intermediate domain-independent MTBR tau for distinguishing CSF samples from subjects with non-AD tauopathy and those with AD. The subjects in this cohort included subjects with AD (n=28), subjects clinically diagnosed with CBD or CBD / PSP (n=20), subjects clinically diagnosed with FTD (n=22), and subjects clinically diagnosed with PSP (n=11). CSF samples from these subjects were processed using the PostIP-IP method generally described in Example 4 and evaluated by mass spectrometry generally described in Example 3. Brain insoluble tau was evaluated as described in Example 3.

[0241] As described in Example 5, analysis of tryptic peptides after processing of CSF PostIP-IP samples identified a low correlation between R1 and early R2 tryptic peptides (e.g., IGST, VQII, LDLS, etc.) and late R2, R3, and / or R4 tryptic peptides (e.g., IGSL, etc.) in subjects with non-AD tauopathies (Figures 43, 44, and 45). It was hypothesized that in CSF, tau species from non-AD tauopathies contain (1) fewer R1 and R2 and (2) more R3 and R4 than tau species from AD, and that this is indicative of brain tau deposition (Figure 46). To test this hypothesis, brain insoluble tau was analyzed. As shown in Figure 47, the tryptic peptide VQII is enriched in brain tau aggregates from 4R-tauopathies. The tryptic peptides FIVPG and IGSL are also enriched in brain tau aggregates, but at lower abundance compared to AD. These data further support the ratio of R1 or R2 to R3 or R4 as a way to distinguish AD from non-AD tauopathies.

[0242] The analysis was then further expanded to include CSF samples from genetically confirmed FTLD cases (R406W, n=7; P301L, n=3), control subjects (n=44), and additional CSF samples from subjects with AD (n=41). CSF samples from these subjects were processed using the PostIP-IP method generally described in Example 4 and evaluated by mass spectrometry generally described in Example 3. Analysis of tryptic peptides after CSF PostIP-IP sample processing similarly identified a low correlation between R1 and early R2 tryptic peptides (e.g., IGST, VQII, LDLS, etc.) and late R2, R3, and / or R4 tryptic peptides (e.g., IGSL, etc.) in subjects with non-AD tauopathies (Figures 48 and 49). These data confirm the use of the ratio of R1 or R2 to R3 or R4 abundance as a method for distinguishing AD from non-AD tauopathies and also demonstrate the ability to distinguish non-AD tauopathies from control subjects. In particular, CSF samples obtained from genetically confirmed FTLD cases further rigorize the analysis.

Claims

1. 1. A method for measuring tau in a biological sample, comprising: (a) providing a biological sample selected from a blood sample or a CSF sample; (b) removing proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid phase extraction; (d) cleaving the purified tau with a protease to obtain a sample containing proteolytic peptides of tau; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect and quantify at least one of the tau proteolytic peptides; method.

2. 2. The method of claim 1, wherein the biological sample is depleted of amyloid beta, N-terminal tau, mid-domain tau, or any combination thereof.

3. 3. The method of claim 2, wherein (i) the biological sample is depleted of amyloid beta, N-terminal tau and mid-domain tau, (ii) the biological sample is depleted of N-terminal tau and mid-domain tau, or (iii) the biological sample is depleted of mid-domain tau.

4. 2. The method of claim 1, wherein the solid phase in step (c) comprises a reversed-phase adsorbent that adsorbs tau.

5. step (b) comprises mixing an acid to precipitate proteins in the biological sample; and / or In step (e), liquid chromatography-mass spectrometry is carried out by a nano-LC / MS system; The method of any one of claims 1 to 4.

6. 1. A method for measuring tau in a biological sample, comprising: (a) reducing N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau in a biological sample by affinity depletion, wherein the biological sample is a blood sample or a CSF sample; (b) enriching MTBR tau by a method comprising (i) removing additional proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, and then purifying tau from the supernatant by solid phase extraction, or (ii) affinity purifying MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) cleaving the enriched MTBR tau with a protease to obtain a sample containing proteolytic peptides of tau; and (d) performing liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides to detect and quantify at least one tau proteolytic peptide; method.

7. 7. The method of claim 6, wherein step (a) comprises reducing (i) N-terminal tau, mid-domain tau, or N-terminal tau and mid-domain tau, and (ii) amyloid beta.

8. the biological sample comprises human tau, and wherein step (a) further comprises: contacting the biological sample with at least one epitope binding agent that specifically binds to an epitope within amino acids 1 to 243 (inclusive) of Tau 441; or contacting the biological sample with a first epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive) of Tau 441 and a second epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of Tau 441; or contacting the biological sample with a first epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive) of Tau 441, a second epitope that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of Tau 441, and a third epitope binding agent that specifically binds to an epitope of amyloid beta; or contacting the biological sample with a first epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive) of Tau 441 and a second epitope binding agent that specifically binds to an epitope of amyloid beta; or contacting the biological sample with a first epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of Tau 441 and a third epitope binding agent that specifically binds to an epitope of amyloid beta; The method of claim 6.

9. The method of claim 8, wherein the epitope binding agent that specifically binds to amyloid beta is HJ5.

1.

10. 9. The method of claim 8, wherein the epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive) of Tau441 is HJ8.

5.

11. 9. The method of claim 8, wherein the epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of Tau441 is Tau1.

12. 12. The method of any of claims 6 to 11, wherein the solid phase extraction comprises a reversed phase adsorbent that adsorbs tau.

13. a method for enriching MTBR tau comprising step (b)(i), wherein protein precipitation comprises mixing the biological sample with an acid to precipitate proteins; and / or In step (d), liquid chromatography-mass spectrometry is carried out by a nano-LC / MS system; The method of any one of claims 6 to 11.

14. 14. The method of claim 13, wherein the solid phase extraction performed in step (b) comprises a reversed phase adsorbent that adsorbs tau.

15. a method for enriching MTBR tau comprising step (b)(ii), wherein affinity purification of MTBR tau comprises contacting the product of step (a) with an epitope binding agent that specifically binds to an epitope that is C-terminal to the epitope of step (a); and / or In step (d), liquid chromatography-mass spectrometry is carried out by a nano-LC / MS system; The method of any one of claims 8 to 11.

16. The epitope binding agent of step (b) within amino acids 221 to 441 (inclusive) of Tau441; or within amino acids 235 to 441 (inclusive) of Tau441; or within amino acids 235 to 368 (inclusive) of Tau441; or within amino acids 244 to 368 (inclusive) of Tau441; or Within amino acids 244 to 299 (inclusive) of Tau441 16. The method of claim 15, wherein the antibody specifically binds to an epitope of

17. 17. The method of claim 16, wherein the epitope binding agent is an antibody selected from the group consisting of 77G7, RD3, RD4, UCB1017, PT76, E2814 and 7G6.

18. 18. The method of claim 15, 16 or 17, wherein step (c) comprises desalting the resulting cleavage products by solid phase extraction, the solid phase extraction comprising a reversed phase adsorbent that adsorbs tau.

19. The method of any one of claims 1 to 5, wherein the protease is trypsin.

20. 20. The method of claim 19, wherein step (d) comprises detecting and quantifying at least one proteolytic peptide of tau, wherein the proteolytic peptide of tau has an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:

9.

21. 20. The method of claim 19, wherein step (d) comprises detecting and measuring the concentration of at least two tau proteolytic peptides, wherein the two tau proteolytic peptides have amino acid sequences selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:

9.

22. 22. The method of claim 21, wherein the at least two tau proteolytic peptides have the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:6, SEQ ID NO:3 and SEQ ID NO:7, SEQ ID NO:3 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:8, SEQ ID NO:4 and SEQ ID NO:7, SEQ ID NO:4 and SEQ ID NO:8, SEQ ID NO:5 and SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:6, SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:9 and SEQ ID NO:

5.

23. The method of any one of claims 6 to 11, wherein the protease is trypsin.

24. 24. The method of claim 23, wherein step (d) comprises detecting and measuring the concentration of at least one proteolytic peptide of MTBR tau, wherein the proteolytic peptide of tau has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:

9.

25. 24. The method of claim 23, wherein step (d) comprises detecting and measuring the concentration of at least two proteolytic peptides of MTBR tau, wherein the two tau proteolytic peptides have amino acid sequences selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:

9.

26. The method of claim 25, wherein the at least two proteolytic peptides of MTBR tau have the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:6, SEQ ID NO:3 and SEQ ID NO:7, SEQ ID NO:3 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:8, SEQ ID NO:4 and SEQ ID NO:7, SEQ ID NO:4 and SEQ ID NO:8, SEQ ID NO:5 and SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:6, SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:9 and SEQ ID NO:

5.

27. 13. The method of claim 12, wherein the protease is trypsin.

28. 14. The method of claim 13, wherein the protease is trypsin.

29. 16. The method of claim 15, wherein the protease is trypsin.

30. 30. The method of any of claims 27, 28 or 29, wherein step (d) comprises detecting and measuring the concentration of at least one proteolytic peptide of MTBR tau, wherein the proteolytic peptide of tau has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:

9.

31. 30. The method of any of claims 27, 28 or 29, wherein step (d) comprises detecting and measuring the concentration of at least two proteolytic peptides of MTBR tau, wherein the proteolytic peptides of tau have amino acid sequences selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:

9.

32. The method of claim 31, wherein the at least two proteolytic peptides of MTBR tau have the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:6 and SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:7, SEQ ID NO:3 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:7, SEQ ID NO:2 and SEQ ID NO:8, SEQ ID NO:4 and SEQ ID NO:7, SEQ ID NO:4 and SEQ ID NO:8, SEQ ID NO:5 and SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:6, SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:9 and SEQ ID NO:

5.

33. 33. The method of any of claims 6 to 32, wherein the method further comprises detecting and measuring the concentration of N-terminal tau, mid-domain tau or amyloid beta removed from the biological sample in step (a).

34. A method for measuring Alzheimer's disease (AD)-related pathology in a subject, comprising measuring tau in a CSF or blood sample by the method of any of claims 6 to 18, wherein the tau to be measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof, and wherein the amount of the measured MTBR tau species or the ratio thereof represents AD-related pathology in the subject's brain. method.

35. 35. The method of claim 34, wherein the AD-related pathology is tau deposition in the brain of the subject.

36. 35. The method of claim 34, wherein the AD-related pathology is amyloid beta deposits in the subject's brain or cerebral arteries.

37. A method for measuring Alzheimer's disease (AD)-associated tau deposits in the brain of a subject, comprising measuring tau in a CSF or blood sample by the method of any of claims 6 to 18, wherein the tau measured is an MTRB tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), and wherein the amount of the measured MTRB tau species represents AD-associated tau deposits in the subject's brain. method.

38. A method for measuring Alzheimer's disease (AD)-associated tau deposits in the brain of a subject, comprising measuring tau in a CSF or blood sample by the method of any of claims 6 to 18, wherein the tau to be measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof, and wherein the amount of the measured MTBR tau species or the ratio thereof represents AD-associated tau deposition in the brain of the subject. method.

39. 1. A method for aiding in the diagnosis of Alzheimer's disease, comprising: The method of any one of claims 6 to 18, comprising measuring tau in a CSF or blood sample, wherein the tau measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof; wherein a difference of 1.5σ or more in the quantified MTBR tau species is indicative of Alzheimer's disease, where σ is the standard deviation defined by a normal distribution measured in a control population that is amyloid-negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF; method.

40. 1. A method for aiding in measuring Alzheimer's disease (AD) progression in a subject, comprising:

19. Measuring tau in the first and second CSF or blood samples by the method of any one of claims 6 to 18, wherein the tau measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof; and calculating a difference between the quantified MTBR tau species in the second sample and the first sample, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates progression of Alzheimer's disease in the subject; method.

41. 41. The method of any of claims 37 to 40, wherein the subject is amyloid-negative.

42. 42. The method of claim 41, wherein the subject does not have dementia.

43. 42. The method of claim 41, wherein the subject has dementia.

44. 41. The method of any of claims 37 to 40, wherein the subject is amyloid positive.

45. 45. The method of claim 44, wherein the subject does not have dementia.

46. 45. The method of claim 44, wherein the subject has dementia.

47. 45. The method of claim 41 or claim 44, wherein the subject has a CDR score of 0.5 to 1.

0.

48. 45. The method of claim 41 or claim 44, wherein the subject has a CDR score of >1.0 to 2.0 (moderate AD).

49. 45. The method of claim 41 or claim 44, wherein the subject has a CDR score of >2.

0.

50. 50. The method of any of claims 37 to 49, further comprising amyloid beta quantification, N-terminal tau quantification, mid-domain tau quantification, tau post-translational modification quantification or ApoE isoform identification in a biological or CSF sample.

51. 20. A method for measuring tau pathology in the brain of a subject, comprising measuring tau in a CSF or blood sample by the method of any of claims 6 to 18, wherein the tau measured is an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 9, or a combination thereof, wherein the amount of the quantified MTBR tau species or the ratio thereof represents tau pathology in the brain of the subject. method.

52. A method for assisting in the identification of a 4R-tauopathy, comprising measuring tau in a biological sample by the method of any of claims 6 to 17, wherein the tau measured is (a) an MTBR tau species comprising the amino acid sequence of SEQ ID NO:9 and (b) an MTBR tau species comprising the amino acid sequence of SEQ ID NO:4, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:5, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO:7, or an MTBR tau species comprising the amino acid sequence of SEQ ID NO:8; wherein the ratio of the quantified MTBR species in (a) to the quantified MTBR species in (b) aids in the identification of a 4R-tauopathy. method.

53. A method for assisting in the discrimination between 3R-tauopathy or 4R-tauopathy and Alzheimer's disease, comprising measuring tau in a biological sample by the method of any one of claims 6 to 18, wherein the tau measured is (a) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5, or a combination thereof; and (b) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof; wherein the ratio of the quantified MTBR species in (a) to the quantified MTBR species in (b) aids in distinguishing between a 3R-tauopathy or a 4R-tauopathy and Alzheimer's disease. method.

54. A method for aiding in the diagnosis of a 4R-tauopathy, comprising: The method of any one of claims 6 to 18 includes measuring tau in a CSF or blood sample, wherein the tau to be measured is (a) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 2, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5, or a combination thereof, and (b) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7, an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8, or a combination thereof; wherein A difference of 1.5σ or more in the quantified MTBR tau species is indicative of a 4R-tauopathy, where σ is the standard deviation defined by a normal distribution measured in a control population without clinical signs or symptoms of tauopathy and who are amyloid-negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF. method.

55. 55. The method of any of claims 51 to 54, wherein the subject does not have dementia.

56. 56. The method of claim 55, wherein the 4R-tauopathy is corticobasal degeneration, frontotemporal lobar degeneration, frontotemporal dementia, or progressive supranuclear palsy.

57. 55. The method of any of claims 51 to 54, wherein the subject has dementia.

58. 58. The method of claim 57, wherein the 4R-tauopathy is corticobasal degeneration, frontotemporal lobar degeneration, frontotemporal dementia, or progressive supranuclear palsy.

59. 1. A method for aiding in determining whether a subject is in need of treatment, comprising: providing a processed CSF or blood sample from a subject that is (a) depleted of mid-domain tau and (b) enriched for MTBR tau; quantitating in the treated sample MTBR tau species comprising the amino acid sequence of SEQ ID NO:2, MTBR tau species comprising the amino acid sequence of SEQ ID NO:3, MTBR tau species comprising the amino acid sequence of SEQ ID NO:4, MTBR tau species comprising the amino acid sequence of SEQ ID NO:5, MTBR tau species comprising the amino acid sequence of SEQ ID NO:6, MTBR tau species comprising the amino acid sequence of SEQ ID NO:7, MTBR tau species comprising the amino acid sequence of SEQ ID NO:8, MTBR tau species comprising the amino acid sequence of SEQ ID NO:9, or combinations thereof; a difference of 1.5σ or more in the quantified MTRB tau species or the ratio of the quantified MTRB tau species indicates that the subject is in need of treatment to modify tau pathology, where σ is the standard deviation defined by a normal distribution measured in a control population that has no clinical signs or symptoms of tauopathy and is amyloid-negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF, and where the amount of quantified MTRB tau species or their ratio is representative of tau pathology in the subject's brain; method.

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