WO2018106889A1

WO2018106889A1 - Diagnosis of alzheimer's disease

Info

Publication number: WO2018106889A1
Application number: PCT/US2017/065060
Authority: WO
Inventors: Joel Steven ROSS
Original assignee: Cogwellin L.L.C.
Priority date: 2016-12-09
Filing date: 2017-12-07
Publication date: 2018-06-14

Abstract

Acetylated tau is detected in the tissue samples and sample of bodily fluids of Alzheimer Disease (AD) patients. Methods of diagnosing AD in subjects have been developed using acetylated tau as the biomarker.

Description

DIAGNOSIS OF ALZHEIMER'S DISEASE

RELATED APPLICATIONS

[0001 ] This application claims the benefit of priority of U.S. Provisional Application 62/432,295, filed December 9, 2016, and U.S. Provisional application 62/447, 167, filed January 17, 2017, both of which are incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] The present disclosure describes a biomarker for diagnosing Alzheimer's disease (AD). The method involves detecting the biomarker using an immunoassay.

BACKGROUND

[0003] The accumulation of tau fibrils in the brain characterizes a group of

neurodegenerative diseases known as tauopathies, which include frontotemporal dementia (FTD) and Alzheimer's disease (AD). Alzheimer's disease (AD) is the sixth- leading cause of death in the United States. AD is a progressive neurodegenerative disease affecting 60-70% of dementia patients. AD is characterized by memory loss and disorientation. The hallmark of the disease is associated with the dysregulation of amyloid beta 42 (Ab-42), total tau (t-tau), and phosphorylated tau (p-tau) proteins, resulting in deposition of amyloid plaques, neuronal death, and accumulation of tangles respectively.

[0004] Recently, it has been suggested that other post-translational modifications may also contribute to the tauopathy associated with AD. In particular, tau has been demonstrated to be modified by lysine acetylation, including lysine 280 (K280) within the microtubule-binding motif. Immunohistochemical and biochemical studies of brains from tau transgenic mice and patients with AD and related tauopathies showed that acetylated tau pathology is specifically associated with insoluble, Thioflavin-positive tau aggregates, and the acetylated form has been detected in diseased tissue, suggesting it may play a role in pathological tau transformation. Collectively, this suggests that tau K280 acetylation is a potential target for drug discovery and biomarker development for AD and may represent a better predictive biomarker for monitoring the progression of Alzheimer's disease in patients.

SUMMARY

[0005] This present disclosure describes a method of diagnosing AD using a biomarker. The biomarker is acetylated tau. In embodiments, the method includes detecting acetylated tau in the tissues of the central nervous system (CNS) and bodily fluids of subjects suspected of having AD. The method also quantitates acetylated tau in the bodily fluids the subjects. In embodiments, the bodily fluid is cerebral spinal fluid (CSF), and acetylated tau is acetylated K280 tau.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the white matter at the superior cerebellar peduncle of a control patient.

[0007] FIG. 1 B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the white matter at the superior cerebellar peduncle of a Braaks Stage 1 AD patient.

[0008] FIG. 2A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the cerebral white matter of a control patient.

[0009] FIG. 2B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the cerebral white matter of a Braaks Stage 1 AD patient.

[0010] FIG. 3A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the putamen of a Braaks Stage 1 AD patient.

[001 1 ] FIG. 3B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the neurons of the putamen of a Braaks Stage 1 AD patient.

[0012] FIG. 4A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the caudate nucleus of a Braaks Stage I AD patient.

[0013] FIG. 4B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the large cholinergic mterneurons in the caudate nucleus of a

Braaks Stage 1 AD patient. [0014] FIG. 5A shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the reticular thalamic nucleus of a Braaks Stage 1 AD patient.

[0015] FIG. 5B shows the results of immunohistochemical staining performed to detect acetylated 280 tau in the neurons of the reticular thalamic nucleus of a Braaks Stage 1 AD patient.

[0016] FIG. 6A shows the results of western blot analysis performed to detect acetylated 280 tau in the cerebrospinal fluid of a control patient (MEC-A-010, Lane 2) and a patient with AD (MEC-X-061 , Lane 3).

[0017] FIG. 6B shows the results of western blot analysis performed to detect acetylated 280 tau in the cerebrospinal fluid of a control patient (Lane 2) and a patient with AD (Lane 3) using a lighter stain than was used in the western blot analysis of FIG. 6A.

[0018] FIG. 7A shows Ponceau S staining of transferred proteins. Lanes 1 - 8 are spinal fluid samples, showing dense protein staining in the 50-75kDa region, the expected location of acetylated tau. Lane 9 shows the location of full-length tau peptide, having a molecular weight of 55-60kda. Lane 10 is pre-stained molecular weight markers, with the position of the 50 kDa protein indicated.

[0019] FIGs. 7B-7D show a band for acetylated tau in the CSF of AD patients MECA 018 (FIG 7B), MEC-M 028 (FIG 7C), and MEC-B 038, MECA 004, and MEC-A-003 (FIG 7D).

[0020] FIG. 8 shows the detection of acetylated K280 tau in the CSF of AD patient. A strong signal in the CSF sample from AD patient (MEC-X-061 ) was detected as compared with the CSF sample from non-AD patient (MEC-A-010).

[0021 ] FIG. 9 shows detection and quantitation of acetylated tau in the CSF patient (MEC-X-061 ) using the Singulex Erenna Immunoassay System.

DETAILED DESCRIPTION

[0022] Tau proteins are soluble proteins that stabilize microtubules and are important in axonal maintenance and axonal transport. Tau proteins are found mostly in neurons. There are six tau isoforms which are formed from alternative splicing in exons 2, 3, and 10 of the tau gene. These six isoforms are distinguished by their number of binding domains. Tau is a phosphoprotein with serine and threonine phosphorylation sites. Accordingly, tau proteins control microtubule stability through the different isoforms and phosphorylation.

[0023] Hyperphosphorylation of tau proteins result in the self-assembly of insoluble neurofibrillary or gliofibrillary tangles in the brain. An abnormal accumulation of neurofibrillary tangles composed of paired helical filaments (PHFs) and straight filaments are found in tauopathy patients including those diagnosed with AD.

[0024] Although the mechanism by which soluble tau accumulates to form insoluble tau fibrils is not fully understood, tau acetylation has been associated with the progression of AD. Min (2010) reported that acetylation of tau prevents its degradation. Min (2010) also showed that tau acetylation was elevated in patients at early and moderate Braak stages of tauopathy. Min (2015) reported the identification of tau acetylation at K174 at an early change in AD brains and a critical determinant in tau homeostasis and toxicity in mice. Min (2015) showed that the acetyl-mimicking mutant K174Q slows tau turnover and induced cognitive deficits in vivo. Salsalate and salicylate have been shown to enhance tau turnover and reduce tau levels. Min (2015) also reported that in the PS 19 transgenic mouse model of frontotemporal dementia (FTD0, administration of salsalate after disease onset inhibited p300 activity, lowered levels of total tau and tau acetylated at K174, rescued tau-induced memory deficits, and prevented hippocampai atrophy. Accordingly, acetylated tau may play a critical role in AD.

[0025] The conventional method for diagnosing AD involves brain imaging studies using magnetic resonance imaging (MRI) or positron emission tomography (PET). U.S. Publication No. 2013/0251731 describes methods of diagnosing tauopathies using tau acetylation as a biomarker for these diseases. In particular, acetylation of the tau at least one of the following lysine residues has been suggested as a biomarker for certain tauopathies: K150, K163, ΚΓ74, K234, K240, K259, K274, K280, K281 , K290, K31 1 , K369, and K395. Furthermore, studies of the brains from Alzheimer's disease patients and related tauopathies show that acetylated K280 tau is specifically associated with insoluble tau aggregates. In these studies, tau K280 acetylation was only detected in diseased tissues, suggesting that acetylated K280 tau may have a role in causing insoluble tau accumulation. As such, acetylated K280 tau can be considered a specific biomarker for AD.

[0026] The presence of a biomarker in a biological sample can be detected by immunohistochemistry, immunocytochemistry, immunofluorescence,

immunoprecipitation, western blotting, and ELISA, depending on the biological sample to be tested. The biological sample to be tested can be obtained from a subject suspected of having AD. The biological sample can be a tissue sample or a sample of bodily fluid obtained from the subject.

[0027] The inventor has confirmed the presence of the acetylated tau in the various types of cells of the central nervous system (CNS) of AD patients. Acetylated K280 tau was shown to be present in the different types of brainstem nuclei from Braaks Stage 1 AD patient (see Table I). Moreover, acetylated K280 tau was also shown to be present in the superior cerebellar peduncle and cerebral white matter from the Braaks Stage 1 AD (see FIG. 1 B and FIG. 2B). Further, acetylated K280 tau was shown to be present in the putamen, caudate nucleus, and reticular thalamic nucleus from the Braaks Stage 1 AD patient (FIG. 3A to FIG. 5B).

[0028] The present disclosure describes the use of acetylated tau as a biomarker for diagnosing AD. In embodiments, the detection of the presence of acetylated tau in various post mortem CNS tissues of subjects indicates the subject has AD. Examples of CNS tissues that can be tested for diagnosing the subject include brainstem, superior cerebellar peduncle, cerebral white matter, cerebral grey matter, putamen, caudate nucleus, and reticular thalamic nucleus, as well as all subthalamic nuclei. In particular embodiments, the acetylated tau is acetylated K280 tau. The probe for detecting the biomarker can be an antibody, such as acetylated K280 tau specific antibody. The antibody can be obtained commercially or by methods known in the art for preparing antibodies.

[0029] The inventor has also confirmed the presence of acetylated tau in the bodily fluid of AD patients. Acetylated tau, particularly acetylated K-280 tau, was shown to be present in the cerebral spinal fluid (CSF) of AD patients. It was also shown that the amount of acetylated tau in CSF of AD patients is very low, in the sub-nanogram to picogram range which makes detection using conventional methods, such as western blotting, very difficult. Therefore, there is a need to develop a new method for detecting acetylated tau.

[0030] When the amount of acetylated tau is low in a sample, as in a CSF sample, techniques including concentrating the sample, increasing the sample volume to run on a gel (for western blot or other immunoassay methods for detection), and collecting a larger volume of sample, could be used to enhance detection of acetylated tau. The sample can be concentrated at least about four times, about five times, about six time, about seven times, about eight times, about nine times, about ten times, about 20 times, about 50 times, or about 100 times. The sample also can be concentrated to dryness and reconstituted to the desired volume for detection by various methods.

Moreover, other techniques, such as isoelectric focusing to separate acetyl tau from other proteins can be used to enhance detection. Accordingly, the present disclosure describes a novel method for detecting acetylated tau in the bodily fluids of subjects which includes a combination of the above steps and others for diagnosing AD in such subjects.

[0031 ] The present disclosure describes a method of diagnosing AD using bodily fluids from subjects. The method includes detecting the presence of acetylated tau in bodily fluids of subjects. Bodily fluids include CSF, urine, semen, saliva, sweat, whole blood, plasma, serum, bile, lymph, tears, and pleural fluid. In embodiments, the method includes detecting the presence of acetylated tau in the CSF of a subject for diagnosing AD in the subject. In particular embodiments, the acetylated tau is acetylated K-280 tau.

[0032] Cerebrospinal fluid (CSF) is a clear, colorless body fluid in the brain and spinal cord. It is produced by the choroid plexuses of the ventricles of the brain, absorbed in the arachnoid granulations, and circulates the subarachnoid space around the brain and spinal cord. It serves as a cushion for the brain within the skull and act as a shock absorber for the central nervous system. The CSF also circulates nutrients and chemicals filtered from the blood and removes waste products form the brain.

Therefore, examining CSF can be useful in diagnosing AD.

[0033] CSF can be obtained from a subject via a lumbar puncture (spinal tap) of a subject. A lumbar puncture involves inserting a long thin hollow needle between two bones in the lower spine and into the space where the CSF circulates and withdrawing CSF using the syringe.

[0034] In embodiments, the bodily fluid includes exosomes. Exosomes are cell-derived vesicles that can be isolated from many biological fluids. Exosomes have been shown to be involved in specialized functions, including intracellular signaling. The method described herein includes isolating exosomes from bodily fluids for detecting the presence of acetylated tau. In embodiments, the method includes isolating exosomes, for example from serum or CSF, and detecting the presence of acetylated tau. In particular embodiments, the method detects the presence of acetylated K280 tau in serum exosomes and CSF exosomes.

[0035] The method for diagnosing AD includes an immunoassay. In an immunoassay, the presence of a molecule (analyte) in a sample solution is detected and measured via a biochemical reaction using a probe that binds the analyte. Immunoassays can be run in multiple steps with reagents being added and washed away or separated at different points in the assay. Immunoassays can be performed by mixing reagents and sample and making a measurement, such as a homogeneous immunoassay, and

immunoassays can involve multiple steps such as heterogeneous or separation immunoassay. In the method of diagnosis described herein, the analyte is the acetylated tau and the probe is the antibody that binds the acetylated tau. In

embodiments, the acetylated tau is acetylated K280 tau and the antibody is the acetylated K280 tau specific antibody.

[0036] Calibrators are often used in an immunoassay. The calibrator contains the analyte in question in a known amount, so that the signal strength of the analyte in the sample and in the calibrator can be compared to for determining the presence and concentration of the analyte in the sample.

[0037] Immunoassays use various labels to allow for detection of antibody binding to analyte. Examples of labels include enzymes (enzyme-linked immunosorbent assays (ELISA)), radioactive isotopes (radioimmunoassay (RIA)), fluorogenic reporters (used in protein microarrays), and electrochemiluminescent tags (light emits in response to electric current). Some immunoassays are label-free, such as surface plasmon resonance which detect binding between an unlabeled antibody and analyte. [0038] Immunoassays include ELISA, RIA, fluorescence immunoassay (FIA) including time-resolved FIA, dot blot, slot blot, western blot, immunoprecipitation, enzyme immunoassay (EIA), immunohistostaining, immunochromatography, chemiluminescent immunoassay, surface plasmon resonance immunoassay, and other known

immunoassays.

[0039] The method of diagnosing AD described herein also includes using

commercially available immunoassay system. Some of these systems provide a kit for ease of testing. Many have enhanced sensitivity for detecting and quantitating very low amounts of analyte in a sample, for example in the picogram range. Examples of some companies that provide such immunoassay platforms include Siemens Healthcare Diagnostic, Randox Laboratories, Abbott Diagnostics, Quanterix Corporation, and EMD Millipore.

[0040] An example of an immunoassay system is the Singulex Erenna© immunoassay system owned by EMD Millipore. The Erenna® is a bench-top analytical instrument that utilizes capillary flow, laser-induced fluorescence, and a highly sensitive detection optics module to achieve single molecule counting (SMC), Thus, it can detect and quantitate analyte in the lower analytical measurement range. Immunoassay reagents are supplied by Singulex in a kit format. Paramagnetic niicroparticles (MPs) are used as the solid phase for immune-capture and detection of analytes in a microplate format. Signal generated by fluorescently labeled detection molecules are counted as digital events, which corresponds to a single analyte molecule. Data is analyzed with the Erenna© software or exported for analysis with standard quantitative ELISA curve fitting software.

[0041 ] The method of diagnosing AD described herein includes detecting and quantitating acetylated tau in a subject suspected of having AD. As an example, the method includes comparing the results obtained from the subject with the results of a control or healthy subject, that is a subject that does not have AD, for determining whether acetylated tau is present and in what quantity in the subject suspected of having AD. The presence of acetyl tau in a subject indicates that the subject has AD. The amount of the acetyl tau can provide some information as to what stage of AD, mild (early), moderate (middle), or severe (advanced). Studies from various groups have shown that up until the very severe stages of AD there is an increasing amount of acetylated Tau (Min et al. 2015 and Irwin et al. 2012). However, the amount of acetylated tau is reduced at the end stages of the disease. The reduction in acetylated tau at the end of the disease is because there are insufficient numbers of brain cells to produce acetylated Tau due to loss of neurons in the brain from acetylated tau damage.

[0042] The terms "tau" and "tau protein" are used interchangeably in this disclosure to refer to the "tau protein." Likewise, the terms "acetylated tau" and "acetylated tau protein" are used interchangeably to refer to the "acetylated tau protein,"

[0043] Methods disclosed herein include diagnosing subjects in need of being diagnosed for AD. Subjects include mammals, for example human, mouse, dog, horse, pig, etc. Subjects in need of diagnosing (in need thereof) are subjects suspected of having AD. Such subjects include human patients diagnosed with mild cognitive impairment (MCI), dementia, cognitive disability in ages over 35, and/or

psychiatric/behavioral issues in ages 35 and over.

[0044] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms "include" or "including" should be interpreted to recite: "comprise, consist of, or consist essentially of." The transition term "comprise" or "comprises" means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase "consisting of" excludes any element, step, ingredient or component not specified. The transition phrase "consisting essentially of" limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.

[0045] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term "about" has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±1 1 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.

[0046] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0047] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is

incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0048] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0049] The following exemplary embodiments and examples illustrate exemplary methods provided herein. These exemplary embodiments and examples are not intended, nor are they to be construed, as limiting the scope of the disclosure. It will be clear that the methods can be practiced otherwise than as particularly described herein. Numerous modifications and variations are possible in view of the teachings herein and, therefore, are within the scope of the disclosure.

EXEMPLARY EMBODIMENTS

[0050] The following are exemplary embodiments:

1 . A method of diagnosing Alzheimer's disease (AD), wherein the method comprises obtaining a sample of bodily fluid from a subject suspected of having AD and detecting acetylated tau in the sample, thereby diagnosing the subject as having AD.

2. The method of embodiment 1 , wherein the bodily fluid is cerebral spinal fluid (CSF), whole blood, plasma, serum, bile, lymph, mucus, pleural fluid, semen, saliva, sweat, tears, or urine.

3. The method of embodiment 1 or 2, wherein the bodily fluid is CSF.

4. The method of any one of embodiments 1 -3, wherein detecting acetylated tau comprises performing an immunoassay.

5. The method of any one of embodiments 1 -4, wherein the immunoassay comprises using an antibody against acetylated tau.

6. The method of any one of embodiments 1 -5, wherein the antibody is acetylated K280 tau antibody.

7. The method of any one of embodiments 1 -6, wherein the immunoassay comprises western blotting, immunoprecipitation, dot blotting, slot blotting, and

immunohistostaining. 8. The method of any one of embodiments 1 -7, wherein the method further comprises quantitating the acetylated tau in the sample.

9. The method of any one of embodiments 1 -8, wherein the method comprises using an immunoassay system that can measure at least picograms quantities of acetylated tau in a sample.

10. The method of any one of embodiments 1 -9, wherein the method further comprises prior to detection of the acetylated tau in the sample, concentrating the sample at least four times, at least five times, at least six time, at least seven times, at least eight times, at least nine times, at least ten times, at least 20 times, at least 50 times, or at least 100 times.

1 1 . The method of any one of embodiments 1 -10, wherein the method further comprises concentrating the sample of CSF to dryness and reconstituting to a desired volume for detection or quantitation.

12. The method of any one of embodiments 1 -1 1 , wherein the method further comprises isolating exosomes from the bodily fluid and detecting acetylated tau in the isolated exosomes.

13. The method of any one of embodiments 1 -12, wherein the exosomes are isolated from serum of the subject.

14. The method of any one of embodiments 1 -13, wherein the exosomes are isolated from CSF of the subject.

15. The method of any one of embodiments 1 -14, wherein the method further comprises performing isolectric focusing to separate acetylated tau from other proteins.

16. A method of diagnosing AD, wherein the method comprises obtaining a CNS tissue from a subject suspected of having AD and detecting the presence of acetylated tau in the CNS tissue of the subject.

17. The method of embodiment 16, wherein the CNS tissue is brainstem, superior cerebellar peduncle white matter, cerebral white matter, putamen, caudate nucleus, or reticular thalamic nucleus.

18. The method of embodiment 16 or 17, wherein the acetylated tau is acetylated K280 tau. 19. The method of one of embodiments 16-18, wherein the method comprises performing immunohistochemistry, immunocytochemistry, or immunofluorescence.

20. The method of one of embodiments 16-19, wherein the probe is acetylated K280 tau antibody.

EXAMPLES

[0051 ] Example 1. Immunohistochemical Staining of Acetylated K280 tau in the CNS of AD Patients.

[0052] Various cell types from the central nervous system of control and Alzheimer's disease patients were tested for the presence of acetylated K280 tau using

immunohistochemical staining.

[0053] The brainstem nuclei from a Braaks Stage 1 AD patient were tested for presence of acetylated K280 tau. Table I shows semi-quantitative data (from +: mild to +++: severe staining) obtained from immunohistochemical staining using an anti- acetylated K280 tau antibody. The data indicate that acetylated K280 tau is present in all of the AD patient brainstem nuclei tested.

Table I

[0054] The white matter at the superior cerebellar peduncle and the cerebral white matter from Braaks Stage 1 AD and control patients were tested for presence of acetylated K280 tau. As shown in FIG. 1 B and 2B, respectively, the superior cerebellar peduncle and cerebral white matter from the Braaks Stage 1 AD patient show staining which indicates the presence of acetylated K280 tau. On the other hand, the control samples do not show such staining (FIG. 1A and FIG. 2A).

[0055] The subcortical nuclei putamen, caudate nucleus and reticular thalamic nucleus from a Braaks Stage 1 AD were tested for presence of acetylated K280 tau. As shown in FIG. 3A and FIG. 3B; FIG. 4A and FIG. 4B; and FIG. 5A and FIG. 5B, respectively, the putamen, caudate nucleus and reticular thalamic nucleus from the Braaks Stage 1 AD patient show distinct staining which indicates the presence of acetylated K280 tau.

[0056] Example 2. Acetylated Tau Screening of CSF Samples of Patients.

[0057] In this Example, CSF samples were obtained from patients. Exosomes were isolated from the CSF and characterized using physical and molecular analysis followed by western blotting for acetylated tau.

[0058] I. Isolation and Characterization of Exosomes from CSF Samples.

[0059] Materials: Beckman Coulter thick-walled polycarbonate centrifuge tubes, 25 x 89 mm (38m L total volume); Beckman Coulter thick-walled polycarbonate bottles, 38 x 102 mm (70ml_ total volume); 50ml_ screw cap tubes; 1 .5ml_ snap cap tubes; 250ml_ screw cap bottles; 500ml screw cap bottles; Beckman Model L8-80M Ultracentrifuge; Beckman Type 50.2 Ti fixed angle rotor (for 25 x 89 mm tubes); Beckman Type 45 Ti fixed angle rotor (for 38 x 102 mm bottles); NanoDrop Spectrophotometer ND-1000; BioRad Dot Blot apparatus (96 well); and CSF samples collected from patients MEC-X- 061 and MEC-B-053, and healthy control volunteers MEC-A-010, and MEC-A-005.

[0060] Methods:

[0061 ] A. Exosome Pelleting by Ultracentrifugation.

1 . Spinal fluid is centrifuged at 3000xg for 20 min at room temperature to pellet cell debris (in 50, 250, or 500m L screw cap vessels).

2. Collect clarified supernate.

3. Centrifuge clarified supernate at 100,000 xg (Avg. RCF) for 2 hrs at 4°C.

4. Aspirate supernate.

5. Leave tubes or bottles upside down at an angle for 2 min to allow excess media to drain.

6. Resuspend pellet(s) in minimum volume of DPBS (100uL). 7. Follow manufacturer's instructions to estimate protein, RNA, and DNA concentration using the NanoDrop spectrophotometer.

8. Aliquot appropriate volume (100uL ) into 1 .5ml snap cap tubes.

9. Store at -80°C.

[0062] B. Dot Blotting.

1 . Exosome samples were denatured in SDS-PAGE sample buffer, heated 70°C 10 min and centrifuged 30s 10,000 xg.

2. Proteins were Dot Blotted according to manufacturer's instructions.

3. Blots were blocked for 30 min at RT in TBST-BSA.

4. The blots were then incubated with primary antibody overnight at 4°C with gentle agitation.

5. The blots were washed 3x 5 min each in TBS-T.

6. Re-blocked for 30 min RT in TBS-T 3% BSA.

7. Incubated with secondary antibody coupled to HRP at 1 :3000 for 60 min at RT.

8. Washed 3x 5 min each in TBS-T.

9. The blots were incubated with chemiluminescent substrate for 5 min according to the manufacturer's protocol.

10. Images were obtained at various times of exposure (5-1 ,200 sec) using a Bio-Rad ChemiDoc station.

[0063] Results and Conclusions: Protein estimations for exosomes were very low - 0.02-0.01 ng/mL. Dot blot did not reveal any signal for any samples. Exosome concentration was too low for the 1 ml_ spinal fluid samples to isolate a meaningful amount for Dot Blot analysis.

[0064] II. Analysis of Concentrated Spinal Fluid Samples for Acetylated Tau.

[0065] Proteins from the CSF samples were concentrated followed by western blotting for acetylated tau.

[0066] Materials: Millipore Ultrafiltration centrifugation tubes, 10,000 Dalton molecular weight cut-off; Beckman centrifuge; and BioRad SDS PAGE Western Blotting apparatus.

[0067] Methods: Western Blotting. 1 . Spinal fluid samples ( MEC-A-01 , MEC-X-061 ) were concentrated to approximately 250 uL.

2. Entire sample was loaded onto SDS polyacrylamide gel after denaturing in SDS- PAGE. sample buffer, heated 70°C 10 min and centrifuged 30s 10,000 xg.

3. Denatured samples were loaded onto 4-12% NuPage Bis-Tris polyacrylamide gels.

4. Proteins were resolved during electrophoresis at 200V in 1x MOPS buffer according to manufacturer's instructions.

5. Prestained molecular weight markers (SeeBlue, Invitrogen) were run in parallel to monitor the progress of electrophoresis as well as transfer.

6. Following electrophoresis proteins were transferred to nitrocellulose filters (0.45 uM Invitrogen) using the NuPage Transfer system.

7. Transfer was performed in 1x Transfer Buffer (Invitrogen) containing 10% methanol for 60 min at 35V.

8. At the end of the transfer the filters were briefly (15 sec) rinsed in MilliQ H2O.

9. Following the water rinse, the blots were blocked for 30 min at RT in TBST- 5% BSA.

10. The blots were then incubated with primary antibody overnight at 4°C with gentle agitation Anti - Tau Antibody (Acetyl K280), Rabbit Polyclonal (AnaSpec; Catalog # AS- 56077).

1 1 . The blots were washed 3x 5 min each in TBS-T.

12. Re-blocked for 30 min RT in TBS-T 1 % BSA.

13. Incubated with secondary antibody coupled to HRP at 1 :3000 for 60 min at RT.

14. Washed 3x5 min each in TBS-T.

15. The blots were incubated with chemiluminescent substrate for 5 min according to the manufacturer's protocol.

16. Images were obtained at various times of exposure (5-300 sec) using a Bio-Rad ChemiDoc station.

[0068] Results and Conclusions: Concentrated cerebrospinal fluid samples from control and Alzheimer's disease patients were tested using western blot analysis for the presence of acetylated K280 tau. Faint but discernable band, with an approximate molecular weight of 60 kilo Daltons, was observed for the MEC-X-061 sample, and not in the MEC-A-01 sample (Control) when probed with the anti-tau antibody (Acetyl K280). As shown in FIG. 6A and FIG. 6B, the AD patient sample (Lane 3) shows a faint but discernable band at the molecular weight expected for acetvlated K280 Tau when probed using the anti-tau antibody (Acetyl K280), Rabbit Polyclonal available from AnaSpec (Catalog #AS-56077). The molecular weight of this protein is between 55,000- 60,000 Daltons, which is the expected molecular weight range for tau family. On the other hand, the control experiment does not show such a band (FIG. 6A and FIG. 6B, Lane 2). Thus, putative acetylated K280 tau has been identified in a concentrated cerebrospinal fluid sample from an Alzheimer's patient by western blot analysis.

[0069] Concentration of the CSF samples enabled the detection of acetylated tau by western blot analysis, using acetyl-K280-specific polyclonal antibody.

[0070] Example 3. Analysis of Concentrated Spinal Fluid Samples for Acetylated Tau.

[0071 ] In this Example, CSF samples were collected from several AD patients. The CSF samples were concentrated. Western blotting was performed for acetylated K280 tau.

[0072] Materials: Millipore Ultrafiltration centrifugation tubes, 30,000 Dalton molecular weight cut-off; Beckman centrifuge; BioRad SDS PAGE Western Blotting apparatus; and CSF samples collected from patients.

MECA 014 2 MEC-B 048 2

MECA 015 2 MEC-B 049 2

MECA 016 2 MEC-B 050 2

MECA 017 2 MEC-X 050 2

MECA 018 2 MECB 052 2

MECA 019 2 MECB 053 2

MECA 020 3 MECB 054 2

MECA 022 3 MECB 058 2

MECA 024 2 MEC-B 059 2

MEC Q 024 2 MEC-X 061 2

MECA 025 2 MEC-B 061 2

MEC-Z 026 4 MEC-B 064 2

MEC-M 026 2 MEC-B 065 2

MEC-M 027 2 MEC-B 072 2

MEC-M 028 2 MECA-021 2

[0073] Methods: Western Blotting.

1 . Spinal fluid samples were concentrated to approximately 250 uL.

2. Entire sample was loaded onto SDS polyacrylamide gel after denaturing in SDS- PAGE sample buffer, heated 70°C 10 min and centrifuged 30s 10,000 xg.

9. Blot was stained with Ponceau S to visualize all proteins transferred.

10. Following another water rinse, the blots were blocked for 30 min at RT in TBST- 5% BSA. 1 1 . The blots were then incubated with primary antibody overnight at 4°C with gentle agitation anti-tau antibody (Acetyl K280), Rabbit Polyclonal (AnaSpec; Catalog # AS- 56077).

12. The blots were washed 3x 5 min each in TBS-T.

13. Re-blocked for 30 min RT in TBS-T 1 % BSA.

14. Incubated with secondary antibody coupled to HRP at 1 :3000 for 60 min at RT.

15. Washed 3x5 min each in TBS-T.

16. The blots were incubated with chemiluminescent substrate for 5 min according to the manufacturer's protocol.

17. Images were obtained at various times of exposure (5-300 sec) using a Bio-Rad ChemiDoc station.

[0074] Results and Conclusions: Discernable bands, with an approximate molecular weight of 55-60 kilo Daltons, and running in parallel with full-length tau peptide, were observed for samples MECA 018, (FIG. 7B) and MEC-M 028 (FIG. 7C). Faint bands were observed for samples MEC-B 038, MECA 004, and MEC-A-003 (FIG. 7D). FIG. 7A shows Ponceau S staining of transferred proteins. Lanes 1 - 8 are spinal fluid samples, showing dense protein staining in the 50-75kDa region, the expected location of acetylated tau. Lane 9 shows the location of full-length tau peptide, having a molecular weight of 55-60kda. Lane 10 is pre-stained molecular weight markers,, with the position of the 50 kDa protein indicated.

[0075] Putative acetylated tau has been identified in concentrated patient spinal fluid samples by western blot analysis. Evidence in support of this statement includes an acetyl-tau immunoreactive band migrating parallel to full-length tau peptide, in the approximate molecular weight range of 50-60kDa. The predicted molecular weight of tau isoforms reported in the literature is 45-65kda. It needs to be pointed out that the abundance of tau/acetyl-tau in spinal fluid is very, very low, in the nanogram to picogram amounts in the different samples which makes detection by western blotting challenging. The limit for detection of this and other immunoreactive methods (i.e.: dot- blotting, immunoprecipitation) is generally in the microgram to high nanogram levels of protein. The results of this Example support the idea that acetylated tau can be detected immunoreactively in the CSF of AD patients. [0076] Below are other ways to enhance detection of the protein: 1 . Further concentrate the spinal fluid samples. In this study, the capability was limited to concentrating the sample 4-fold (from 1000uL to 250uL). Additional technologies exist to concentrate the sample to dryness, then reconstitute to a desired volume for gel electrophoresis; 2. Increase sample volume run on the gel. In this Example, capability was limited to 60uL of sample. Larger gel units with larger well capacities would enable the entire sample to be run on the gel and western blotted; 3. Immunoprecipitate from a larger sample volume of spinal fluid. In this Example, volumes of sample received were approximately 1 ml_. Larger volumes should contain more tau, and concentrating by immunoprecipitation may increase the possibility of detection by western blotting or other methods; 4. Isoelectric focusing to further separate acetyl tau/tau from other proteins. Separating proteins by charge, and not molecular weight, may further separate the observed putative immunoreactive acetyl-tau from surrounding contaminating proteins currently detected by standard SDS-PAGE.

[0077] AD patients' spinal fluid samples were concentrated. Western blot analysis was performed to detect acetylated tau, using acetyl-K280-specific polyclonal antibody. A protein band was detected in two of these samples tested, and a possible band detected in an additional 3 samples (FIGs. 7B, 7C, and 7D). This band had an approximate molecular weight of 55,000-60,000 Daltons, which is the expected molecular weight range for tau family members. Full-length tau peptide was run as a molecular weight control, and the immunoreactive band was found to run at the same position.

[0078] Example 4. Detecting Acetylated Tau in CSF of AD Patient by Dot Blot

[0079] This Example confirms that acetylated K280 tau can be detected in the CSF of AD patients.

[0080] CBP-Mediated Acetylation:

1 . Incubate 5 uM tau for 1 h at 37 °C with 0.5 ug CBP and 1 mM acetyl CoA in the following buffer: 50 mM Tris-CI, pH 8.0, 1 mM DTT, 10% v/v glycerol, 0.1 mM EDTA.

2. Reaction products aliquoted and stored at -80 °C

[0081 ] Dot Blot: 1 . Load acetylated tau (acTau) standards onto nitrocellulose membrane at 100, 50, 10, 5, and 1 ng tau/dot in duplicate (reaction products diluted in TBS) in 200 μΙ_ volume using dot blot concentrator

a. CSF samples: Load 1 mL onto membrane (2 x 500 μί aliquots)

2. Rinse wells of dot blot concentrator 2x with 200 μί TBS

3. Block for 1 h/RT with 5% milk/TBST (blocking buffer)

4. Incubation overnight at 4 °C with anti-actau K280 diluted 1 :1000 in blocking

buffer

5. Wash 3 x 10 min with TBST

6. Incubate 1 h/RT with rabbi-HRP-conjugated secondary antibody diluted 1 : 10,000 in blocking buffer

7. Wash 3 x 10 min with TBST

8. Wash briefly 3x with ddhbO

9. Incubate ~1 min with full-strength West Femto Max (Pierce)

10. Image on Kodak Imaging Station

[0082] Results and Conclusion: FIG 8 shows a strong signal in the CSF sample from AD patient (MEC-X-061 ) as compared with the CSF sample from non-AD patient (MEC- A-010).

[0083] Example 5: Detecting Acetylated Tau in the CSF of AD Patient using the Singulex Erenna® Platform.

[0084] In this Example, CSF from patient with advanced AD is obtained and probed with the K280 tau antibody using the Erenna® Immunoassay system from Singulex. FIG. 9 shows acetylated K280 tau detected in the CSF of advanced AD patient. The dot blot sensitivity with the Erenna® Immunoassay system is about 1 ng. The predicated concentration of acetylated tau in the CSF of the AD Patient is about 10 pg/mL.

[0085] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context [0086] The subject matter described above is provided by way of illustration only and should not be construed as limiting. Various modifications and changes may be made to the subject matter described herein without following the example embodiments and applications illustrated and described, and without departing from the true spirit and scope of the present invention, which is set forth in the following claims.

[0087] All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entireties as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof.

[0088]

REFERENCES

Min et al., Neuron. 2010 September, 67(6): 953-966.

Min et al., Nature Medicine, 2015 October, 21 (10): 1 154-1 162. (Published online September 21 , 2015)

Irwin et al., Brain, 2012, 135: 807-818. U.S. Publication No. 2013/0251731

Claims

2. The method of claim 1 , wherein the bodily fluid is cerebral spinal fluid (CSF), whole blood, plasma, serum, bile, lymph, mucus, pleural fluid, semen, saliva, sweat, tears, or urine.

3. The method of claim 2, wherein the bodily fluid is CSF.

4. The method of claim 1 , wherein detecting acetylated tau comprises performing an immunoassay.

5. The method of claim 4, wherein the immunoassay comprises using an antibody against acetylated tau.

6. The method of claim 5, wherein the antibody is acetylated K280 tau antibody.

7. The method of claim 5, wherein the immunoassay comprises western blotting, immunoprecipitation, dot blotting, slot blotting, and immunohistostaining.

8. The method of claim 5, wherein the method further comprises quantitating the acetylated tau in the sample.

9. The method of claim 5, wherein the method comprises using an immunoassay system that can measure at least picograms quantities of acetylated tau in a sample.

10. The method of claim 1 , wherein the method further comprises prior to detection of the acetylated tau in the sample, concentrating the sample at least four times, at least five times, at least six time, at least seven times, at least eight times, at least nine times, at least ten times, at least 20 times, at least 50 times, or at least 100 times.

1 1 . The method of claim 10, wherein the method further comprises concentrating the sample of CSF to dryness and reconstituting to a desired volume for detection or quantitation of acetylated tau.

12. The method of claim 1 , wherein the method further comprises isolating exosomes from the bodily fluid and detecting acetylated tau in the isolated exosomes.

13. The method of claim 12, wherein the exosomes are isolated from serum of the subject.

14. The method of claim 12 wherein the exosomes are isolated from CSF of the subject.

15. The method of claim 1 , wherein the method further comprises performing isolectric focusing to separate acetylated tau from other proteins.

17. The method of claim 16, wherein the CNS tissue is brainstem, superior cerebellar peduncle white matter, cerebral white matter, putamen, caudate nucleus, or reticular thalamic nucleus.

18. The method of claim 16, wherein the acetylated tau is acetylated K280 tau.

19. The method of claim 16, wherein the method comprises performing

immunohistochemistry, immunocytochemistry, or immunofluorescence.

20. The method of claim 19, wherein the probe is acetylated K280 tau antibody.