Tau Protease Compositions And Methods

Abstract

Tau protein has a causative role in Alzheimer's disease and multiple other neurodegenerative disorders exhibiting tau histopathology collectively termed tauopathies. The primary function of tau protein is to facilitate assembly and maintenance of microtubules in neuronal axons. In the disease process tau protein becomes modified, loses its affinity to microtubules and accumulates in the cell body where it forms aggregates. The large neurofibrillary tangles formed from tau protein assembled into filaments were thought to be the pathological structure of tau. However, more recent work indicates that smaller, soluble oligomeric forms of tau are best associated with neuron loss and memory impairment. A novel and unexpected protease activity has been discovered to be associated with tau in oligomeric but not monomeric structures. Methods have been developed to form and purify tau protease and to assay its activity. Tau protease activity constitutes a totally novel mechanism for tau-mediated neurodegenerative disease by causing tau loss of function, as it cleaves itself, and gain of toxic function as it can cleave other proteins and facilitate cell death through apoptotic and/or senescence pathways. Tau protease presents a novel and unique target for the development of therapeutics that may be achieved by several strategies including by inhibiting the tau oligomer enzymatic activity.

Description

[0001] This application claims the benefit of the filing date of U.S. Provisional Application No. 61/189,679, filed Aug. 20, 2008, entitled "Methods And Composition Comprising Tau Oligomers." The entire disclosure is hereby incorporated by reference into the present disclosure.

BACKGROUND

[0002] Alzheimer's disease (AD) is the most common cause of dementia in the elderly that affects an estimated 15 million people worldwide and 40% of the population above 85 years. The disease is characterized by progressive loss of memory, speech and movement with a total incapacitation of the patient and eventually death. AD takes a terrible toll on those with the disease as well as their families, friends and caregivers.

[0003] The symptoms of AD manifest slowly and the first symptom may only be mild forgetfulness. In this stage, individuals may forget recent events, activities, the names of familiar people or things and may not be able to solve simple math problems. As the disease progresses into moderate stages of AD, symptoms are more easily noticed and become serious enough to cause people with AD or their family members to seek medical help. Moderate-stage symptoms of AD include forgetting how to do simple tasks such as grooming, and problems develop with speaking, understanding, reading, or writing. Severe stage AD patients may become anxious or aggressive, may wander away from home and ultimately need total care.

[0004] No cure is currently available for AD. Today, medication therapy focuses on controlling the symptoms of AD and its various stages. For example, mild to moderate AD can involve treatment with cholinesterase inhibitors such as Cognex.RTM. (tacrine), Aricept.RTM. (donepezil), Exelon.RTM. (rivastigmine), or Razadyne.RTM. (galantamine). Whereas moderate to severe AD can be treated with Namenda.RTM. (memantine). These medications may help delay or prevent AD symptoms from becoming worse for a limited period of time. So early AD treatment is warranted. However, there is no clear evidence that these medications have any effect on the underlying progression of the disease.

[0005] There is a large and rapidly growing unmet need for disease modifying drugs for Alzheimer's disease (AD). The classical hallmarks of AD are inter-neuronal plaques consisting of precipitates or aggregates of amyloid beta protein (A.beta.), and intra-neuronal neurofibrillary tangles (NFTs) of tau protein. Tau protein promotes microtubule assembly and stability and is critical for the function of axons, whereas the normal function of A.beta. is not fully understood. The amyloid cascade hypothesis has been widely accepted as the pathological pathway of AD. It holds that the generation of A.beta. and accumulation of A.beta. aggregates in the brain initiate the disease process. It is supported by genetic evidence that mutations leading to increased accumulation of A.beta. aggregates leads to familial AD. However, there are a number of weaknesses in the A.beta. cascade hypothesis in that it does not address the importance of other pathways that can cause neurodegeneration (Seabrook et al. 2007). The accumulation and distribution of NFTs in the brains of AD patients is highly correlated with disease progression and can be used to stage AD by post-mortem brain histopathology, whereas there is poor correlation between AD and the accumulation of neuritic plaques composed of beta amyloid. This has been used to challenge the amyloid hypothesis (Josephs et al. 2008). Lackluster results for A.beta. directed therapeutics in late stage clinical trials has increased interest in exploring alternative targets for drug discovery such as tau (Iqbal et al. 2009).

[0006] While extensive research in the past decade has identified possible biomarkers for AD, there is still an urgent need for composition and methods that are specifically useful in diagnosing, stratifying, or monitoring the progression or regression of AD. New compositions and methods are also needed that serve as drug targets for the identification of new medication therapies to treat AD and to monitor different medications therapeutic effect when used to treat AD, as well as compositions that are useful as immunotherapeutic agents.

SUMMARY

[0007] The present application provides methods and compositions comprising tau protease. These tau proteases, in some embodiments, can be a cysteine protease, which can cleave tau resulting in tau fragments or peptide fragments that can be used in diagnostic and prognostic assays, allowing AD to be diagnosed earlier (while the patient is alive) and more accurately than was previously possible. These tau fragments can better help the clinician stratify, or monitor the progression or regression of AD, than currently available assays. In addition, tau proteases identified according to the composition and methods disclosed can serve as drug targets for the identification of new therapeutic agents for the treatment of AD or other tauopathies and allow monitoring of different medication therapies benefit when used to treat AD or other tauopathies. In some embodiments, these tau oligomers, fragments or tau proteases can be used as immunotherapeutic agents to stimulate the immune system.

[0008] In some embodiments, the tau itself acquires proteolytic activity (autoproteolytic activity) that leads to its own fragmentation. This activity accounts for the tau protease activities. The structure of the autoproteolytic dimer and trimer of tau comprises 4R tau with one or two free thiols indicates that it is a cysteine protease. In context of the disease process, tau self-association leads to formation of autoproteolytic oligomers that cause loss of tau function through fragmentation of the protein and at the same time facilitate aggregation of the cleaved microtubule binding domain into toxic structures.

[0009] In some embodiments, the tau protease is an enzyme for drug discovery and to reduce, prevent or reverse Alzheimer's disease. Blocking this tau protease enzymatic function may halt or reverse AD. In some embodiments, the substrates or breakdown products for the tau protease (i.e. the 17 kDa fragment) can be used as biomarkers for disease progression because they are directly proportional to the tau protease enzymatic activity.

[0010] In one embodiment, there is a tau protease associated with AD.

[0011] In another embodiment, there is a tau protease, wherein the tau protease is a cysteine protease.

[0012] In another embodiment, there is a protease inhibitor that inhibits or blocks tau protease activity.

[0013] In some embodiments, a method is provided for screening an agent for modulation or disruption of purified tau dimer, tau trimer, tau tetramer, tau pentamer, tau hexamer or a combination thereof, the method comprising: a) contacting a sample containing tau dimer, tau trimer, tau tetramer, tau pentamer, tau hexamer or a combination thereof with an agent suspected of being capable of modulating tau oligomer formation or disrupting tau oligomers; and b) detecting the amount of tau dimer, tau trimer, tau tetramer, tau pentamer, tau hexamer or a combination thereof, wherein a decrease in tau dimer, tau trimer, tau tetramer, tau pentamer, tau hexamer or a combination thereof indicates that the agent modulates tau oligomer formation or disrupts tau oligomer.

[0014] In some embodiments, a reagent composition is provided used to perform high resolution NMR, X-ray crystallography, generating antibodies to specific oligomer sizes, or for immunotherapy.

[0015] Additional features and advantages of various embodiments will be set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of various embodiments. The objectives and other advantages of various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In part, other aspects, features, benefits and advantages of the embodiments will be apparent with regard to the following description, appended claims and accompanying drawings where:

[0017] FIG. 1 illustrates the six tau protein isoforms that result from alternate splicing with the N-terminal inserts in the acidic domain (E2 and E3), the repeat domains of the microtubule binding region (R1, R2, R3 and R4) and the tau pseudo repeat (R') indicated. Note that there are three tau protein isoforms with three repeats (tau352, tau381 and tau410 only contain repeats R1, R3 and R4) and three tau protein isoforms with four repeats (tau383, tau412 and tau441) in the microtubule binding domain (R1, R2, R3 and R4).

[0018] FIG. 2 illustrates tau441 structural features including the projection domain (N-terminal), the proline rich region, and assembly domain (C-terminal). Also indicated is the beta-sheet forming regions (PHF6* and PHF6) and the two cysteines (Cys291 in R2 and Cys322 in R3). All 4R tau protein isoforms contain four repeat regions as shown (R1, R2, R3 and R4), whereas 3R tau protein isoforms only contain three repeat regions (R1, R3 and R4).

[0019] FIG. 3 illustrates microtubule binding domain structural features through the repeat and pseudo repeat regions. The amino acid charge is indicated above the amino acid abbreviation. The beta-sheet forming regions and cysteines are highlighted in bold. Potential proline or glycine mediated bending regions are underlined. The microtubule binding regions are defined as follows: R1=Q244 to K274; R2=V275 to S305; R3=V306 to Q336; R4=V337 to N368; and R'=K369 to S400. In 3R tau isoforms, R2 is absent.

[0020] FIG. 4 illustrates intrinsic differences in tau isoform oligomerization that provides the basis for the disulfide mediated tau oligomer model. 3R tau can only form dimers and has a single cysteine (Cys322); whereas 4R tau forms n+1 oligomers without termination and has two cysteines (Cys291 and Cys322). Oxidative conditions caused rapid oligomer formation due to oxidation of free thiols in monomeric tau.

[0021] FIG. 5 illustrates the tau oligomers formed using various ratios of 3R to 4R tau protein isoforms during reaction. The accumulation of higher order aggregates indicates that the dimer is reactive consistent with the formation of only a single disulfide in the initial 4R/4R structure. Incorporation of 3R tau limits oligomer extension. Higher order oligomers formed with increased levels of 4R tau. The table shows the calculated and apparent molecular weight for the 3R/3R 4R/4R and 3R and 4R tau oligomers formed.

[0022] FIG. 6 illustrates that tau oligomers can be purified in stable form as shown for a mixture of tau412 oligomers that was purified. To prepare specific tau oligomers at high purification Tau412 oligomer enriched preparations (lane 2) were size-fractionated to isolate monomer (lane 3), dimer (lane 4), trimer (lane 5) and tetramer (lane 6) and stabilized in buffer.

[0023] FIG. 7 illustrates two potential cut sites in the tau monomer for cutting at the N-terminal at position Gly 164, and cutting at the C-terminal at position Gly302. The model contains the active site residues important for either a R2 or R3 protease (Cys291, and Cys322 or His299 or His329/330) together with the target amino acid which makes up the catalytic triad of the tau oligomer cysteine protease, as well as important residues that may stabilize the substrate and or facilitate its interaction with the thiol group including Lys163, Lys298, and in the case of C-terminal cleavage, His299.

[0024] FIG. 8 illustrates a PAGE analysis of the purified, inactive tau monomer, the purified tau dimer protease, and the purified tau trimer protease showing the cleavage products that result in a 28 kDa and a 14 kDa fragment.

[0025] FIG. 9 illustrates analysis of the C-terminal fragment for expected MW and charge for tau412 and tau441 which shows that the tau oligomer protease results in a 17 kDa fragment for cleavage of the tau441 consistent with the fragment size consistently observed in Alzheimer's disease as a tau breakdown product.

[0026] FIG. 10 illustrates the comparison of the tau oligomer protease with other known cysteine proteases that indicates that it is a papain-like cysteine endoprotease (CAT B-like).

[0027] FIG. 11 illustrates schematic of the tau oligomer protease dimer showing the two active site regions and the catalytic triad (grey triangle) for the R2 active site and the R3 active site.

[0028] FIG. 12 illustrates a proposed mechanism for the tau oligomer cysteine protease.

[0029] FIG. 13 illustrates the model of a disulfide mediated structure for a 3R/3R tau dimer showing alignment of the peptides in parallel to form a beta sheet structure.

[0030] FIG. 14 illustrates the model of a disulfide mediated structure for a 4R/4R tau dimer showing alignment of the peptides in parallel to form a beta sheet. The formation of an intermolecular disulfide linkage traps the tau proteins into an aggregation template that contains two reactive free thiol groups.

[0031] FIG. 15 illustrates the model of a disulfide mediated structure for a 4R/4R/3R tau trimer showing alignment of the peptides in parallel to form a beta sheet. The reaction of 3R tau protein to the 4R/4R tau dimer eliminates occurs via additional formation of a disulfide linkage. This results in the case of the 4R/4R/3R tau trimer with a single remaining reactive free thiol group. At this time, there is no way to predict whether the disulfide is between 3R R3 and 4R R2 or 4R R3. The trimer is shown with the disulfide linkage to 4R R3 strictly for illustrative purposes.

[0032] FIG. 16 illustrates the model of a disulfide mediated structure for a 3R/4R/4R/3R tau tetramer, showing alignment of the peptides in parallel to form an additional beta sheet structure. The 3R/4R/4R/3R tau tetramer contains no reactive free thiol groups and cannot propagate disulfide mediated tau oligomers.

[0033] FIG. 17 illustrates that disulfide-linked tau oligomers are present in the cerebrospinal fluid (CSF) of AD patients illustrating the biological relevance of extracellular tau cysteine protease and highlight its importance in Alzheimer's disease. It should be noted that the 17 kDa tau breakdown fragment observed is consistent with the fragment produced from tau441 from the tau oligomer protease autocatalytic activity. It should be further noted that the extracellular location of tau oligomer protease puts the enzyme in a position to cleave APP and may tie tau and A-beta pathologies together.

[0034] FIG. 18 illustrates that tau in CSF is "reactive" in that it contains reactive thiols and forms higher order aggregates if accelerated by temperature elevation as shown in the left panel. The panel on the right shows the higher order aggregates that are stable to reducing conditions, SDS, and heat potentially due to interaction with the 28 kDa polyanionic N-terminal fragment of tau after it is cleaved.

[0035] It is to be understood that the figures are not drawn to scale. Further, the relation between objects in a figure may not be to scale, and may in fact have a reverse relationship as to size. The figures are intended to bring understanding and clarity to the structure of each object shown, and thus, some features may be exaggerated in order to illustrate a specific feature of a structure.

DETAILED DESCRIPTION

[0036] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values 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 following 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.

[0037] 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. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range of "1 to 10" includes any and all subranges between (and including) the minimum value of 1 and the maximum value of 10, that is, any and all subranges having a minimum value of equal to or greater than 1 and a maximum value of equal to or less than 10, e.g., 5.5 to 10.

[0038] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," include plural referents unless expressly and unequivocally limited to one referent. Thus, for example, reference to "tau protein" includes one, two, three or more tau proteins.

[0039] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.

[0040] The references, mentioned in the specification, are incorporated herein by reference for all that they disclose.

[0041] The headings below are not meant to limit the disclosure in any way; embodiments under any one heading may be used in conjunction with embodiments under any other heading.

[0042] In various embodiments, the activity and composition of tau protease is provided and methods for its production, and its use for the development of therapeutics and biomarkers for Alzheimer's disease and other tauopathies.

[0043] The present disclosure includes methods and compositions comprising substantially purified and stabilized tau oligomer or a fragment or a peptide derivative thereof. These tau oligomers and compositions are useful for the identification of biomarkers associated with Alzheimer's disease (AD) or tauopathies. Biomarkers identified according to the methods and compositions disclosed can be used in diagnosing, stratifying, or monitoring the progression or regression of AD or tauopathies. The biomarkers may be used as drug targets to develop new drugs and monitor different medication therapies to treat AD or tauopathies.

DEFINITIONS

[0044] Tauopathies are a class of neurodegenerative diseases resulting from the pathological aggregation of tau protein in so-called neurofibrillary tangles (NFT) in the brain. Some examples of tauopathies include, but are not limited to, frontotemporal dementia, Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, frontotemporal lobar degeneration, also known as Pick's disease, or the like.

[0045] "Alzheimer's patient", "AD patient", and all refer to an individual who has been diagnosed with AD (for example, by MMSE score or post mortem by autopsy or has been given a probable diagnosis of Alzheimer's disease (AD). AD includes individuals with a probable diagnosis of mild AD, moderate AD, or severe AD. Non-AD patient refers to a "normal" individual or sample from a "normal" individual who has or would be assessed by a physician as not having AD or mild cognitive impairment (MCI). In various embodiments, a non-AD patient may have a Mini-Mental State Examination (MMSE) (referenced in Folstein et al., J. Psychiatr. Res 1975; 12:1289-198) score or would achieve a MMSE score in the range of 27 or above or assessed by another mental examination method. On average people with Alzheimer's disease who do not receive treatment lose 2 to 4 points each year on the MMSE. An "individual" is a mammal, more preferably a human. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets.

[0046] An "individual with mild AD" or "mild AD" is an individual who has been diagnosed with AD (for example, post mortem by autopsy) or has been given a diagnosis of probable AD. In various embodiments, this individual has either been assessed with the Mini-Mental State Examination (MMSE) (referenced in Folstein et al., J. Psychiatr. Res 1975; 12:1289-198) and scored 20-26 or would achieve a score of 20-26 upon MMSE testing or assessed by another mental examination method.

[0047] An "individual with moderate AD" or "moderate AD" is an individual who has been diagnosed with AD (for example, post mortem by autopsy) or has been given a diagnosis of probable AD. In various embodiments, this individual has either been assessed with the MMSE and scored 10-19 or would achieve a score of 10-19 upon MMSE testing or assessed by another mental examination method.

[0048] An "individual with severe AD" or "severe AD" is an individual who has been diagnosed with AD (for example, post mortem by autopsy) or has been given a diagnosis of probable AD. In various embodiments, this individual has either been assessed with the MMSE and scored below 10 or would achieve a score of below 10 upon MMSE testing or assessed by another mental examination method.

[0049] As used herein, methods for "aiding diagnosis" refer to methods that assist in making a clinical determination regarding the presence, or nature, of the AD or MCI or tauopathy, and may or may not be conclusive with respect to the definitive diagnosis. Accordingly, for example, a method of aiding diagnosis of AD or tauopathy can comprise measuring the amount of one or more AD or tauopathy biomarkers in a biological sample from an individual.

[0050] The term "stratifying" refers to sorting individuals into different classes or strata based on the features of AD or tauopathy. For example, stratifying a population of individuals with Alzheimer's disease involves assigning the individuals on the basis of the severity of the disease (e.g., mild, moderate, severe, etc.).

[0051] As used herein, the term "treatment" refers to the alleviation, amelioration, and/or stabilization of symptoms, as well as delay in progression of symptoms of a particular disorder. For example, "treatment" of AD includes any one or more of: elimination of one or more symptoms of AD, reduction of one or more symptoms of AD, stabilization of the symptoms of AD (e.g., failure to progress to more advanced stages of AD), and delay in progression (e.g., worsening) of one or more symptoms of AD, and regression (e.g., reverting back to the earlier stage of AD).

[0052] As used herein, the term "predicting" refers to making a finding that an individual has a significantly enhanced probability of developing AD or tauopathy. The term "prognosis" includes the likely outcome or course of AD or tauopathy.

[0053] In various embodiments of the present application, we disclose novel purified and stabilized tau oligomer or a fragment or a peptide derivative thereof. Tau protein exists in 6 isoforms of 352-441 amino acid residues in the adult brain. The term "tau protein" refers to any protein of the tau protein family including, but not limited to, native tau protein monomer, precursor tau proteins, tau peptides, tau intermediates, metabolites, tau derivatives that can be antigenic, or antigenic or non-antigenic fragments thereof. Fragments include less than entire tau protein provided the fragment is antigenic and will cause antibodies or antibody binding fragments to react with the tau fragment.

[0054] Non-reactive tau oligomer includes one composed of 3R tau (i.e. 3r/3R tau dimer or 3R/4R/3R tau trimer), or one in which further oxidation has rendered the sulfonic derivative, or one where truncation has occurred removing the cysteine thiol.

[0055] The tau protein family in addition is characterized by the presence of a characteristic N-terminal segment which is shared by all members of the family, sequences of approximately 50 amino acids inserted in the N-terminal segment, which are developmentally regulated in the brain, a characteristic tandem repeat region consisting of 3 or 4 tandem repeats of 31-32 amino acids, and a C-terminal tail. In various embodiments, tau protein has the following amino acid sequence shown SEQ ID NOS. 1-6. Embodiments of tau protein may have an amino acid sequence that is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or substantially identical to the sequence given in SEQ ID NOS: 1-6.

[0056] As applied to any of the disclosed tau protein, peptides, the term "substantially identical" means that two peptide or protein sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 70 percent sequence identity, such as at least 90 percent sequence identity, or at least 95 percent sequence identity, or at least 99 percent sequence identity. Residue positions, which are not identical, in various embodiments, differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. In various embodiments, conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

[0057] In some embodiments, tau protein is provided. The six tau protein isoforms can be expressed by alternated splicing of the primary transcript of the MAPT gene on chromosome 17q21. Tau isoforms can be expressed in the central nervous system (FIG. 1); SEQ ID NO. 1-6. The convention used for amino acid numbering is based on the longest of the six tau isoforms containing 441 amino acids. R1 includes the first microtubule-binding repeat: 244qtapvpmpdlknvkskigstenlkhqpgggk274 (FIGS. 2 and 3). R2 includes the second microtubule-binding repeat: 275gggkvqiinkkldlsnvqskcgskdnikhvpgggs305 (FIGS. 2 and 3). R3 includes the third microtubule-binding repeat: 306 pgggsvqivykpvdlskvtskcgslgnihhkpgggq336 (FIGS. 2 and 3). R4 includes the fourth microtubule-binding repeat: 337vevksekldfkdrvqskigsldnithvpgggn368 (FIGS. 2 and 3). R' includes the pseudo microtubule-binding repeat: 369kkiethkltfrenakaktdhgaeivykspvvs400 (FIGS. 2 and 3).

[0058] 3R tau includes tau isoforms 3R/0N, 3R/1N, 3R/2N, containing 410, 381 or 352 amino acids, lacking the second microtubule-binding repeat (R2) due to alternate splicing of exon 10, and containing 0, 1 or 2 N-terminal inserts due to alternate splicing of exons 2 or 3 (FIGS. 1 and 2). 3R tau also includes fragments or peptides of tau including the 3R microtubule binding domain.

[0059] 4R tau includes tau isoforms 4R/0N, 4R/1N, 4R/2N containing 441, 412 or 383 amino acids containing all microtubule-binding repeats including R2, and containing 0, 1 or 2 N-terminal inserts due to alternate splicing of exons 2 or 3 (FIG. 1). 4R tau also includes fragments or peptides of tau including the 4R microtubule binding domain.

[0060] PHF6* includes hexapeptide motif 275vqiink280, which exhibits propensity to form bet-sheet structure.

[0061] PHF6 includes hexapeptide motif 306vqivyk311, which exhibits propensity to form bet-sheet structure.

[0062] Tau monomer includes any individual 3R or 4R tau.

[0063] Tau oligomer includes an aggregate of tau protein subunits. The minimal size of a tau oligomer is two subunits, and the maximal size of a tau oligomer referred to in this application is 12 tau subunits. These tau oligomers are tau dimer, tau trimer, tau tetramer, tau pentamer, tau hexamer, tau septamer, tau octamer, tau nonamer, tau decamer, tau unadecamer, tau dodecamer. In some embodiments, the subunits may be composed of any 3R or 4R tau.

[0064] The tau oligomer can be substantially purified and/or isolated (FIG. 6). In some embodiments, tau protein can be purified by cation exchange using SP Sepharose, heat denaturation in Laemmli sample buffer 5 min at 95.degree. C., and fraction collection from continuous SDS-PAGE. Tau oligomers can be formed by incubation of tau subunits in buffer (50 mM Tris pH 7.4) at 37.degree. C. The size range of the oligomers may be controlled by modulation of tau concentration, length of incubation, buffer composition, and/or choice of tau isoforms, fragment or peptide and/or mixtures thereof.

[0065] The tau oligomer subunits may or may not be linked by disulfide bonds. In some embodiments, the tau oligomer can be stabilized by disulfide bonds and is stable for at least two months in a non-reductive environment.

[0066] In some embodiments, the tau subunits interact in a specific orientation such that: during 3R tau-3R tau intermolecular association the hexapeptide motif PHF6 in R3 of one 3R subunit aligns with the hexapeptide motif PHF6 in R3 of the other 3R tau subunit in the same amino to carboxyl orientation such that they may form cross beta strand structure; such that cysteine322 in R3 in one 3R tau subunit and cysteine322 in R3 in the other 3R tau subunit are aligned enabling disulfide bond formation; such that there are no free thiol groups and hexapeptide motifs in each of the 3R tau subunits precluding similar associations with additional 4R or 3R tau subunits (FIG. 13). During 4R tau-4R tau intermolecular association the hexapeptide motif PHF6* in R2 of one tau subunit aligns with the hexapeptide motif PHF6 in R3 of the other 4R tau subunit in the same amino to carboxyl orientation such that they may form cross beta strand structure; such that cysteine291 in R2 and cysteine322 in R3 are aligned enabling disulfide bond formation; such that there are a free thiol group and hexapeptide motif in each of the 4R tau subunits enabling similar associations with additional 4R or 3R tau subunits facilitating oligomer extension from either end (FIG. 14). During 4R tau-3R tau intermolecular association the hexapeptide motif PHF6* in R2 of the 4R tau subunit aligns with the hexapeptide motif PHF6 in R3 of the 3R tau subunit in the same amino to carboxyl orientation such that they may form cross beta strand structure; such that cysteine291 in R2 of 4R tau and cysteine322 in R3 of 3R tau are aligned enabling disulfide bond formation; such that there is unidirectional oligomer extension from the 4R tau end of the oligomer containing one free thiol group and one hexapeptide motif and termination of oligomer extension from the 3R end with no free hexapeptide motifs or thiols (FIG. 15); such that addition of 3R tau to both ends of an oligomer terminates oligomer extension through this mechanism (FIG. 16). In some embodiments, the cross beta sheet structures in the oligomer may stack in antiparallel orientation. In some embodiments, oligomer size may be controlled by modulation of the ratio of 3R to 4R tau concentrations (FIGS. 4 and 5).

[0067] In various embodiments, tau monomeric units (tau proteins (e.g., SEQ ID NOS: 1-6), peptides, or fragments thereof) may oligomerize and form tau oligomers, which are soluble in bodily fluids (e.g., CSF, blood, urine, cytoplasmic fluid, etc.). It has been found by Applicants that extracellular soluble tau monomeric units and/or tau oligomers increase in AD or tauopathies.

[0068] Tau oligomerization includes multimerizing two or more tau proteins, tau peptides, tau intermediates, tau metabolites, tau derivatives that can be antigenic, tau antigenic fragments, or tau-tau complexes. The multimer can contain any desired number of tau peptide/protein complexes and thus can form any multimer, such as but not limited to, a dimer, a trimer, a tetramer, a pentamer, a hexamer, octamer, decamer, dodecamer, or the like. However, in order to be soluble in bodily fluids (e.g., CSF, blood, urine, etc.) the multimer cannot be too long as it may become insoluble in bodily fluids. By "insoluble" is meant that the tau oligomer will precipitate out of the bodily fluid. In various embodiments, tau oligomer that comprises 50 tau monomer units is too long and may be insoluble in bodily fluids. In other embodiments, tau oligomer that comprises 100 tau monomer units is too long and may be insoluble in bodily fluids. By "soluble" is meant that the tau oligomer will dissolve in the bodily fluid. For example, soluble tau may be extracellular and appear in the CSF. By associated is meant covalent or non-covalent, hydrophobic or hydrophilic interactions, H bonding, or van der Waals attachment.

[0069] Tau proteins, peptides, or fragments thereof may undergo truncation at one or more sites (e.g., carboxy and/or amino truncations). For example, tau proteins may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid truncation at the N and/or C terminus. Truncation typically occurs by enzymes, which cleave at the carboxy or amino terminus. Such enzymes, include, but are not limited to caspases that comprise cysteine proteases.

[0070] "Tau protease" includes an enzyme that can cleave, digest, or degrade tau oligomer (e.g., tau dimmer, trimer, tetramer, etc.) into the lower order structure or even monomeric units to fragments thereof. In some embodiments, the tau itself can act as a protease and cleave itself when it is in the oligomeric form. This type of activity is referred to, in some embodiments, as autoproteolytic activity.

[0071] In some embodiments, a tau protease is an oligomer of tau composed of at least one 4R tau and additional 3R and 4R tau molecules. The minimal structure of a tau protease is 4R tau-4R tau dimer or 4R tau-3R tau dimer. Tau dimer and tau trimer have proteolytic activity as evidenced by the fragmentation of tau in these preparations during incubation, whereas reduction of tau oligomer to tau monomer inhibited tau fragmentation during incubation. Additionally, tau monomer preparations do not exhibit proteolytic activity (FIG. 8). Tau protease can be formed and purified and/or isolated following the methods for isolating tau oligomers.

[0072] In one embodiment, tau protease is an endopeptidase and member of the class of cysteine proteases that have a common catalytic mechanism involving a nucleophilic cysteine thiol in a catalytic triad (FIGS. 7 and 11). In one embodiment the tau oligomer protease is a papain-like cysteine protease (CAT B-like endoprotease) based on analysis of its active site and sequence similarities to tau (FIG. 10).

[0073] In some embodiments, tau-tau interaction enables formation of a cleft that contains the active site, which contains the catalytic triad. Tau oligomers with two terminal 4R tau subunits can form two active sites, whereas a terminal 3R tau subunit precludes activity at that end of the oligomer. The availability of cysteine thiols is enabled by the interaction of R2 of 4R tau with R3 of 3R or 4R tau such that there is at least one free thiol available in one or both of the 4R tau subunits from cysteine291 in R2 and/or cysteine322 in R3. Additional structures of tau dimers, not included in the tau oligomer definitions above, enabling thiol availability are interaction of tau subunits by alignment of R3 of one subunit with R3 of the other subunit such that cysteines at position 322 in each of the subunits form a disulfide bond, and such that there are free thiols from cysteines at position 291 in each subunit in R2 or by alignment of R2 of one subunit with R2 of the other subunit such that cysteines at position 291 in each of the subunits form a disulfide bond, and such that there are free thiols from cysteines at position 322 in each subunit in R3. The histidine residues in the tau protease active site required for deprotonation of the thiol/s are histidine299 in R2 and histidine239 or histidine330 in R3. In one embodiment, derived from analysis of tau proteolytic fragments (FIG. 8), the carbonyl carbon of glycine at position 302, and possibly glycine303 or glycine304 is the substrate of the nucleophilic attack by the deprotonated cysteine's anionic sulfur. In another embodiment glycine164 is the carbonyl carbon of glycine at position 164 is the substrate of the nucleophilic attack by the deprotonated cysteine's anionic sulfur.

[0074] In one embodiment, during nucleophilic attack a 28 kDa fragment of the substrate is released with an amine terminus, the histidine residue in the protease is restored to its deprotonated form, and a thioester intermediate linking the new carboxy-terminus of the substrate to the cysteine thiol is formed. The thioester bond is subsequently hydrolyzed to generate a carboxylic acid moiety on the remaining substrate 14 kDa fragment, while regenerating the free enzyme thiol group.

[0075] In some embodiments, proteases can cleave tau. These proteases include, for example, cellular proteases, such as for examples, caspase, cathepsin B, CAT B-like endoprotease, or other cysteine proteases. These proteases can be added by exogenous administration to an animal or can be produced endogenously in vivo by the animal.

[0076] "Cleaved tau protein" or "cleaved tau products" includes for example, tau proteins that may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids cleaved from the N and/or C terminus of tau. These can be also referred to as tau fragments and result from a separate protease or when tau cleaves itself.

[0077] Any protease inhibitors may be used to prevent or reduce tau cleavage. Some examples of protease inhibitors include AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), which is a serine protease inhibitor that inhibits trypsin, chymotrypsin, plasmin, kallikrein, thrombin; or E-64 (N--[N-(L-3-transcarboxyirane-2-carbonyl)-L-Leucyl]-agmatine), which is an irreversible and highly selective cysteine protease inhibitor; or N-ethylmaleimide, which is a cysteine protease inhibitor like calpain; or phenylene dimaleimide, which is a cysteine protease inhibitor or a combinations thereof. Some additional cysteine protease inhibitors that can prevent tau cleavage, include, CA074Me, amelimide, or combination thereof. These cysteine proteases can prevent or inhibit tau cleavage and allow the oligomer to remain intact or prevent or inhibit tau protein from acting as a protease itself.

[0078] Cysteine protease inhibitors include aldehydes, semicabazones, methyl ketones, trifluoromethyl ketones, alpha keto acids, alpha esters, alpha keto amides, and diketones, nitriles, halomethyl ketones, diazomethanes, acyloxymethylketones, methysulfonium salts, epoxysuccinyl derivatives, unsaturated derivatives, disulfides, azapeptides, azobenzenes, o-acylhydroxamates, lysosomotropic bases, calmodulin antagonists, aziridines, thiiranes, or the like as discussed by Otto et al. in Chem Reviews 1997, 97, 133-171. This entire disclosure is herein incorporated by reference into the present disclosure.

[0079] An "isolated" tau oligomer or tau peptide derivative, or tau fragment, as used herein, means a naturally-occurring tau oligomer or peptide or fragment that has been separated or substantially separated from the cellular components (e.g., CSF, brain cells, other peptides, etc.) that naturally accompany it by purification, recombinant synthesis, or chemical synthesis, and also encompasses non-naturally-occurring recombinantly or chemically synthesized oligomers or peptides or fragments that have been purified or substantially purified from cellular components, biological materials, chemical precursors, or other chemicals. In vitro methods of making tau oligomer are described in U.S. application Ser. No. 11/704,079, filed Feb. 8, 2007 and U.S. Publication No. 20070218491, which is hereby incorporated by reference into the present disclosure.

[0080] "Purified" as used herein includes that the protein, peptide derivative or fragment thereof is free not only of other proteins, but also of other materials used in the isolation and identification of the protein, such as, for example, sodium dodecyl sulfate and other detergents as well as the support material. The protein is at least 90% free, preferably at least 95% free and, more preferably, at least 98% or 99% free of such materials.

[0081] Tau oligomer can be cleaved into a fragment and be in a peptide form. "Peptide" comprises a string of at least three amino acids linked together by peptide bonds. Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides may contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, http://www.cco.caltech.edu/.about.dadgrp/Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.

[0082] "Peptide derivatives" includes derivatives (chemically functionalized protein molecules obtained starting with the disclosed protein sequences) or mimetics (three-dimensionally similar chemicals) of the native tau oligomer, as well as proteins sequence variants (such as mutants), genetic alleles, fusions proteins of tau or combinations thereof.

[0083] A "free cysteine amino acid" refers to a cysteine amino acid residue which has a thiol functional group (--SH), and is not paired as an intramolecular or intermolecular disulfide bridge. Accordingly, the free cysteine amino acid can be antigenic for generation of antibodies to it.

[0084] A "free thiol moiety" refers to the amino acid (e.g., cysteine) which has a thiol functional group (--SH), and is not paired as an intramolecular or intermolecular disulfide bridge.

[0085] "Tau-A.beta. complex" includes interactions, aggregates, and/or coupling between tau, tau intermediates, metabolites, tau derivatives, or antigenic fragments of tau and .beta.-amyloid protein (A.beta.1-42), A.beta.1-42 intermediates, A.beta.1-42 metabolites, A.beta.1-42 derivatives, or antigenic fragments of A.beta.1-42. A.beta.1-42 comprises 42 amino acids (SEQ ID NO: 7). In various embodiments, tau-A.beta. complex comprises at least tau and .beta.-amyloid protein (A.beta.1-42) or tau and A.beta.1-40 protein. In various embodiments, tau-A.beta. complex comprises at least tau and .beta.-amyloid protein (A.beta.1-42) or tau and A.beta.1-40 protein and tau and A.beta.1-42 or A.beta.1-40 molecules, such as, for example, amyloid oligomer, other amyloid protein, tau-oligomer-A.beta.1-42 or tau and A.beta.1-40 protein.

[0086] "Amyloid" refers to amyloidogenic proteins, peptides, or fragments thereof which can be soluble (e.g., monomeric or oligomeric) see, e.g., Lambert et al., Proc. Natl. Acad. Sci. U.S.A. 95, 6448-6453 (1998)). .beta.-amyloid protein (A.beta.) may comprise 39-43 amino acids. Typically, the A.beta.1-42 peptide is produced by sequential proteolytic cleavage of the amyloid precursor protein (APP) by the enzyme(s) beta and gamma secretases. The length of the A.beta. peptide appears to dramatically alter its biochemical/biophysical properties. Specifically, the additional two amino acids at the C-terminus of A.beta.1-42 are very hydrophobic, presumably increasing the propensity of A.beta.1-42 to aggregate. For example, Jarrett et al. demonstrated that A.beta.1-42 aggregates vary rapidly in vitro as compared to A.beta.1-40, suggesting that the longer forms of A.beta. may be the important pathological proteins that are involved in the initial seeding of the neuritic plaques in Alzheimer's disease (Jarrett et al., Biochemistry 32, 4693-4697 (1993); Jarrett et al., Ann. NY Acad. Sci. 695, 144-148, (1993)).

[0087] As used herein, the term ".beta.-amyloid" or "A.beta." or "amyloid beta" refer to amyloid beta proteins or peptides, amyloid beta precursor proteins or peptides, intermediates, and modifications and immunologic fragments thereof, unless otherwise specifically indicated. In particular, "A.beta." refers to any peptide produced by proteolytic processing of the APP gene product or peptides that are associated with amyloid pathologies, including A.beta.1-39, A.beta.1-40, A.beta.1-41, A.beta.1-42, and A.beta.1-43. Embodiments of A.beta.1-42 may have an amino acid sequence that is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or substantially identical to the sequence given in SEQ ID NO: 7.

[0088] In some embodiments, the "aggregation core", contains all of the important structural elements for the tau dimer binding interface between the two tau monomers and will be used to isolate tau oligomer specific antibodies that recognize this structural epitope.

[0089] The tau oligomer or a cleaved tau fragment or a peptide thereof can be used as a biomarker. A "biomarker" includes, protein(s), peptide(s), protein-protein complexes or aggregates, protein-peptide complexes or aggregates, peptide-peptide complexes or aggregates, immunogenic fragments, or metabolite(s) (e.g., glycated, truncated, phosphorylated peptide, protein, complex, aggregates) whose presence, absence, or, level of expression is a measure of the progression or regression of tauopathy, AD or of the likelihood of developing tauopathy, or AD. A biomarker may comprise a single protein, peptide, protein-protein complex, protein-peptide complex, peptide-peptide complex or metabolite, or it may comprise a plurality of proteins, peptides, complexes, immunogenic fragments and/or metabolites whose presence, absence, or levels of expression collectively provide a measure of the progression or regression of tauopathy, AD or of the likelihood of developing a tauopathy or AD.

[0090] The tau oligomer or a fragment or a peptide thereof can be used as an epitope or antigen to generate antibodies thereto. The term "epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond or a site on a molecule against which an antibody will be produced and/or to which an antibody will bind. For example, an epitope can be recognized by an antibody defining the epitope. A "linear epitope" is an epitope where an amino acid primary sequence comprises the epitope recognized. A linear epitope typically includes at least 3, and more usually, at least 5, for example, about 8 to about 10 amino acids in a unique sequence. A "conformational epitope", in contrast to a linear epitope, is an epitope wherein the primary sequence of the amino acids comprising the epitope is not the sole defining component of the epitope recognized (e.g., an epitope wherein the primary sequence of amino acids is not necessarily recognized by the antibody defining the epitope). Typically a conformational epitope comprises an increased number of amino acids relative to a linear epitope. With regard to recognition of conformational epitopes, the antibody recognizes a 3-dimensional structure of the peptide or protein or fragment thereof. For example, when a protein molecule folds to form a three dimensional structure, certain amino acids and/or the polypeptide backbone forming the conformational epitope become juxtaposed enabling the antibody to recognize the epitope. Methods of determining conformation of epitopes include but are not limited to, for example, x-ray crystallography 2-dimensional nuclear magnetic resonance spectroscopy and site-directed spin labeling and electron paramagnetic resonance spectroscopy.

[0091] The term "antibody" is used to include intact antibodies and binding fragments thereof, including but not limited to, for example, full-length antibodies (e.g., an IgG antibody) or only an antigen binding portion (e.g., a Fab, F(ab').sub.2 or scFv fragment). The fragment can also be generated by phage display technology known in the art.

[0092] Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen. Optionally, antibodies or binding fragments thereof, can be chemically conjugated to, or expressed as, fusion proteins with other proteins. The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab').sub.2 and Fv fragments. In various embodiments, papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily. In various embodiments, pepsin treatment yields an F(ab').sub.2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc'). As used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab').sub.2 fragments.

[0093] An "Fv" fragment is the minimum antibody fragment, which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V.sub.H-V.sub.L dimer). It is in this configuration that the three complementarity determining regions (CDRs) of each variable domain interact to define an antigen binding site on the surface of the V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. The Fab fragment (also designated as F(ab)) also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains have a free thiol group. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge-cysteines of the F(ab').sub.2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.

[0094] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies, directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), or may be made by recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567. "Polyclonal antibodies" include different antibodies directed against different determinants (epitopes).

[0095] "Specific binding" or "binding specifically" between two entities, may include a binding affinity of at least 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9M.sup.-1, or 10.sup.10 M.sup.-1. Affinities greater than 10.sup.8 M.sup.-1 are preferred for specific binding. Antibodies to tau are described in U.S. patent application Ser. No. 12/069,399, filed Feb. 8, 2008 and published as US-2008-0220449. The entire disclosure is hereby incorporated by reference into the present disclosure.

[0096] The tau oligomer or a fragment or a peptide thereof can be used as an immunotherapeutic agent. The term "immunotherapeutic agent" is intended to mean an agent that stimulates anti-tau oligomer immunity. Agents that stimulate anti-tau oligomer activity are preferably those that directly or indirectly stimulate T-cells and/or NK cells to stop the oligomerization of tau to achieve a prophylactic and/or therapeutic goal. The tau oligomer or a fragment or a peptide derivative thereof can be used to make antibodies for passive immunotherapies or the tau oligomer or a fragment or a peptide derivative thereof can be used for active immunity, such as for example, in a vaccine composition when administered to the mammal.

[0097] The tau oligomer or a fragment or a peptide derivative thereof can be used with adjuvants and administered to the animal. Any adjuvant may be used in accordance with the present invention. A large number of adjuvant compounds is known; a useful compendium of many such compounds is prepared by the National Institutes of Health and can be found on the world wide web (http:/www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf, incorporated herein by reference; see also Allison Dev. Biol. Stand. 92:3-11, 1998; Unkeless et al. Annu. Rev. Immunol. 6:251-281, 1998; and Phillips et al. Vaccine 10:151-158, 1992, each of which is incorporated herein by reference). Hundreds of different adjuvants are known in the art and could be employed in the practice of the present invention.

[0098] The tau oligomer or a fragment or a peptide derivative thereof can be administered to the animal in a pharmaceutical composition. Those of ordinary skill in the art will appreciate that pharmaceutical composition can be administered to individuals via any of a variety of routes, protocols, and dosing regimens. Known routes of administration include, for example, intravenous (IV), intraperitoneal (IP), intragastric (IG), subcutaneous (SQ), intramuscular (IM), oral (PO), rectal (PR), intrathecal, vaginal, intranasal, transdermal, intradermal, subcutaneous, intrathecal, epidural, or the like.

[0099] The tau oligomer or a fragment or a peptide derivative thereof can be part of a pharmaceutical composition designed for administration to the mammal. Pharmaceutical compositions for use in accordance with the present application may include a pharmaceutically acceptable excipient or carrier. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The pharmaceutical compositions of this invention can be administered to humans and/or to animals, orally, rectally, parenterally, intracistemally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), bucally, or as an oral or nasal spray.

[0100] Any dose can be administered to an animal. Dosages can vary depending on the relative potency of individual compositions, and can generally be estimated based on data obtained from in vitro and in vivo animal models. Typically, the dosage may be from about 0.01 micrograms to about 100 g per kg of body weight, and may be given once or more daily, weekly, or even less often. Following successful administration, it may be desirable to have the subject undergo additional booster administrations to maintain a suitable level of the anti-tau oligomer, tau fragment, or tau-peptide activity. For example, an additional dosage can be administered 6, 12, 24, 36, 48, 60 or more months after an initial dosage. In some cases, additional dosages can be administered every 6, 12, 18, 24, 30, 36, 42, 48, 54, 60 or more months after an initial dosage. Additional dosages also can be administered as needed.

[0101] The term "animal", as used herein, refers to humans as well as non-human animals, including, for example, mammals, birds, reptiles, amphibians, and fish. Preferably, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). An animal may be a transgenic animal.

[0102] The tau oligomer or a fragment or a peptide derivative thereof can be used a drug target in drug discovery. In various embodiments, methods of screening for candidate agents for the treatment of AD or tauopathies are provided by assaying prospective candidate agents for activity in modulating tau oligomer or a fragment or a peptide derivative of tau oligomer. The screening methods may utilize tau oligomer or a fragment or a peptide derivative of tau oligomer described herein as "drug targets." Prospective agents are tested for activity in modulating a drug target in an assay system. As will be understood by those of skill in the art, the mode of testing for modulation activity will depend on the biomarker and the form of the drug target used (e.g., complete protein or peptide fragment).

[0103] When the tau oligomer or a fragment or a peptide derivative of tau oligomer itself is the drug target, prospective agents are tested for activity in modulating levels or activity of the protein/peptide itself (e.g., soluble tau oligomer). Modulation of levels of tau oligomer or a fragment or a peptide derivative of tau oligomer can be accomplished by, for example, increasing or reducing half-life of the biomarker protein or drug candidate concentration. Modulation of activity of tau oligomer or a fragment or a peptide derivative of tau oligomer can also be accomplished by increasing or reducing the availability of the tau oligomer or a fragment or a peptide derivative of tau oligomer to bind to its receptor(s) or ligand(s). In various embodiments, a method of screening an agent for modulation or disruption of tau oligomer or a fragment or a peptide derivative of tau oligomer is provided, the method comprising: a) contacting a sample containing tau oligomer or a fragment or a peptide derivative of tau oligomer with an agent suspected of being capable of modulating tau oligomer formation or disrupting tau oligomers; and b) detecting the amount of tau oligomer or a fragment or a peptide derivative of tau oligomer in the sample, wherein a decrease in soluble tau oligomer indicates that the agent modulates tau oligomer formation or disrupts tau oligomer. In various embodiments, the mechanism that the agent disrupts tau oligomer formation is by inhibiting tau-tau binding.

[0104] The screening assay can also be used to determine effective doses of agents to optimize disruption or modulation of tau oligomer. For example, the method of screening compounds may involve comparing the sample being analyzed to a sample that does not contain the agent or less agent to determine if the agent modulates or inhibits tau oligomer. "Disruption" of tau oligomer includes the interruption of tau oligomer formation.

[0105] Prospective agents for use in the screening methods may be chemical compounds and/or complexes of any sort, including both organic and inorganic molecules (and complexes thereof). As will be understood in the art, organic molecules are most commonly screened for AD biomarker modulatory activity. In some situations, the prospective agents for testing will include the target AD biomarker (e.g., soluble tau oligomer).

[0106] Suitable agents for screening include, but are not limited to, antioxidants (vitamin E and vitamin C), anti-inflammatory agents (e.g., curcumin, demethoxycurcumin, bisdemethoxycurcumin, and/or morin), antibiotics, chelation agents, cliouinol, ergoloids, estrogen, herbal agents (e.g., ginko biloba, huperzine A, melissa officinalis (lemon balm), etc.), statins, vitamin B, or proteases, protease inhibitors, cysteine proteases, combinations thereof or derivatives thereof. Derivatives include analogs of the above agents that are pharmacologically active.

[0107] It has been found the new observation that tau protein has protease activity and that tau protease is an endopeptidase. It has further been found that tau protease is a cysteine protease. In some embodiments, it has been found that the tau protease is a cysteine protease with similarities to papain. In some embodiments, the composition of tau protease has been discovered. It has also been found: the mechanism for tau protease activity; that tau protease requires tau oligomer formation for activity; tau oligomer formation enables formation of a cleft for the active site; tau oligomer formation is necessary for protease activity; 4R tau subunits enable greater possibilities for the formation of oligomers with free thiols than 3R tau subunits; methods to modulate tau protease activity by modulating the 3R and 4R tau subunit composition; the toxicity associated with the relative overexpression of 4R tau to 3R in multiple neurodegenerative diseases is caused by the increased formation of tau proteases; a specific alignment of tau subunits such that there are free thiols available for the catalytic triad; catalytic triads are composed of a.) Cysteine 291 or Cysteine322, b.) Histidine 299 or Histidine329 or Histidine330, and c.) Glycine164 or 302, 303 or 304; the tau protease can cleave itself; the tau protease can cleave other tau proteins; tau protease cleavage of tau facilitates its aggregation; the tau protease dependent cleavage of tau causes loss of tau function; tau protease formation enables gain of toxic function by cleaving tau and proteins other than tau; tau protease cleaves intracellular proteins facilitating apoptosis and/or senescence of neurons and/or other cell types; tau protease cleaves extracellular proteins such as receptors or neurotansmitters; tau protease cleaves membrane proteins; tau protease facilitates apoptosis by activating proteins in apoptosis such as caspases; tau protease cleaves amyloid precursor protein (APP) facilitating production of beta amyloid; methods to form tau protease structures; methods to purify and/or isolate tau proteases; tau protease activity is a target for small molecule drug discovery for AD and other tauopathies; tau protease is a target for immunotherapy; protein fragments generated by tau protease are biomarkers for AD and other neurodegenerative diseases; and/or the tau proteolytic fragment with apparent molecular weight of .about.17 kDa is a biomarker for disease.

Tau Protein Structure and Function

[0108] Tau is encoded by the MAPT gene located on the long (q) arm of chromosome 17 at position 21.1. Alternative splicing of the second and third exons in the N-terminal portion of tau and the tenth exon yields a total of six protein isoforms. Tau is an intrinsically unstructured protein due to its very low hydrophobic content and has been characterized to have a projection domain, a basic proline-rich region, and an assembly domain. The domain in the carboxyl-terminal portion of the protein critical for tau self-association into oligomers and fibrils, the assembly domain, contains either three or four repeats (3R or 4R) of a conserved tubulin-binding motif depending on alternative splicing of exon 10 encoding the second repeat (Lee et al. 1989). Hexapeptide motifs PHF6* and PHF6 in the second and third repeats, respectively, have propensity to form .beta.-sheet structures which are involved in tau interaction with tubulin to form MTs and tau self-interaction to form pathological aggregates such as paired helical filaments (PHF) (von Bergen et al. 2000; Amos 2004). Tau 4R isoforms have greater microtubule stabilizing ability than 3R isoforms. Only the shortest 3R tau isoform is expressed during fetal development where there are dynamic changes in the cytoskeleton, whereas adult brain normally has a ratio of about 1:1 for 3R to 4R isoforms. Expression of 4R/2N in the hippocampus of tau knockin/knockout mice suppresses proliferation and promotes neuronal differentiation (Sennvik et al. 2007). Hyperphosphorylation of tau, particularly in the assembly domain, decreases the affinity of tau to MTs to regulate MT dynamics and axonal transport (Konzack et al. 2007; Dubey et al. 2008). Additional regions in the basic proline-rich domain and the pseudo-repeat also stabilize MTs by interacting with its negatively charged surface (Mukrasch et al. 2007). The projection domain facilitates interaction with the plasma membrane (Brandt et al. 1995; Maas et al. 2000). Interaction of tau with membranes is also thought to facilitate tau aggregation (Chirita et al. 2003). For recent reviews of tau structure and function see (Wang and Liu 2008; Ballatore et al. 2007).

Tau in Neurodegenerative Diseases

[0109] Genetic evidence has shown that abnormal forms of tau are sufficient for neurodegeneration causing memory loss and other neurological deficits in multiple frontotemporal dementia and sporadic tauopathies (for a recent review of the role of tau in neurodegeneration see Gendron and Petrucelli 2009; Iqbal et al. 2009). The 32 different mutations found in the study of over 100 families can be grouped into categories influencing splicing of the primary transcript and causing changes in amino acid sequence of tau. Most missense mutations are located in the assembly domain and generally reduce the affinity of tau to MTs. Several of these mutations promote aggregation of tau in vitro and in vivo such as P301L and P301S. Mutations in the stem-loop structure at the border of exon 10 and the following intron alter splicing causing aberrations in the ratio of 4R to 3R isoforms demonstrating that maintenance of the proper ratio of tau isoforms is necessary to prevent neurodegeneration and dementia. Sporadic tauopathies such as progressive supranuclear palsy, corticobasal degeneration, Pick's disease and argyrophilic grain disease are characterized by pathology with tau filaments composed predominantly of 4R isoforms and are linked to MAPT mutations (Goedert and Jakes 2005).

[0110] Recent reports also indicate aberrant splicing of tau transcripts in AD demonstrating a common defect with AD and Parkinson disease. Increases in the ratio of 4R to 3R tau mRNAs were found in individual human cholinergic basal forebrain neurons in nucleus basalis and CA1 hippocampal neurons in AD (Ginsberg et al. 2006). Aberrant alternative splicing in sporadic AD was also shown using polymerase colony, a single-molecule-based technology. A trans mechanism involving the reduction of splicing factor htra2-beta-1 in AD was linked to the increase in four-repeat tau isoforms (Conrad et al 2007). Further genetic support for a causative role for tau in AD comes from the observation that tau haplotypes driving higher levels of tau expression increase AD risk (Myers et al. 2005) and from the report of linkage of tau haplotypes with increased CSF tau in people with A.beta. deposition accelerated AD progression (Kauwe et al. 2008). Similarly, increased expression of 4R tau and linkage to MAPT haplotypes has been reported for Parkinson disease (Tobin et al. 2008), the second most common form of neurodegenerative disease following AD. A recent study of tau in primary cultures of human cortical neurons indicated that the 4R tau isoforms are principally involved in tau oligomerization (Deshpande et al. 2008). There are also multiple post-translational modifications to tau protein in AD and other tauopathies that cause both loss of function and gain of toxicity (Mazanetz et al., 2007).

[0111] In some embodiments, the tau proteins have the following isoforms:

TABLE-US-00001 Spliced Residues exons Nomenclature 352 -- 3R/0N 381 3 3R/1N 410 2, 3 3R/2N 383 10 4R/0N 412 3, 10 4R/1N 441 2, 3, 10 4R/2N

[0112] Recent advances in research in AD have highlighted the importance of tau in pathogenesis (Marx, 2007) and its use as a target for the development of disease modifying therapeutics. Evidence from mouse models indicates that tau reduction reverses disease phenotypes (Santacruz et al. 2005; Oddo et al. 2006; Asuni et al. 2007) and that tau is necessary for the development of cognitive deficits in AD models caused by over-expression of A.beta. (Roberson et al. 2007).

[0113] The pathological structures of tau most closely associated with AD progression are tau oligomers in mouse models that also accumulate in human neurodegenerative diseases collectively termed "tauopathies" (Berger et al. 2007; Maeda et al. 2006; Sahara et al. 2007, 2008). NFTs have been implicated in mediating neurodegeneration in AD and tauopathies as it correlates well with cognitive deficits and neuron loss (Arriagada et al., 1992; Bancher 1993; Guillozet et al., 2003; Iqbal et al. 2009). However, the study of animal models of tauopathy has shown that memory impairment and neuron loss is dissociated from accumulation of NFT (Brunden et al. 2008; Rocher et al. 2009). Strong support for this contention came from the analysis of transgenic mice rTg4510 that express tau P301L in the forebrain under control of a tetracycline-regulated promoter. These mice developed memory impairment, neuron loss and NFT when the construct was expressed. However, suppression of expression caused improvement in memory and reduction in neuron loss even as NFTs continued to accumulate clearly demonstrating that pretangle tau species were responsible for the neurodegenerative phenotype (Santacruz et al. 2005). Additionally, there was regional dissociation of neuron loss and NFT pathology in this model (Spires et al. 2006) and in another mouse model expressing all six human isoforms (Andorfer et al. 2003) showing that tangles are not acutely neurotoxic.

[0114] Incongruence between tangle formation, neurodegeneration and behavioral deficits were found in other mouse models of tauopathy and AD. Transgenic mice expressing a human mutant tau P301S construct prone to aggregation developed hippocampal synapse loss and dysfunction, as well as, microglial activation months before the accumulation of filamentous tau inclusions (Yoshiyama et al. 2007). Similarly, a transgenic mouse model expressing human tau protein with two mutations found in FTDP-17 (P301S and G272V) exhibited axonopathy before tangle formation (Leroy et al. 2007). The triple transgenic AD mouse model accumulating both tau and A.beta. pathology was used to study the effects of immuno-reduction of tau and A.beta.. Antibodies against both proteins were needed to improve learning and memory behavior in these mice. Soluble tau, but not NFT, was reduced by the treatment again showing the dissociation between the neurodegenerative phenotype and insoluble tau aggregates (Oddo et al. 2006).

Role of Tau Cleavage in Neurodegenerative Diseases

[0115] Cleavage of tau plays a role in the oligomerization and formation of a pathological tau species in AD (Gamblin et al. 2003; Rissman et al. 2004). Although a number of cellular proteases can cleave tau it is unclear which proteases are facilitating disease progression. Tau can be cleaved by caspase-3 at Asp421 and results in a highly fibrillogenic tau isoform aggregates better than full-length tau and facilitates aggregate formation of full length tau (Gamblin et al. 2003; Rissman et al. 2004). Immunological studies indicate that tau truncated at Asp421 co-localize with fibrillar tau pathologies in AD patient brains (Gamblin et al. 2003; Rissman et al. 2004). Asp-421-cleaved tau is toxic to cultured neurons (Fasulo et al. 2005; Chung et al. 2001; Matthews-Roberson et al. 2008) by causing mitochondrial dysfunction (Quintanilla et al. 2009). However, in a recent publication (Zhang et al. 2009), there was a lack of activated caspase-3 in truncated tau-positive neurons in tauP301L single transgenic and APP/PS1/tauP301L triple transgenic mouse models. Caspase-3 levels in the brain dramatically decreased early in development and remained consistently low in adulthood. Hyperphosphorylation of tau was also found to inhibit caspase-3 cleavage of tau in vivo. Thus, these animal studies indicate that the results from cell-free and cell-based assays (Gamblin et al. 2003; Rissman et al. 2004) do not reflect in vivo conditions and lead to the conclusion that an unidentified, caspase-3 independent pathway is responsible for tau truncation at Asp421 (Zhang et al. 2009). In another study of the appearance of phosphorylated and truncated tau in the brain and spinal cord of mice transgenic for mutant human P301S tau protein it was found that the late appearance and low abundance of tau truncated at Asp421 made it unlikely that truncation at this site was necessary for tau aggregation (Delobel et al. 2008). Using an inducible cell model expressing the microtubule binding domain of tau with the .DELTA.K280 mutation, an unidentified cytosolic protease activity was identified that cleaves tau towards the N-terminus of the microtubule binding repeat domain. Subsequent targeting of the cleaved product to the lysosome led to cathepsin L truncation of tau at the carboxyl terminus creating fragments highly prone to aggregation and subsequent degradation by macroautophagy (Wang et al. 2009).

[0116] Here we show the highly novel mechanism in neurodegenerative diseases that upon self-association tau acquires proteolytic activity that leads to its own fragmentation. This activity accounts for the unknown tau protease activities described above. The structure of the autoproteolytic dimer and trimer composed of 4R tau with one or two free thiols indicates that it is a cysteine protease. In context of the disease process, tau self-association leads to formation of autoproteolytic oligomers that cause loss of tau function through fragmentation of the protein and at the same time facilitate aggregation of the cleaved microtubule binding domain into toxic structures. The proteolytic activity of oligomers formed from 4R tau helps explain the toxicity of 4R tau overexpression common in neurodegenerative diseases. Other proteins may also be targeted by this activity augmenting its toxic gain of function both in the intra- and extra-cellular environments. This proteolytic activity is a novel target that is amenable to small molecule drug discovery, and the autoproteolytic fragments are direct biomarkers for disease progression as there level indicates activity of this pathological tau protease.

[0117] Having now generally described the invention, the same may be more readily understood through the following reference to the following examples, which are provided by way of illustration and are not intended to limit the present invention unless specified.

EXAMPLES

Example 1

[0118] Purified preparations of tau352 or tau441 were incubated at 37.degree. C. in neutral buffer with a very low concentration of hydrogen peroxide to create an oxidizing environment that would not lead to protein degradation. Non-reducing sample buffer was used to prepare samples for SDS-PAGE using a 4-20% gradient gel (BioRad) to resolve purified tau monomer, dimer, trimer and tetramer. Silver staining was performed with the Silver Express kit (Invitrogen) shown in FIG. 4.

Example 2

[0119] Purified preparations of tau352 and tau441 were incubated at a 1:1 ratio or 1:4 ratio of tau352:tau441 at 37.degree. C. in pH neutral buffer for 45 min. or 2 days. Non-reducing sample buffer was used to prepare samples for SDS-PAGE using a 4-20% gradient gel (BioRad) to resolve purified tau monomer, dimer, trimer and tetramer. Silver staining was performed with the Silver Express kit (Invitrogen) Shown in FIG. 5.

Example 3

[0120] Purified Tau412 monomer, dimer and trimer were incubated in 50 mM Tris-HCl pH 7.4 overnight at 37.degree. C. Preparations were buffer exchanged into artificial CSF buffer (in mM: 124.0 NaCl, 4.4 KCl, 1.0 Na2HPO4, 25.0 NaHCO3, 2.0 CaCl2, 2.0 MgSO4, and 10.0 glucose) using PD10 columns (GE Lifesciences) and the first five fractions (10 .mu.l) were analyzed by SDS-PAGE (4-20% gradient gel) and stained with Coumassie blue shown in FIG. 8.

Example 4

[0121] Five post-mortem CSF specimens (10 .mu.l each) from individuals with advanced AD were analyzed by SDS PAGE using a 4-20% gradient gel (BioRad). Specimens were provided by New York Brain Bank, Taub Center, Columbia University Medical Center. Samples were prepared with standard Laemmli buffer (sample buffer containing reductant) or sample buffer without reductant. Proteins were transferred to a nitrocellulose membrane and immunoblot was performed with mAb HT7 recognizing all tau isoforms and independently of tau phosphorylation (shown in FIG. 17).

Example 5

[0122] Five post-mortem CSF specimens (15 .mu.g each) from individuals with advanced AD were analyzed by SDS PAGE using a 4-20% gradient gel (BioRad). Samples were prepared with standard sample buffer containing reductant or sample buffer without reductant. Specimens were provided by New York Brain Bank, Taub Center, Columbia University Medical Center. The samples with reductant were heated 5 min at 95.degree. C. before loading on gel. Proteins were transferred to a nitrocellulose membrane and immunoblot was performed with mAb HT7 recognizing all tau isoforms and independently of tau phosphorylation (shown in FIG. 18).

Example 6

[0123] This example illustrates the amino acid positions where tau oligomer cuts itself.

Tau Oligomer Protease Fragment Analysis for Tau412 and Tau441 (Note that the Sequence Number Used are for Tau441 for the Cut Sites)

[0124] Tau412 Cut at Glycine164 N-terminal fragment

Fragment 1

MW=13,989.9

[0125] Estimated pI=4.62 Estimated charge at pH 7.40=-13.9

TABLE-US-00002 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPPQT PTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVS KSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKG

[0126] Tau412 Cut at Glycine164 C-terminal fragment

Fragment 2

MW=28,978.8

Estimated PI=9.98

[0127] Estimated charge at pH 7.40=20.5

TABLE-US-00003 QANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPS LPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTE NLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVD LSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVP GGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSST GSIDMVDSPQLATLADEVSASLAKQGL

[0128] Tau 412 Cut at Glycine302 N-terminal fragment

Fragment 3

MW=28,323.2

Estimated PI=8.73

[0129] Estimated charge at pH 7.40=3.2

TABLE-US-00004 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPPQT PTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVS KSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAP KTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVV RTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIIN KKLDLSNVQSKCGSKDNIKHVPG

[0130] Tau 412 Cut at Glycine302-C-terminal fragment

Fragment 4

MW=14,645.5

Estimated PI=8.95

[0131] Estimated charge at pH 7.40=3.4

TABLE-US-00005 GGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQS KIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGD TSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0132] Tau441 Cut at Glycine164 N-terminal fragment

Fragment 5

MW=16,889.0

[0133] Estimated pI=4.56 Estimated charge at pH 7.40=-17.8

TABLE-US-00006 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQT PTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEG TTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTK IATPRGAAPPGQKG

[0134] Tau441 Cut at Glycine302 C-terminal fragment

Fragment 6

MW=28,978.8

Estimated PI=9.98

[0135] Estimated charge at pH 7.40=20.5

TABLE-US-00007 QANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPS LPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTE NLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVD LSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVP GGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSST GSIDMVDSPQLATLADEVSASLAKQGL

[0136] Tau441 Cut at Glycine302 N-terminal fragment

Fragment 7

MW=31,222.3

[0137] Estimated pI=7.14 Estimated charge at pH 7.40=-0.6

TABLE-US-00008 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQT PTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEG TTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTK IATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSP GSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPM PDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHV PG

[0138] Tau441 Cut at Glycine302 C-terminal fragment

Fragment 8

MW=14,645.5

Estimated PI=8.95

[0139] Estimated charge at pH 7.40=3.4

TABLE-US-00009 GGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQS KIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGD TSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0140] The references disclosed below are hereby incorporated by reference into the present disclosure for all that they disclose.

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[0216] It will be apparent to those skilled in the art that various modifications and variations can be made to various embodiments described herein without departing from the spirit or scope of the teachings herein. Thus, it is intended that various embodiments cover other modifications and variations of various embodiments within the scope of the present teachings.

Sequence CWU 1

1

71352PRTHomo sapiens 1Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala 35 40 45Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 50 55 60Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp65 70 75 80Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 85 90 95Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 100 105 110Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 115 120 125Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 130 135 140Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro145 150 155 160Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys 165 170 175Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 180 185 190Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu 195 200 205Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val 210 215 220Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His225 230 235 240His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp 245 250 255Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr 260 265 270His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr 275 280 285Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val 290 295 300Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser305 310 315 320Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu 325 330 335Ala Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 340 345 3502381PRTHomo sapiens 2Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60Asp Ala Lys Ser Thr Pro Thr Ala Glu Ala Glu Glu Ala Gly Ile Gly65 70 75 80Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala 85 90 95Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys 100 105 110Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala 115 120 125Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala 130 135 140Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro145 150 155 160Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr 165 170 175Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg 180 185 190Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser 195 200 205Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu 210 215 220Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln225 230 235 240Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser 245 250 255Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro 260 265 270Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp 275 280 285Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro 290 295 300Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu305 310 315 320Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser 325 330 335Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser 340 345 350Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu 355 360 365Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 3803383PRTHomo sapiens 3Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala 35 40 45Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 50 55 60Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp65 70 75 80Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 85 90 95Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 100 105 110Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 115 120 125Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 130 135 140Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro145 150 155 160Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys 165 170 175Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 180 185 190Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu 195 200 205Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu 210 215 220Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys225 230 235 240His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp 245 250 255Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His 260 265 270Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe 275 280 285Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His 290 295 300Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe305 310 315 320Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr 325 330 335Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn 340 345 350Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala 355 360 365Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 3804410PRTHomo sapiens 4Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Pro 35 40 45Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60Asp Ala Lys Ser Thr Pro Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr65 70 75 80Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met 85 90 95Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys 100 105 110Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro 115 120 125Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr 130 135 140Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser145 150 155 160Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly 165 170 175Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro 180 185 190Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala 195 200 205Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn 210 215 220Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly225 230 235 240Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val 245 250 255Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly 260 265 270Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr 275 280 285Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly 290 295 300Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln305 310 315 320Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly 325 330 335Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys 340 345 350Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val 355 360 365Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly 370 375 380Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu385 390 395 400Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 405 4105412PRTHomo sapiens 5Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Pro 35 40 45Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60Asp Ala Lys Ser Thr Pro Thr Ala Glu Ala Glu Glu Ala Gly Ile Gly65 70 75 80Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala 85 90 95Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys 100 105 110Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala 115 120 125Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala 130 135 140Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro145 150 155 160Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr 165 170 175Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg 180 185 190Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser 195 200 205Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu 210 215 220Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln225 230 235 240Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser 245 250 255Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro 260 265 270Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys 275 280 285Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly 290 295 300Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg305 310 315 320Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly 325 330 335Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn 340 345 350Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro 355 360 365Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser 370 375 380Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala385 390 395 400Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 405 4106441PRTHomo sapiens 6Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val65 70 75 80Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val 115 120 125Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly 130 135 140Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro145 150 155 160Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys225 230 235 240Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val 245 250 255Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly 260 265 270Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln 275 280 285Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly 290 295 300Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser305 310 315 320Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln 325 330 335Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340 345 350Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn 355 360 365Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala 370 375 380Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser385 390 395 400Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser 405 410 415Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val 420 425 430Ser Ala Ser Leu Ala Lys Gln Gly Leu 435 440742PRTHomo sapiens 7Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40

Claims

1. A tau protease associated with AD.

2. (canceled)

3. A protease inhibitor that inhibits or blocks tau protease activity.

4. A protease inhibitor of claim 3, wherein the protease inhibitor is a cysteine protease inhibitor.

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. A composition comprising tau oligomer having a staggered alignment comprising one or two free thiol moieties from a cysteine amino acid of the tau oligomer, wherein the tau oligomer comprises 3R or 4R isoforms of tau monomer.

13. (canceled)

14. A composition comprising tau protease, wherein the tau oligomer protease is a cysteine protease.

15. A composition according to claim 14, wherein the cysteine protease is adapted for screening drug compounds that enhance or inhibit the tau oligomer proteolytic activity, wherein the tau monomeric unit comprises SEQ ID NO. 1-6. or derivatives.

16. A composition according to claim 15, wherein the cysteine protease is inhibited by a cysteine protease inhibitor that blocks or prevents its activity and is useful in treating tauopathies or Alzheimer's disease.

17. A composition according to claim 15, wherein the tau oligomer protease cleaves tau oligomer to form tau cleavage products and the tau cleavage products function as biomarkers for tauopathies or Alzheimer's disease.

18. A composition according to claim 17, wherein the cysteine protease comprises 4R tau oligomer, 4R and 3R tau oligomer or a combination thereof.

19. A composition comprising extracellular tau oligomer protease, wherein the extracellular tau oligomer protease is a target for immunotherapy.

20. (canceled)

21. (canceled)

22. A cleavage product of tau protein, wherein the cleavage product is formed from tau protease and the cleavage product comprises fragments of tau oligomer, and the tau oligomer is in at least one conformation comprising tau dimer, tau trimer, tau tetramer, tau pentamer, tau hexamer, tau septamer, tau octamer, tau nonamer, tau decamer, tau unadecamer, tau dodecamer, 3R tau, 4R tau, or mixtures of 3R tau and 4R tau or a combination thereof.

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)