WO2002010770A2

WO2002010770A2 - Improved methods for identifying reagents which inhibit tau polymerization

Info

Publication number: WO2002010770A2
Application number: PCT/US2001/041512
Authority: WO
Inventors: T. Chris Gamblin; Lester I. Binder
Original assignee: Neuronautics, Inc.
Priority date: 2000-07-31
Filing date: 2001-07-31
Publication date: 2002-02-07
Also published as: AU2001283528A1; WO2002010770A3

Abstract

The current invention provides novel methods for stimulating the polymerization of tau protein by altering oxidative conditions for the polymerization reaction and/or by altering the oxidative state of a fatty acid used to stimulate the polymerization reaction. The current invention provides a method for screening reagents for the ability to effect tau polymerization using the novel methods of the invention for inducing tau polymerization. Finally, the current invention provides methods for regulating the assembly of tau protein in the brain of mammals in need of such regulation, by administering to the mammal, an effect amount of an inhibitor of fatty acid oxidation.

Description

IMPROVED METHODS FOR IDENTIFYING REAGENTS WHICH INHIBIT TAU POLYMERIZATION

RELATED APPLICATION

This application is based on, and claims benefit of, United States Provisional Application Serial Number 60/221 ,777 filed on July 31 , 2000.

FIELD OF THE INVENTION

The current invention relates to methods for identifying reagents that may be effective for treating certain neurological disorders and methods for treating these neurological disorders. More specifically, the current invention relates to methods for identifying reagents that inhibit tau polymerization and methods for treating tau polymerization in vivo by treating a subject with a reagent that inhibits fatty acid oxidation.

BACKGROUND

The microtubule-associated protein tau is a soluble cytosolic protein that is believed to contribute to the maintenance of the cytoskeleton (Johnson et al., Alzheimer's Disease Review 3: 125 (1998); Buee et al., Brain Research Reviews 1 : Citation in progress (2000)). However, in many disease states, tau protein is induced by unknown cellular conditions to self-associate into filamentous structures (Spillantini et al., Trends Neurosci. 21 : 428 (1998)). These filamentous forms of tau can be found in such varied neurodegenerative disorders as Alzheimer's disease (AD) (Wood et al., Proc. Natl. Acad. Sci. U.S.A. 83: 4040 (1986); Kosik et al., Proc. Natl. Acad. Sci. U.S. A 83: 4044 (1986); Grundke-lqbal et al., J. Biol. Chem. 261 : 6084 (1986)), corticobasal degeneration (CBD) (Feany et al., Am. J. Pathol. 146: 1388 (1995)), progressive supranuclear palsy (PSP) (Tabaton et al., Ann. Neurol. 24: 407 (1988)), Pick's disease (PD) (Murayama et al., Ann. Neurol. 27: 394 (1990)), Down syndrome (Papasozomenos et al., Lab Invest. 60: 123 (1989)), and frontotemporal dementias and Parkinsonism linked to chromosome 17 (FTDP-17) (Spillantini et al., Proc. Natl. Acad. Sci. U.S.A. 94: 4113 (1997)). There remains a need for the identification of effective therapies for these neurodegenerative disorders.

There is still debate as to the involvement of tau fibril formation in the onset of neurodegeneration. It is not known whether abnormal tau polymerization causes or modulates the neurodegeneration process or whether it is simply a byproduct of the process. For example, in AD it is hotly debated whether the dementia-causing pathological structures are the amyloid-beta positive senile plaques, the tau-positive neurofibrillary tangles, or a combination of both (Hardy et al., Nat. Neurosci. 1 : 355 (1998)). In order to understand the etiopathogenesis of AD, there remains a need to identify molecular mechanisms which lead to the polymerization of the pathological structures themselves.

Much of what is currently known regarding tau polymerization stems from in vitro assembly assays. However, with few exceptions, the conditions that have been used to achieve tau polymerization have been extremely nonphysiological. The first experiment describing the self-association of tau protein into AD-like filaments involved 60 hours of incubation in 8M urea (Montejo de Garcini et al., J. Biochem. (Tokyo) 102: 1415 (1987)). Other experiments have required significant truncations of the molecule followed by chemical cross-linking (Wille et al., J. Cell. Biol. 118: 573 (1992)), extremely high protein concentrations (40μM) (Goedert et al., Nature 383: 550 (1996)), incubation periods up to six weeks (Schweers et al., Proc. Natl. Acad. Sci. U.S.A. 92: 8463 (1995)), or combinations of these techniques. Although relatively mild conditions have been described which result in the polymerization of low concentrations of biochemically purified tau protein (Wilson et al., J. Biol. Chem. 270: 24306 (1995)), this process can be greatly enhanced by the addition of polyanionic compounds under oxidative conditions (Goedert et al., Nature 383: 550 (1996); Kampers et al., FEBS Lett. 399: 344 (1996); Hasegawa et al., J. Biol. Chem. 272: 33118 (1997); Friedhoff et al. , Biochemistry 37: 10223 (1998); Friedhoff et al. , Proc. Natl. Acad. Sci. U.S.A. 95: 15712 (1998); Nacharaju et al., FEBS Lett. 447: 195 (1999)) and the addition of free fatty acids under reducing conditions (Nacharaju et al., FEBS Lett. 447: 195 (1999); Wilson et al., Am. J. Pathol. 150: 2181 (1997); King et al., Biochemistry 38: 14851 (1999); King et al., J. Neurochem. 74: 1749 (2000); Gamblin. et al., Biochemistry 39: 6136 (2000)). However, there remains a need to identify improved methods for further enhancing the polymerization of tau protein in vitro in order to help facilitate the identification of reagents which can be used to treat diseases involving tau polymerization in vivo.

Various in vitro polymerization techniques have been used to investigate the in vitro polymerization of tau. For example, it has been shown that the fatty acid induction of tau polymerization proceeds through a ligand- dependent mechanism under reducing conditions (King et al., Biochemistry 38: 14851 (1999)). Another set of experiments showed that, contrary to expectations, extensive phosphorylation of the tau molecule with various protein kinases inhibited the polyanion induction of polymerization (Schneider et al., Biochemistry 38: 3549 (1999)). Some of the factors leading to tau polymerization in the disease state are now being studied. As mentioned above, extensive tau pathology is observed in a class of neurodegenerative disorders called FTDP-17. These disease states have been linked to mutations in the tau gene that lead to missense point mutations or changes in the isoform expression of the tau protein. In vitro experiments have shown that several of the single amino acid missense point mutations found in FTDP-17 can lead to increased filament formation (Nacharaju et al., FEBS Lett. 447: 195 (1999); Gamblin et al., Biochemistry 39: 6136 (2000); Goedert et al., Nat. Med. 5: 454 (1999)). It has also been shown that tau isoforms have different polymerization characteristics, which could lead to increased tau pathology in cases of FTDP-17 with altered isoform compositions (King et al., J. Neurochem. 74: 1749 (2000)). However, a strong link between the risk factors associated with the most common neurodegenerative disorder, AD, ^■ and increased tau polymerization has not been established. Therefore, there remains a need to identify AD risk factors that are associated with tau polymerization in order to accelerate the development of effective AD therapies.

A number of risk factors have been identified which have the common characteristic of being potential contributors to oxidative stress. Thus, oxidative stress may play a major role in the etiology of Alzheimer's disease (AD). The normal aging process, head trauma, increased levels of heavy metals (e.g., Fe, Al, Hg), and, especially in the case of AD, aggregation of the β-amyloid protein (Aβ) are all thought to be potential contributors to increased oxidative stress. In the oxidative stress hypothesis for AD, free radicals generated by these risk factors, possibly in the form of reactive oxygen species, would then attack biological molecules that are sensitive to oxidation, such as proteins, DNA, and lipids/fatty acids, causing a cascade that would eventually lead to neurodegeneration (see, e.g., Markesbery et al., Free Radic. Biol. Med. 23: 134 (1997)).

There is direct evidence that sensitive molecules in vulnerable AD brains are modified by oxidative stress. Free radicals can lead to the carbonyl derivatization of enzymes such as glutamine synthetase and creatine kinase. This process is quickly followed by protease degradation of the enzymes. DNA is also sensitive to oxidative stress. Increases in the adduct 8-hydroxy-2'-deoxyguanisine have been reported for mitochondrial DNA, and to a lesser extent nuclear DNA, in AD brains when compared to age-matched controls. In addition, a two-fold increase in oxidative damage to DNA through strand breaks has been described in the brains of AD patients (see, e.g., Markesbery et al., Free Radic. Biol. Med..23: 134 (1997)).

Polyunsaturated fatty acids (FA) are especially vulnerable to oxidative stress since their double bonds make the removal of H+ by free radicals relatively easy. Although some reports disagree on the location of FA oxidation in AD brain (see, e.g., Markesbery, Brain Pathol. 9: 133 (1999)), it is clear that thiobarbituric acid reactive substances (a marker for FA oxidation) are elevated in these patients. In addition, many FA breakdown products including malondialdehyde (MDA) and hydroxynonenal (HNE) can be detected at greater than normal levels in AD patients. Increased amounts of specific FA metabolites, such as the F2-isoprostanes and F4-neuroprostanes, can also be found in the affected brain regions of AD patients and even in the cerebrospinal fluid of probable AD patients (Montine et al., Neurology 52: 562 (1999)). In addition to the toxicity of some of the FA breakdown products (such as HNE), the alterations in membrane fluidity as a result of FA oxidation may also have deleterious effects in AD patients.

While it is becoming clear that oxidative stress is likely a significant contributor to the neurodegenerative process, it is not clear how these factors are related to the two major pathological structures found in AD, senile plaques and neurofibrillary tangles. Senile plaques (SPs), which consist mainly of polymerized Aβ protein, may contribute to oxidative stress though the generation of free radicals, but their involvement in the neurodegenerative process is not clear. While the addition of Aβ to cultured neurons results in increased protein oxidation and cell death (Busciglio et al., Neuron 14: 879 (1995)), animal models that contain elevated amounts of SPs do not show signs of neurodegeneration (Takeuchi et al., Am. J. Pathol. 157: 331 (2000)). In addition, the presence of SPs does not correlate well with the degree of dementia in AD patients (Arriagada et al., Neurology 42: 631 (1992)).

In contrast, neurofibrillary tangles (NFTs) consisting primarily of polymerized tau molecules do correlate well with the degree of dementia in AD (Arriagada et al., Neurology 42: 631 (1992)). In addition, an emerging class of neurodegenerative disorders that involve the fronto-temporal regions of the brain appear to be caused by pathological tau inclusions in the absence of SPs (Spillantini et al., Proc. Natl. Acad. Sci. U.S.A. 94: 4113 (1997)). Finally, the formation of tau filaments appears to directly cause neurodegeneration in an animal model. Overexpression of the tau protein in lamprey ABC neurons leads to filament formation and subsequent neuronal death (Hall et al., Proc. Natl. Acad. Sci. U.S.A. 94: 4733 (1997); Hall et al., J. Cell Sci. 113: 1373 (2000)).

Although the formation of NFTs may be relevant to the neurodegenerative process, it is not clear how they are involved with the oxidative stress hypothesis for_.AD. Previously, the only link between oxidative stress and tau filament formation has been the reports which describe the prerequisite oxidation of the tau molecule for its polymerization in vitro. The oxidation of a specific cysteine that results in disulfide-linked dimers of tau has been shown to be a necessary first step before the induction of tau filament formation (Schweers et al., Proc. Natl. Acad. Sci. U.S.A. 92: 8463 (1995)). It should be noted, however, that these results required special conditions to be effective. First, the experiments were performed on tau molecules truncated at both the amino- and carboxy-terminal regions so that only the microtubule binding repeat (MTBR) regions remained. Secondly, only three microtubule binding repeats could be used. This was due to the fact that there are two cysteines in the tau molecule, one in MTBR2 and one in MTBR3. If both cysteines were left in the tau constructs, they preferentially formed intramolecular disulfides instead of forming dimers.

The tau oxidation theory does not seem tenable for several reasons. The cellular markers for protein oxidation that have been identified in AD as a result of oxidative stress are the creation of protein carbonyls and the nitration of tyrosine residues (see, e.g., Markesbery et al., Brain Pathol. 9: 133 (1999)). It is not clear whether oxidative stress would actually result in the cysteine oxidation and subsequent dimerization of tau molecules. The filamentous tau structures found in AD consist of all six isoforms of the tau molecule, including those with four MTBR (see, e.g., Spillantini et al., Trends in Neurosciences, 21 :428 (1998)). Therefore, tau molecules containing two cysteines are capable of polymerizing in vivo. If cysteine oxidation of the tau molecule is a prerequisite and the intramolecular disulfide formation is favored over dimerization, one would not expect the four MTBR isoforms of tau to be present in the filaments that make up the NFTs. Therefore, there remains a need to determine the effects of oxidation on tau polymerization in vivo and the mechanism by which oxidative stress induces neurodegeneration in AD.

The current invention describes effective methods for identifying reagents that inhibit tau polymerization. These methods rely on the discovery described herein of vastly improved protocols for inducing tau polymerization in vitro . The improved protocols utilize modifications of the oxidative environment of the polymerization reaction or modifications of the oxidative state of components of the in vitro polymerization reaction. Furthermore, the current invention identifies novel methods for treating disorders involving tau polymerization based on the discoveries related to the improved protocols and an in vivo mechanism of tau polymerization that follows from the in vitro polymerization discoveries.

SUMMARY OF THE INVENTION In one aspect, the present invention provides a method of stimulating polymerization of tau. The method generally comprises:

(a) providing a polymerization buffer;

(b) adding an effective amount of a free fatty acid to the polymerization buffer to form a fatty acid-containing polymerization buffer; (c) adding substantially purified tau protein to the fatty acid- containing polymerization buffer to form a tau polymerization reaction mixture; and

(d) incubating the tau polymerization reaction mixture to permit the formation of tau filaments, wherein the free fatty acid is effectively oxidized during or before the incubation, thereby stimulating the polymerization of tau.

In one embodiment, the method comprises:

(a) providing a polymerization buffer;

(b) adding an effective amount of a free fatty acid and a substantially purified tau protein to the polymerization buffer to form a tau polymerization reaction mixture; and

(c) incubating the tau polymerization reaction mixture to permit the formation of tau filaments, wherein the free fatty acid is effectively oxidized during or before the incubating, thereby stimulating the polymerization of tau.

In one embodiment, the method comprises:

(a) providing a polymerization buffer with an oxidative environment;

(b) adding an effective amount of a free fatty acid to the polymerization buffer to form a fatty acid-containing polymerization buffer; (c) adding substantially purified tau protein to the fatty acid- containing polymerization buffer to form a tau polymerization reaction mixture;

(d) incubating the tau polymerization reaction mixture to permit the formation of tau filaments, thereby stimulating the polymerization of tau.

In one embodiment, the method of this aspect of the invention further comprises (e) detecting the tau filaments in the tau polymerization reaction mixture. In another embodiment, the method further comprises (f) analyzing the tau filaments in the tau polymerization reaction mixture. In another embodiment of this aspect of the invention, steps (d), (e), and (f) are repeated over time to provide a determination of a rate of tau filament formation.

In one embodiment, the free fatty acid is selected from the group consisting of arachadonic acid, palmitoleic acid, oleic acid, linoleic acid, docosahexaenoic acid, and stearic acid. In one embodiment, the fatty acid is arachidonic acid. In another embodiment, the fatty acid is docosahexaenoic acid.

In another embodiment of this aspect of the invention, the substantially purified tau protein is selected from the group consisting of mammalian brain tau protein and a recombinant mammalian tau protein capable of forming filaments. In one embodiment, the recombinant mammalian tau protein is a recombinant wild type tau protein. In another embodiment, the recombinant mammalian tau protein is recombinant tau protein 2N4R. In another embodiment, the recombinant mammalian tau protein is a tau mutant having no cysteine residues. In another embodiment, the recombinant mammalian tau protein is a combination of the above- mentioned substantially purified tau proteins.

In another embodiment, after adding an effective amount of fatty acid, the fatty acid-containing buffer is incubated for at least 5 minutes before adding the substantially purified tau protein. In another embodiment, the incubation period is at least 15 minutes. In another embodiment, the incubation period is at least 30 minutes. In another embodiment, the incubation period is at least 60 minutes. In another embodiment, the method further comprises adding an oxidizer to the fatty acid-containing polymerization buffer before the incubating step. In one embodiment, the oxidizer is selected from the group consisting of a solution of FeCI₃, ADP, and ascorbic acid, a solution of iron citrate, a solution of enzymes that generate specific metabolites of fatty acids, and ultraviolet radiation. In one embodiment, the oxidizer is an enzyme that generates a specific metabolite of fatty acids, wherein the enzyme is selected from the group consisting of cyclooxygenases and lipoxygenases. In another embodiment, the oxidizer is about 50 μM FeCI₃, about 20 mM ADP, and about 10 mM ascorbic acid.

In another embodiment, the method further includes the step of adding an effective amount of a reducing agent to the fatty acid-containing polymerization buffer before the incubating step. In one embodiment, the method comprises: (a) providing a polymerization buffer;

(b) adding an effective amount of a free fatty acid to the polymerization buffer to form a fatty acid-containing polymerization buffer;

(c) adding substantially purified tau protein to the fatty acid- containing polymerization buffer to form a tau polymerization reaction mixture; and

(d) adding an effective amount of a reducing agent to the fatty acid-containing polymerization buffer;

(e) incubating the tau polymerization reaction mixture to permit the formation of tau filaments. In one embodiment, the reducing agent is selected from the group consisting of dithiothreitol (DTT), dithioerythreitol, 2-mercaptothanol, and reduced gluthathione. In another embodiment, the reducing agent is DTT present at a concentration of between about 0.5 mM and about 50 mM. In another embodiment, the DTT is present at a concentration of between about 1 mM and about 25 mM. In another embodiment, the DTT is present at a concentration of between about 2.5 mM and about 10 mM. In another embodiment, the DTT is present at a concentration of about 5 mM. In another aspect, the current invention provides a method for identifying reagents that effect tau polymerization. Typically, these reagents inhibit tau polymerization. The method of this aspect of the invention includes the general method and all of the specific embodiments for stimulating polymerization of tau protein described above and additional steps as described below. The method for identifying reagents that effect tau polymerization comprises:

(a) providing a polymerization buffer;

(c) adding substantially purified tau protein to the fatty acid- containing polymerization buffer to form a tau polymerization reaction mixture;

(d) adding a test reagent to the tau polymerization reaction mixture to form a test reagent/tau polymerization reaction mixture;

(e) incubating the test reagent/tau polymerization reaction mixture to permit the formation of tau filaments, wherein the free fatty acid is effectively oxidized during or before the incubating; and

(f) detecting the tau filaments in the test reagent/tau polymerization reaction mixture, thereby screening the test reagent for the ability to effect tau polymerization. Preferably, this method is used to identify reagents that inhibit tau polymerization.

This aspect of the invention includes all of the embodiments described above for the method of stimulating polymerization of tau protein. The method for identifying reagents that effect tau polymerization can be used to screen for reagents which effect, and preferably prevent and/or inhibit, tau polymer formation in the brains of patients with AD and other related neurodegenerative diseases characterized, at least in part, by tau polymer formation. Another aspect of the current invention is a method for regulating the assembly of the protein tau in the brain of a mammal in need of such a method, wherein the method comprises administering to the mammal a pharmacologically effective amount of an inhibitor of fatty acid oxidation. In one embodiment, the mammal is a human. In another embodiment, the inhibitor of fatty acid oxidation is selected from the group consisting of vitamins with antioxidative properties and non-steroidal anti-inflammatory drugs (NSAIDS). In one embodiment, the vitamin with anti-oxidative properties is selected from the group consisting of Vitamin E, beta carotene, and Vitamin C. In another embodiment, the inhibitor of fatty acid oxygenation is a non-steroidal anti-inflammatory drug selected from the group consisting of aspirin, dilofenic, and ibuprofen. In another embodiment, the inhibitor of fatty acid oxygenation is a selective inhibitor of cyclooxygenase-2. In one embodiment, the cyclooxygenase inhibitor is administered in an amount selected from about 1000 mg per day to about 2500 mg per day. In one embodiment, the cyclooxygenase inhibitor is ibuprofen administered in an amount selected from about 1000 mg per day to about 2500 mg per day. In one embodiment, the administering is performed repeatedly over a period of at least one week. In one embodiment, the administering is performed repeatedly over a period of at least one month. In one embodiment, the administering is performed repeatedly over a period of at least three months. In one embodiment, the administering is performed repeatedly over a period of at least one year. In another embodiment, the administering is performed at least once monthly. In another embodiment, the administering is performed at least once weekly. In another embodiment, the administering is performed at least once daily. In another embodiment, the administering is performed at least once weekly . for at least one month. In another embodiment, the administering is performed at least once per day for at least one month.

In one embodiment, the method for regulating the assembly of the protein tau in the brain of a patient, comprises: identifying a patient in need of a method for inhibiting tau polymerization in the brain; and administering to the patient a pharmacologically effective amount of an inhibitor of fatty acid oxidation.

In one embodiment, the identifying being based on identifying mutant genomic subtypes of tau in the patient. In another embodiment, the identifying is other than a diagnosis of Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of intensity of scattered light versus time for tau polymerization reactions in the presence or absence of dithiothreitol (DTT). Wild type HT40 tau (4uM) plus 75uM arachidonic acid (AA) in the presence (open circles) and absence (closed circles) of 5mM DTT. Reactions were done at room temperature and monitored by the increase in the intensity of scattered light using a laser light scattering (LLS) assay. The data points are the average of eight trials +/- one standard error of the mean. FIG. 2 is a graph of intensity of scattered light versus time for polymerization reactions in the presence or absence of DTT using a tau protein having no cysteine residues. C291 A/C322A double mutant tau protein (4μM) and 75μM AA were incubated at room temperature in the presence (open circles) and absence (closed circles) of 5mM DTT. The reactions were monitored by LLS. The data points are the average of four trials +/- one standard error of the mean.

FIG. 3 is a graph of intensity of scattered light versus early time points for tau polymerization reactions in the presence or absence of DTT using HT40 tau (FIG. 1 ) and a double mutant cysteine-less tau (FIG. 2). Data from the light scattering experiments of FIGS. 1 and 2 are plotted for only the initial 20 minutes to facilitate comparisons of apparent initial velocities of polymerization. HT40 in the absence of DTT is shown as open circles. HT40 in the presence of DTT is represented by closed circles. The double mutant protein is symbolized by closed triangles. FIG. 4 is a series of electron micrographs showing the morphological . differences of filaments formed under different cysteine oxidative conditions. Wild type tau protein in (A) the presence or (B) the absence of 5mM DTT and C291A/C322A double mutant protein (Cysless) and in (C) the presence or (D) the absence of 5mM DTT were incubated with 75μM AA for 5 hours. Samples were removed and viewed by electron microscopy. The images were taken at a magnification of 20,000x. The size bar represents 200nm. FIG. 5 is a series of graphs showing the mass distribution of filaments formed from HT40 and the double mutant tau protein in the presence and absence of DTT. The relative distribution of filament mass is plotted in histograms (bin size = 50nm) for (A) HT40 plus DTT, (B) HT40 in the absence of DTT, (C) double mutant plus DTT, and (D) double mutant in the absence of DTT. Filament lengths were measured manually from regions of micrographs similar to those shown in FIG. 3. Regions from several different micrographs were measured and the relative mass distributions of the filaments were averaged together to generate the histograms; 656, 385, 94, and 204 total filaments were measured for A, B, C, and D, respectively.

FIG. 6 is a graph of filament length/field for polymerization reactions carried out with several FA inducers in the presence and absence of an anti-oxidant. Two different FA inducers: arachidonic acid (AA) and docosahexaenoic acid (DHA), were added to 4μm tau solutions at a final concentration of 75 μm either in the presence or absence of 0.1 % BHT. Samples were analyzed by quantitative electron microscopy since the relative insolubility of BHT interferes with LLS. Error bars represent one standard deviation from the mean (five representative fields were measured for each condition). The amount of tau polymerization after 1 hour at 37°C was greatly reduced for both FA employed in the presence of 0.1% BHT.

FIG. 7 is a series of micrographs showing the morphology of filaments formed in the presence and absence of the antioxidant BHT for both wild type tau and Cysless tau. (A) wild type tau protein and (C) Cysless mutant at concentrations of 4μM were incubated in the presence of 75μM AA and 5mM DTT at 37°C for five hours. Both forms of tau were induced to form filaments. The addition of BHT greatly reduced the amount of filament formation for both (B) wild type tau (HT40) and (D) Cysless tau. Images were taken at a magnification of 20,000x. The size bar represents 200nm. FIG. 8 is a graph of the total length of filaments per field for various oxidative environments. 4 μM tau solutions + 75 μM fatty acid (AA = black bars, DHA =-gray bars) were incubated for 3 hours at 37°C under different oxidative conditions. The conditions are numbered on the x-axis: Condition 1 : polymerization buffer; Condition 2: one hour pre-incubation of FA in polymerization buffer at 37°C before the addition of tau; Condition 3: polymerization buffer + oxidation mixture of 50 μm FeCI₃, 20 mM ADP, and 10 mM /ascorbic acid (Fe/ADP/ascorbate); Condition 4: one hour pre-incubation of FA in polymerization buffer + oxidation mixture of Fe/ADP/ascorbate at 37°C before the addition of tau. Samples were quantified by electron microscopy to be consistent with the BHT inhibition experiment. Error bars represent one standard deviation from the mean of five representative fields.

FIG. 9 is a schematic diagram of a proposed model for oxidative stress-induction of neurodegeneration in AD.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Methods for stimulating tau polymerization. In one aspect, the present invention provides a method of stimulating polymerization of tau protein. The method generally comprises: (a) providing a polymerization buffer;

(d) incubating the tau polymerization reaction mixture to permit the formation of tau filaments, wherein the free fatty acid is effectively oxidized during or before the incubating, thereby stimulating the polymerization of tau. In one embodiment, the method of this aspect of the invention further comprises (e) detecting the tau filaments in the tau polymerization reaction mixture. In another embodiment, the method further comprises (f) analyzing the tau filaments in the tau polymerization reaction mixture. In another embodiment of this aspect of the invention, steps (d), (e), and (f) are repeated over time to provide a determination of a rate of tau filament formation. An "effective amount" of fatty acids is an amount that is sufficient to induce polymerization of tau. This concentration is typically about 1 to about 1000 μM, preferably about 10 to about 100 μM. However, the effective amount of fatty acid may vary depending on the specific reaction conditions, the specific fatty acid, and the oxidative state of the fatty acid.

"Substantially purified" tau protein is tau protein that is separated sufficiently from other proteins and macromolecules isolated during tau protein production to permit the tau protein to polymerize into tau filaments. Methods for obtaining substantially purified tau protein are described below. A wide range of pH, temperature, time, and ionic strength can be used with the current invention. Acceptable ranges for these factors are summarized below and described in Wilson et al., "Regulation of Alzheimer's disease related proteins and uses thereof," WO9705780, (1997). The particular buffer component of the polymerization buffer is not critical for the polymerization reaction. Suitable buffers include, but are not limited to, tris (Tris(hydroxymethyl)aminomethane), HEPES (N-(2-Hydroxyethyl)piperazine- N'-(2-ethanesulfonic acid)), and MES (2-[N-morpholino]ethanesulfonic acid). Buffers are preferably used within their effective pH ranges. For example, tris is preferably used in the polymerization buffer for reactions carried out above about pH 7. MES is preferably used where the polymerization is carried out at below about pH 7. HEPES is preferably used at a pH range of 6.8 to 8.2. Typically, the buffer is at a concentration of about 1 mM to about 1 M, preferably about 100 mM, although other concentrations can be used provided the buffer is effective at the concentration chosen. Tau protein can be used in the polymerization reaction at a wide concentration range. Generally this range is from about 1 to about 100 μM, preferably from about 1.6 to about 6.5 μM. In one embodiment, tau protein is used at about 4 μM. The incubating step can be carried out for a time equal to or greater than about 1 minute but is typically carried out for at least about 1 hour.

The temperature range for the polymerization reaction is from between about 4°C and about 45°C, preferably between about 22°C and about 37°C. The pH range of the reaction is typically between about 6 and about 1 1.

Typically, the ionic strength of the buffer is between 0 and about 200 mM, preferably between about 25 and about 125 mM. When present, the salt used in the polymerization buffer is not critical; examples include NaCI and KCI.

In one example, tau polymerization is induced by incubating about 4 μM tau protein HT40 in the presence of about 75μM free fatty acid (FA) in buffer containing about 10 mM HEPES, about pH 7.4, and about 100 mM NaCI (polymerization buffer) at about 37° C.

Fatty acids can be obtained from a variety of commercial sources including Sigma Chemical Company (St. Louis, MO) and Cayman Chemicals (Ann Arbor, Ml). Typically free fatty acids are used in the cis conformation and at maximum available purity. Before use, free fatty acids are typically diluted into tau from a 200X ethanolic stock, such that the final ethanol concentration in all samples and controls is 0.5%. Values for the critical micellar concentration (CMC) can be obtained based on the phase partitioning of 10 mM phenylnaphthylamine (Kovatchev et al., (1981 )) when free fatty acids are diluted into polymerization buffer. Many free fatty acids are known and can be used in the methods of the present invention. These fatty acids include, but are not limited to, 5,8, 1 1 , 14, 17-eicosapentaenoic acid (20:5); 5,8, 1 1 , 14-eicosatetraenoic acid (20:4) (arachadonic acid); 8,1 1 ,14-eicosatrienoic acid (20:3); 1 1 ,14-eicosadienoic acid (20:2); 11 -eicosenoic acid (20:1 ); eicosanoic acid (20:0); 9,12,15-linolenic acid (18-3); 9;12-linoleic acid (18:2); 9-oleic acid (18-1 ); stearic acid (18:0); 9-palmitoleic acid (16-1 ); palmitic acid (16:0); and myristic acid (14:0). Preferably, the fatty acid is used in an amount from about 1 micromolar to about 100 micromolar. Preferred fatty acids include arachidonic acid and docosahexaenoic acid. The methods of the current invention require that fatty acids are

"effectively oxidized." A fatty acid is "effectively oxidized" when the fatty acid is capable of inducing tau polymerization in a first polymerization reaction procedure, but not when the antioxidant BHT is present during the first polymerization reaction procedure. The first polymerization reaction procedure includes any steps which are necessary for the polymerization of tau including any steps in which fatty acids are oxidized.

Fatty acids may be isolated in an "effectively oxidized" state following synthesis or isolation from natural sources without further treatment before being added to the polymerization buffer. Fatty acids may be "effectively oxidized" by exposure of the fatty acids to an oxidizing environment before addition of the fatty acid to the polymerization buffer.

In certain embodiment, fatty acids may be "effectively oxidized" by the polymerization buffer, for example during the incubation step. The method of this embodiment comprises:

(a) providing a polymerization buffer with an oxidative environment;

In certain embodiments, fatty acids are oxidized by incubating the fatty acids in a polymerization buffer that provides an oxidative environment, before adding substantially purified tau protein. In one embodiment, after adding an effective amount of fatty acid, the fatty acid-containing buffer is incubated for at least about 5 minutes before adding the substantially purified tau protein. In another embodiment, the incubation period is at least about 15 minutes. In another embodiment, the incubation period is at least about 30 minutes. In another embodiment, the incubation period is at least about 60 minutes. In another embodiment, the method further comprises the step of adding an effective amount of an oxidizer to the fatty acid-containing polymerization buffer before the incubating step. In one embodiment, the oxidizer is selected from the group consisting of a solution of FeCI₃, ADP, and ascorbic acid, a solution of iron citrate, a solution of enzymes that generate specific metabolites of fatty acids, and ultraviolet radiation. In one embodiment, the oxidizer is an enzyme that generates a specific metabolite of fatty acids, wherein the enzyme is selected from the group consisting of cyclooxygenases and lipoxygenases. In another embodiment, the oxidizer is about 50 μM FeCI₃, about 20 mM ADP, and about 10 mM ascorbic acid.

Any form of tau which retains the ability to polymerize can be used alone or in combination, for the methods of the current invention. In one embodiment, the substantially purified tau protein is selected from the group consisting of mammalian brain tau protein and recombinant tau protein that has the ability to polymerize. One recombinant tau protein that has the ability to polymerize, and thus can be used with the current invention, is recombinant wild type tau protein. "Wild type tau" includes all naturally- occurring forms of tau that have not been mutated. Thus far, six total wild type isoforms of human tau have been cloned that are expressed in the CNS (Reviewed in Spillantini et al., (1998)). Other recombinant tau proteins retaining the ability to polymerize include, but are not limited to, recombinant tau protein HT40 and a recombinant tau mutant having no cysteine residues. In one preferred embodiment of the current invention, recombinant human tau protein HT40 (also called 2N4R htau and htau40 (Goedert et al., 1989)) is used. HT40 is the wild type protein expressed by a recombinant tau cDNA, Htau40, that encodes a 441 amino acid polypeptide containing exons 2, 3, and 10 that is polyhistidine tagged at its amino terminus. Recombinant tau HT40 was produced in E. coli as a fusion protein with a polyhistidine tag, and purified to near homogeneity by nickel-chelate and gel filtration chromatography (Carmel et al., (1994)). Following purification, HT40 protein is dialyzed against buffer A (20 mM morpholinoethanesulfonic acid pH 6.8, 80 mM NaCI, 2 mM EGTA, 1 mM MgC12, 0.1 mM EDTA) and stored at -80° C. In one preferred embodiment of the current invention, a recombinant tau protein is utilized in which cysteine residues have been replaced with other amino acid residues. Such a protein can be obtained by creating a polynucleotide encoding tau in which residues encoding cysteine residues are mutated to encode other amino acids. Methods for constructing a polynucleotide in which cysteine-encoding residues have been changed to encode a different amino acid are well known in the art (see, e.g., Sambrook et al., "Molecular cloning, a laboratory manual." Cold Spring Harbor Laboratory Press (1989)) and can be created using commercial kits (e.g., QuickChange, Stratagene, La Jolla, CA) and appropriately chosen oligonucleotides.

For example, the nucleotide sequence of htau40, described above, can be modified to encode alanine residues in place of the 2 cysteine residues in the tau protein encoded by htau40. Mutations in htau40 (C291A and C322A) are prepared from a plasmid containing the htau40 sequence, pT7c-htau40 (Goedert et al., (1989)), using a commercial kit (QuickChange, Stratagene, La Jolla, CA) and synthetic oligonucleotides 5'- agcaacgtccagtccaaggctggctcaaaggataatatc [SEQ ID NO:1] and 5'- gcaaggtgacctccaaggctggctcattaggcaac [SEQ ID NO:2] (underlined residues encode cysteine residues, amino acid residues 291 and 322, respectively). The double mutant is created by ligating a Ss EII/EcoRI fragment from pT7c- htau40-C322A into BsfEII/EcoRI digested pT7-htau40-C291A. The double- mutated protein, htau40 (C291A and C322A) [SEQ ID NO:3], called Cysless, is purified by conventional techniques, such as those used to purify wild type HT40 htau described above.

In certain embodiments of the current invention, native mammalian tau is used. Protocols for isolating and substantially purifying tau from assembled microtubules for use in the present invention are found in Wilson et al., "Regulation of Alzheimer's disease related proteins and uses thereof," WO9705780 (1997). For this protocol, microtubules are purified from brain by two cycles of temperature dependent assembly essentially as previously described (Shelanski et al., (1973)), with glycerol added to 25% during the first warm incubation only. Tubulin is further purified by phosphocellulose chromatography (Weingarten et al., (1975)), using precycled phoshocellulose (Sloboda et al., (1976)). Taxol stabilized microtubules (Vallee, (1982)) are prepared by incubating purified tubulin, such as porcine tubuline, at 5 mg/ml with 10 μM taxol for 30 minutes at 37°C. Pellets are resuspended in cycling buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgCI₂, pH 6.9) supplemented with 0.8 M NaCI and 2 mM DTT stirred on ice for 30 minutes, boiled for 10 minutes, stirred on ice for 30 minutes, and centrifuged at 100,000 x g for 45 minutes. Supernatants are concentrated over an ultrafiltration membrane, such as an Amicon YM10 ultrafiltration membrane, loaded on a sieve column, such as a Bio-Gel A-1.5 sieve column (32 x 430 mm, run at 15 ml/hour), equilibrated with buffer A (20 mM MES, 80 M NaCI, 2 mM EGTA, 1 mM MgCI₂, 0.1 mM EDTA, pH 6.8) supplemented with 0.8 M NaCI and 2 mM DTT (buffer A+). Fractions containing tau are brought to about 2.5% perchloric acid, stirred on ice for 30 minutes, and centrifuged at 100,000 x g for 30 minutes. Supernatants are dialyzed against buffer A, and concentrated by ultrafiltration. All procedures except boiling are carried out at 4°C.

In certain embodiments, the method further includes the step of adding an effective amount of a reducing agent to the fatty acid-containing polymerization buffer before the incubating step. In one embodiment, the method comprises:

(a) providing a polymerization buffer;

(e) incubating the tau polymerization reaction mixture to permit the formation of tau filaments.

In one preferred embodiment, the reducing agent is present in the same solution as the tau protein when the tau protein is added to the fatty acid-containing polymerization buffer. In one embodiment, the method involves contacting an already formed tau polymer with a reducing agent to stimulate further polymerization. Many reducing agents are known in the art and can be used with the current invention. These include, but are not limited to, dithiothreitol (DTT), dithioerythreitol, 2-mercaptothanol, and reduced gluthathione. In a preferred embodiment, the reducing agent is DTT. In one embodiment, the DTT is present at a concentration of between about 0.2 mM and about 100 mM. In another embodiment, the DTT is present at a concentration of between about 1 mM and about 25 mM. In another embodiment, the DTT is present at a concentration of between about 2.5 mM and about 10 mM. In another embodiment, the DTT is present at a concentration of about 5 mM.

Tau filaments can be detected and analyzed using a number of techniques well known in the art. These techniques include, but are not limited to measurements of laser light scattering and analysis of electron micrographs. For measurements of laser light scattering, tau polymerization reactions (250μL) in 5mm fluorimeter cells are illuminated with 488nm vertically polarized laser light generated by an ion laser, such as a Lexel model 65 ion laser at a 5mW setting. Images are collected at an angle of 90° to the incident light and perpendicular to the direction of polarization with an Electrim Corp. Model EDC1000HR digital camera with a 25mm lens controlled by HiCam '95 (for example, using the program written by Dr. Guenter Albrecht-Buehler of Northwestern University Medical School, and available at http://www.basic.nwu.edu/g-buehler/hicam.htm). The intensity of scattered light (i_s) is obtained using the histogram feature of Adobe Photoshop. For simple comparisons between experiments, lines are drawn through the polymerization curves and are not meant to imply any particular model of polymerization (Gamblin, Biochemistry 39: 6136 (2000)). For electron microscopic analysis, aliquots are taken at different time points during the polymerization reactions and fixed with about 2% glutaraldehyde. Samples are prepared for electron microscopy by floating a carbon coated formvar grid on 10μL of glutaraldehyde fixed sample for one minute followed by staining with 2% uranyl acetate for one minute. A transmission electron microscope (e.g., JEOL 1220 transmission electron microscope operating at 60 kV) is used to view the grids. Images are captured at 20,000x using a digital camera (e.g., MegaPlus Model 1.61 AMT Digital Kodak camera controlled by the AMT Camera Controller software package). Images can be processed and quantified as previously described (King et al., J. Neurochem. 74: 1749 (2000)). Filament lengths are measured manually from prints of micrographs. Regions from several different micrographs are measured and the mean and standard error of the mean of the relative mass distributions of the filaments was determined.

Methods for isolating reagents that inhibit tau polymerization. In another aspect, the current invention provides a method for identifying reagents that effect tau polymerization. Typically, these reagents inhibit tau polymerization. The method of this aspect of the invention includes the general method and all of the specific embodiments for stimulating polymerization of tau protein described above and additional steps as described below. The method for identifying reagents that effect tau polymerization comprises:

(a) providing a polymerization buffer; (b) adding an effective amount of a free fatty acid to the polymerization buffer to form a fatty acid-containing polymerization buffer; (c) adding substantially purified tau protein to the fatty acid- containing polymerization buffer to form a tau polymerization reaction mixture; (d) adding a test reagent to the tau polymerization reaction mixture to form a test compound/tau polymerization reaction mixture;

(e) incubating the reagent/tau polymerization reaction mixture to permit the formation of tau filaments, wherein the free fatty acid is effectively oxidized during or before the incubating; and (f) detecting the tau filaments in the reagent/tau polymerization reaction mixture, thereby screening the test reagent for the ability to effect tau polymerization.

This aspect of the invention includes all of the embodiments described above for the method of stimulating polymerization of tau protein. The method of the method for identifying reagents aspect of the invention can be used to screen for reagents which prevent tau polymer formation in the brains of patients with AD and other related neurodegenerative diseases characterized in part by in vivo tau polymer formation. The term "reagent" as used herein describes any molecule (e.g., protein, nucleic acid, polypeptide, or pharmaceutical) with the capability of effecting the polymerization of tau. Generally a plurality of assay mixtures are run in parallel with different reagent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control (i.e., zero concentration or below the level of detection). In one embodiment, the reagent inhibits the polymerization of tau. ^' In another embodiment, the reagent increases, accelerates, or enhances the the polymerization of tau. Candidate reagents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic molecules having a molecular weight of more than about 50 and less than about 2,500 daltons. Candidate reagents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least one of the group consisting of an amine, carbonyl, hydroxyl, or carboxyl group, and preferably at least two of such functional chemical groups. The candidate reagents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate reagents are also found among biomolecules including peptides, proteins, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives thereof, structural analogs, or combinations thereof.

Candidate reagents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, and the like to produce structural analogs. In addition, compounds can be obtained from commercial sources.

Compounds with identified structures from commercial sources can be efficiently screened for activity against a particular reaction by first restricting the compounds to be screened to those with preferred structural characteristics. As an example, compounds with structural characteristics causing high gross toxicity can be excluded. Similarly, once a number of inhibitors of a specific target have been found, a sub-library may be generated consisting of compounds which have structural features in common with the identified inhibitors. In order to expedite this effort, the ISIS computer program (MDL Information Systems, Inc.) is suitable to perform a 2D-substructure search of the Available Chemicals Directory database (MDL Information Systems, Inc.). This database contains structural and ordering information on approximately 175,000 commercially available chemical compounds. Other publicly accessible chemical databases may similarly be used.

Gross acute toxicity of an identified reagent may be assessed in a suitable animal model such as, for example, a mouse model. The inhibitor is administered at a range of doses, including high doses, (typically 0 - 100 mg/kg, but preferably up to at least 100 times the expected therapeutic dose) subcutaneously or orally, as appropriate, to healthy mice. The mice are observed for 3-10 days. In the same way, a combination of such an inhibitor with any additional therapeutic components is tested for possible acute toxicity. Method for treating tau polymerization in vivo with inhibitor of fatty acid oxidation. Another aspect of the current invention is a method for regulating the assembly of the protein tau in the brain of a mammal in need of such a regulation, wherein the method comprises administering to the mammal a pharmacologically effective amount of an inhibitor of fatty acid oxidation in a pharmaceutically-acceptable carrier.

In one embodiment, the mammal is a human. In another embodiment, the inhibitor of fatty acid oxidation is selected from the group consisting of vitamins with antioxjdative properties and non-steroidal anti-inflammatory drugs (NSAIDS). In one embodiment, the vitamin with anti-oxidative properties is selected from the group consisting of Vitamin E, beta carotene, and Vitamin C. In another embodiment, the inhibitor of fatty acid oxygenation is a non-steroidal anti-inflammatory drug selected from the group consisting of aspirin, dilofenic, and ibuprofen. In another embodiment, the inhibitor of fatty acid oxygenation is a selective inhibitor of cyclooxygenase-2. In one embodiment, the cyclooxygenase inhibitor is administered in an amount selected from about 1000 mg per day to about 2500 mg per day. In one embodiment, the cyclooxygenase inhibitor is ibuprofen administered in an amount selected from about 1000 mg per day to about 2500 mg per day.

In one embodiment, the administering is performed repeatedly over a period of at least one week. In one embodiment, the administering is performed repeatedly over a period of at least one month. In one embodiment, the administering is performed repeatedly over a period of at least three months. In one embodiment, the administering is performed repeatedly over a period of at least one year. In another embodiment, the administering is performed at least once monthly. In another embodiment, the administering is performed at least once weekly. In another embodiment, the administering is performed at least once daily. In another embodiment, the administering is performed at least once weekly for at least one month. In another embodiment, the administering is performed at least once per day for at least one month.

This aspect of the invention provides for treatment and/or prevention of various diseases and disorders associated with induction of tau polymerization by oxidized fatty acids. The invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a therapeutic of the invention. In a preferred aspect, the therapeutic is substantially purified. The subject is preferably an animal, including, but not limited to, cows, pigs, horses, chickens, cats, dogs, and the like, and more preferably is a mammal, and most preferably is a human. Various delivery systems are known and can be used to administer a therapeutic of the invention. Such systems include, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic (see, e.g., Wu and Wu, "Receptor-mediated in vitro gene transformation by a soluble DNA carrier system," J. Biol. Chem. 262:4429 (1987)), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The therapeutics may be administered by any convenient route, including, for example, infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, and the like) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed (e.g., by an inhaler or nebulizer) using a formulation containing an aerosolizing agent. In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment, such as the brain. This may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application (e.g., wound dressing), injection, catheter, suppository, or implant (e.g., implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like. In one embodiment, administration can be by direct injection at the site (or former site) of a tissue that is subject to damage by oxidation, such as the brain. In another embodiment, the therapeutic can be delivered in a vesicle, in particular a liposome (see, e.g., Langer, "New methods of drug delivery," Science 249: 1527 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365 (1989)). In yet another embodiment, the therapeutic can be delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, (1990); Sefton, "Implantable pumps ' Crit. Rev. Biomed. Eng. 14:201 (1987); Buchwald et al., "Long-term, continuous intravenous heparin administration by an implantable infusion pump in ambulatory patients with recurrent venous thrombosis," Surgery 88:507 (1980); and Saudek et al., "A preliminary trial of the programmable implantable medication system for insulin delivery," N. Engl. J. Med. 321 :574 (1989)). In another embodiment, polymeric materials can be used (see, e.g., Ranger et al., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., "Inhibition of calcification of bioprosthetic heart valves by local controlled-release diphosphonate," Science 228:190 (1985); During et al., "Controlled release of dopamine from a polymeric brain implant: in vivo characterization," Ann. Neurol. 25:351 (1989); and Howard et al., "Intracerebral drug delivery in rats with lesion- induced memory deficits," J. Neurosurg. 71 :105 (1989)). Other controlled release systems discussed in the review by Langer et al. (1990) can also be used.

This aspect of the present invention typically includes a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The therapeutic, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These therapeutics can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. The therapeutic can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such therapeutics will contain a therapeutically effective amount of the active ingredient, preferably in purified form, together with a suitable amount of carrier so as to provide proper administration to the patient. The formulation should suit the mode of administration. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water or saline can be provided so that the ingredients may be mixed prior to administration. ^■ The amount of the therapeutic of the invention which will be effective depends on the nature of the tau-related disorder or condition, as well as the stage of the disorder or condition. Effective amounts can be determined by standard clinical techniques. In addition, in vitro assays, such as those described below, may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20 to about 500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to about 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of about 0.5% to about 10% by weight; oral formulations preferably contain about 10% to about 95% active ingredient. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the therapeutics of the invention.

In one embodiment, the method for regulating the assembly of the protein tau in the brain of a patient comprises: identifying a patient in need of a method for inhibiting tau polymerization in the brain; and administering to the patient a pharmacologically effective amount of an inhibitor of fatty acid oxidation.

In one embodiment, the identifying being based on identifying mutant genomic subtypes of tau in the patient. Typically, these mutant subtypes are involved with increased Tau protein polymerization. (Reviewed by Spillantini et al., (1998).) In another embodiment, the identifying is other than a diagnosis of Alzheimer's disease. For this embodiment, the identifying may be, but is not limited to, the diagnosis of another disorder involving tau polymerization, such as Pick's disease, progressive supranuclear palsy, corticobasal degeneration and familial frontotemporal dementia, and parkinsonism linked to chromosome 17 (FTDP-17).

The following examples describe and illustrate the methods and compositions of the invention. These examples are intended to be merely illustrative of the present invention, and not limiting thereof in either scope or spirit. Unless indicated otherwise, all percentages are by weight. Those skilled in the art will readily understand that variations of the materials, conditions, and processes described in these examples can be used.

EXAMPLE 1: Analysis of the effects of a reducing agent on fatty acid-induced tau polymerization. The effects of cysteine oxidation on the induction of tau polymerization was investigated by analyzing the effects of a reducing agent on tau polymerizations. The tau protein used for this experiment was the protein expressed by a recombinant tau cDNA, Htau40 (Goedert et al., 1989), that encodes a 441 amino acid polypeptide containing exons 2, 3, and 10 that is polyhistidine tagged at its amino terminus.

Recombinant tau HT40 was produced in E. coli as a fusion protein with a polyhistidine tag, and purified to near homogeneity by nickel-chelate and gel filtration chromatography (Carmel et al., 1994). Following purification HT40 protein is dialyzed against buffer A (20 mM morpholinoethanesulfonic acid pH 6.8, 80 mM NaCI, 2 mM EGTA, 1 mM MgCI₂, 0.1 mM EDTA) and stored at -80°C.

Arachidonic acid (AA) and docosahexaenoic acid (DHA) were purchased from Cayman Chemicals (Ann Arbor, Ml). All other free fatty acids were purchased in the cis conformation and at maximum available purity from Sigma Chemical Company (St. Louis, MO). Free fatty acids were diluted into tau from a 200X ethanolic stock, such that the final ethanol concentration in all samples and controls was 0.5%. Values for the critical micellar concentration (CMC) were obtained based on the phase partitioning of 10 mM phenylnaphthylamine (Kovatchev et al., 1981) when free fatty acids were diluted into polymerization buffer.

Tau polymerization was induced by incubating 4 μM tau protein HT40 • in the presence of 75 μM arachidonic Acid (AA) in buffer containing 10 mM HEPES, pH 7.4, and 100 mM NaCI (polymerization buffer) at 37° C. Dithiothreitol, when present in a reaction, was used at a final concentration of 5mM unless otherwise noted.

Tau polymerization reactions (250μL) in 5mm fluorimeter cells were illuminated with 488nm vertically polarized laser light generated by a Lexel model 65 ion laser at a 5mW setting. Images were collected at an angle of 90° to the incident light and perpendicular to the direction of polarization with an Electrim Corp. Model EDC1000HR digital camera with a 25mm lens controlled by HiCam '95 (written by Dr. Guenter Albrecht-Buehler of Northwestern University Medical School, and available at http://www.basic.nwu.edu/g-buehler/hicam.htm). The intensity of scattered light (i_s) was obtained using the histogram feature of Adobe Photoshop. For simple comparisons between experiments, lines were drawn through the polymerization curves and are not meant to imply any particular model of polymerization (Gamblin et al., Biochemistry 39: 6136 (2000)). The role of cysteine-mediated oxidation in the arachidonic acid (AA) induction of tau polymerization was investigated by several methods. First, the effect of dithiothreitol (DTT) was established. It has been shown previously that the removal of this sulfhydryl protecting agent from solutions of truncated tau constructs allows cysteine specific dimerization to occur by air oxidation and is required for the induction of polymerization by polyanionic compounds (Schweers et al., Proc. Natl. Acad. Sci. USA 92:8463 (1995)). AA was added to solutions of full length tau either in the presence or absence of DTT. The reactions were monitored by right angle laser light scattering (LLS). The presence of DTT in the tau polymerization reactions greatly enhanced the amount of LLS when compared to reactions without DTT (Fig. 1 ). This suggested that cysteine oxidation of full length tau protein is not required for tau polymerization, and may be inhibitory for the process.

EXAMPLE 2. Effects of eliminating cysteine oxidation on fatty acid-induced tau polymerization. An analysis was performed of the polymerization of a Tau protein in which cysteine residues were replaced with amino acids which are not oxidized. Mutations in htau40 (C291 A and C322A) were prepared from pT7c-htau40 using a commercial kit (QuickChange, Stratagene, La Jolla, CA) and synthetic oligonucleotides 5'- agcaacgtccagtccaaggctggctcaaaggataatatc [SEQ ID NO:1] and 5'- gcaaggtgacctccaaggctggctcattaggcaac [SEQ ID NO:2] (underlined residues encode amino acid residues 291 and 322, respectively). Double mutant pT7c-htau40-C291A/C322A was created by ligating a BsfEII/EcoRI fragment from pT7c-htau40-C322A into SsfEII/EcoRI digested pT7-htau40-C291A. The double-mutated protein, htau40 (C291 A and C322A) [SEQ ID NO:3], called Cysless, was purified in the same manner as wild type 2N4R htau described above.

Polymerization reactions were carried out as described in Example 1 except that Cysless was used in the polymerization reactions in place of 2N4R htau.

The effect of cysteine oxidation was further tested by changing the only two cysteine residues present in the full length tau molecule (Cys291 and Cys322) into non-reactive alanines through site-directed mutagenesis. The double mutant (Cysless) formed filaments upon the addition of AA to the solution as indicated by the increase in LLS (FIG. 2). This result further supported the supposition that cysteine oxidation is not required for filament formation. In addition, Cysless tau protein was no longer dependent on the presence of DTT to achieve filament formation (FIG. 2). Cysteine oxidation alters the apparent rate of polymerization. FIG. 3 was prepared using only the initial data of FIGS. 1 and 2 (i.e., first 20 minutes). Major differences in the polymerization of the wild type protein plus DTT, wild type protein without DTT, and Cysless are more easily discerned in FIG. 3. The wild type protein polymerized more quickly in the absence of DTT than in the presence of the reducing agent at these early time points. The apparent rate of polymerization for Cysless (irrespective of the presence of DTT) was much lower than for the wild type protein.

EXAMPLE 3. Cysteine oxidation results in morphologically distinct filaments. The morphology of tau filaments polymerized by fatty acid induction under various oxidation conditions, was compared.

Polymerization reactions were carried out as described in Examples 1 and 2. Aliquots were taken at different time points during the polymerization reactions and fixed with 2% glutaraldehyde for quantitative electron microscopy. Samples were prepared for electron microscopy by floating a carbon coated formvar grid on 10μL of glutaraldehyde fixed sample for one minute followed by staining with 2% uranyl acetate for one minute. A JEOL 1220 transmission electron microscope operating at 60 kV was used to view the grids. Images were captured at 20,000x using a MegaPlus Model 1.61 AMT Digital Kodak camera controlled by the AMT Camera Controller software package. All images were processed and quantified as previously described (King et al., (2000)). Filament lengths were measured manually using digital micrographs and image analysis software (Optimas 6.1 , Media Cybernetics, Silver Spring, MD). Regions from several different micrographs were measured and the mean and standard error of the mean (indicated as the +/- value below) of the relative mass distributions of the filaments was determined.

The differences observed in the rates of polymerization discussed in Example 2 above prompted an investigation into the resulting morphology of the filaments. Samples were taken at the end of polymerization reactions and viewed by electron microscopy (FIG. 4). Wild type protein polymerized most rapidly in the absence of DTT. However, only aggregates and a few very short filaments were formed (FIG. 4B). Still, these short filaments and particles of tau only were observed upon addition of AA (data not shown). Filaments consisting of the slowly polymerizing Cysless mutant appeared much longer than those formed from the wild type protein (FIG. 4C and 4D). The wild type protein, which exhibited an intermediate rate of polymerization, appeared to have significant populations of both the very short filaments and longer filaments.

These apparent morphological differences were confirmed by measuring the mass distribution of the filaments in each of the four populations. 91.7% ± 1.2 of the filament mass formed by wild type protein in the absence of DTT consisted of filaments below 50nm in length (FIG. 5B). In the presence of DTT, only 52.4% ± 14,7 of the filament mass formed by the wild type protein were in this category (FIF. 5A). In stark contrast to these results, the filaments consisting of the Cysless protein had only 11.3% ± 1.7 and 23.1 % ± 7.1 of the filament mass below 50nm in the presence and absence of DTT, respectively (FIGS. 5C and 5D). EXAMPLE 4. An anti-oxidant can block the fatty acid induction of tau polymerization. An analysis was performed of the ability of the antioxidant butylated hydroxytoluene (BHT) to inhibit fatty acid induction of tau polymerization. Butylated-hydroxytoluene (BHT) (Sigma-Aldrich, St. Louis, MO) was diluted into 100% ethanol to make a 4% stock solution. Polymerizations were carried out as described in Examples 1 and 2 above except that for the BHT-treated samples, BHT was added to the polymerization buffer to a final concentration of 0.1 % before the addition of 75 mM free fatty acid and HT40 tau. Additionally, for certain experiments, as indicated below, two different fatty acid inducers, arachidonic acid (AA) and docosahexaenoic acid (DHA), were added to 4 μM tau solutions at a final concentration of 75 μM either in the presence or absence of 0.1 % BHT. Samples were analyzed by quantitative electron microscopy as described above in Example 3, since the relative insolubility of BHT interferes with LLS. The mean and standard deviation of the mean was determined for each sample by measuring five representative fields for each condition.

The experiments described in Examples 1-3 above show that the cysteine-mediated oxidation of the tau molecule is not necessarily conducive for tau polymerization. However, the fatty acids that are used in this in vitro polymerization assay are also potential targets of oxidative stress in AD. Therefore, if oxidation of the tau molecule does not enhance polymerization, perhaps the oxidation of the inducer molecule does. A supporting rationale for this approach was derived from the work which correlated the extent of saturation of free fatty acids (FAs) to their efficacy of induction of tau polymerization (Wilson, Am. J. Pathol. 150: 2181 (1997)). In general, for any given chain length of fatty acids, unsaturated FAs were more potent than saturated ones. Since the degree of unsaturation directly reflects the ease of oxidation, this suggested a potential role for oxidation of the FAs in the induction process. To test this notion, polymerization reactions were performed with the two major FAs of phosphatidylethanolamine in human gray matter: 20:4 w6 (AA) and 22:6 w3 (docosahexaenoic acid, or DHA). DHA, which has two more double bonds than AA and therefore four more sites for oxidation, induced the polymerization of tau to a greater degree than did arachidonic acid (FIG. 6). In addition, when polymerization reactions with these two FAs were performed in the presence of a strong antioxidant, BHT, tau polymerization was inhibited. This suggested that oxidation of the FA is a necessary step for the induction of tau filament formation. Experiments were therefore performed to determine whether the BHT inhibition of tau filament formation was dependent on the oxidation of the tau molecule itself. The addition of BHT to polymerization reactions consisting of the Cysless mutant also inhibited the process (FIG. 7), indicating that the addition of BHT was not inhibiting polymerization in a tau-oxidation-depehdent fashion.

EXAMPLE 5. Increases in oxidative environments can influence tau polymerization. An analysis was performed of the effects on tau polymerization of increased oxidative environments of the polymerization reaction and of increased oxidative environments of fatty acids before they were added to the tau polymerization reaction. Polymerization experiments were performed under four different sets of oxidation conditions. The first set of conditions was fatty acid plus tau in polymerization buffer alone. These are the same conditions employed for previous tau polymerization reactions. It was assumed that this is an oxidative environment since the addition of BHT inhibited the reaction (FIGS. 6 and 7). The second set of conditions was simply to increase the amount of time the fatty acid was in the oxidative environment of the polymerization reaction before the addition of tau. This was accomplished by preincubating 75 mM FA in polymerization buffer at 37°C before adding HT40 tau to the FA containing polymerization buffer. The third set of conditions involved the incubation of FA and tau in polymerization buffer supplemented with 50 mM FeCI₃, 20 mM ADP, and 10 mM ascorbic acid (Fe/ADP/ascorbate). Similar conditions have been used previously to generate oxidation products of AA and DHA, the

F2-isoprostanes and the F4-neuroprostanes respectively (Roberts et al., J. Biol. Chem. 273: 13605 (1998); Longmire et al., Biochem. Pharmacol. 47: 1173 (1994)). The fourth set of conditions involved preincubation of the fatty acids in polymerization buffer supplemented with Fe/ADP/ascorbate for one hour at 37° before the addition of tau.

Samples were analyzed by quantitative electron microscopy as described above in Examples 3 and 4. The mean and standard deviation of the mean was determined for each sample by measuring five representative fields for each condition.

Arachidonic acid induced tau polymerization under the first oxidative condition (in polymerization buffer), whereas the second (increased incubation time in polymerization buffer) and the third (addition of

Fe/ADP/ascorbate) conditions greatly increased the amount of tau filament formation (FIG. 8). If the AA was left in buffer alone for one hour at 37°C (condition 2), the increased exposure time of the potential oxidizers in the polymerization buffer would increase the chances that the AA would become oxidized. By the addition of the Fe/ADP/ascorbate system (condition 3), free radicals production should increase, thereby increasing the chances that the AA would become oxidized. The fourth oxidative environment (incubation with Fe/ADP/ascorbate for one hour at 37°C), however, greatly diminished the amount of tau filament formation. This suggests that a favorable oxidative product may be susceptible to further oxidation, resulting in compounds which are incapable of inducing tau polymerization.

Docosahexaenoic acid (DHA) behaved differently than AA (FIG. 8). Under the first oxidative condition (polymerization buffer alone), DHA induced the formation of tau filaments to a greater extent than did AA under the same conditions. However, the increased oxidative potentials in conditions 2, 3, and 4 had a detrimental effect on DHA-induced tau filament formation. This reduced polymerization-promoting efficacy of DHA is most likely due to its increased sensitivity to oxidation since it contains six double bonds as opposed to the four double bonds in AA. Its mere introduction into an unprotected aqueous environment would appear to be sufficient to oxidize it into its active form.

EXAMPLE 6. Conclusions and further considerations of the effects of oxidation on tau polymerization induced by fatty acids. The role of oxidative stress factors was also investigated using a different in vitro paradigm for tau polymerization. (Wilson et al., "Regulation of Alzheimer's disease related proteins and uses thereof," WO9705780 (1997).) In this system, FAs are used to induce full length tau molecules to form filamentous structures. These tau filaments form efficiently in a few hours and are morphologically similar to the straight filaments (SFs) observed in AD and other neurodegenerative diseases. In addition, these filaments are similar to tau-positive AD structures in their reactivity to immunological reagents such as conformationally sensitive antibodies and thioflavin-S. The synthetic filaments may also be structurally related to authentic paired-helical filaments (PHFs), in that PHFs purified form AD brain can be used as "seeds" for synthetic filament formation. If monomeric tau is added to PHF fragments in the presence of FAs, synthetic tau filaments will actually grow from the ends of the PHF, suggesting a structural similarity.

Role of Tau Oxidation in the Fatty Acid Induction of Polymerization. With regard to the involvement of cysteine oxidation, it was found that the removal of a sulfhydryl protective agent, dithiothreitol (DTT), from the FA-induced polymerization reactions did not result in filament formation, but rather in the formation of small (<50nm) structures whose relationship to longer filaments is currently unknown. These structures were present in filament populations that were formed in the presence of DTT, but only accounted for about 50% of the total filament mass. When the reactive cysteines of tau were mutated into non-reactive alanines, the mutants were still capable of filament formation, but at a reduced apparent velocity. The filaments formed, however, were on average much longer than those that formed with wild type protein. This is most likely due to the fact that the formation of shorter structures (<50nm) was greatly reduced. When the apparent rate of filament formation is compared with the length distributions of the filaments, it appears that the formation of the apparent cysteine-oxidation enhanced short structures (<50nm) is energetically favored over the formation of longer, true filamentous tau structures. It is possible that this process is actually in competition with true polymerization even in the presence of DTT. However, since these aggregates do not elongate further, even after several days of incubation (data not shown), it is not clear if they are ordered structures or non-specific aggregations of disulfide cross-linked tau molecules.

Claims

The cysteine-mediated oxidation of the tau molecule does not appear to be required for in vitro tau polymerization. It is also unlikely that this event is the mechanism by which oxidative stress induces tau filament formation in vivo. We have not analyzed the effects on in vitro polymerization of tau of oxidative changes in the tau molecule, such as carbonyl formation or tyrosine nitration, affect. These experiments would provide further insight on the effects of tau oxidation on its polymerization.Role of Fatty Acid Oxidation in Tau Polymerization. While the experiments described above indicate that the cysteine-oxidation of the tau molecule is not a major contributing factor to its polymerization, the possibility that oxidation could be affecting other parameters of in vitro polymerization cannot be ruled out. FAs are a major target for oxidative stress in vivo, and therefore could be affected in our in vitro system. The notion that FA oxidation could affect the induction of polymerization is not without precedence. Previous experimentation found a rough correlation between the increased sites of unsaturation in FA and increased efficacy of polymerization. In fact, DHA, which contains two more double bonds and hence four more sites for oxidation than AA, stimulates tau filament formation to a greater extent than AA. The addition of a free-radical scavenging anti-oxidant (butylated hydroxytoluene, or BHT) to the FA-induced tau polymerization reactions inhibited the process to the point that no filament formation was observed in the presence of BHT. This phenomenon was most likely due to the inhibition of AA and DHA oxidation, since the addition of BHT also inhibited the polymerization of the Cysless mutant that should be insensitive to oxidation. It appears, therefore, that the oxidation of polyunsaturated fatty acids is at least greatly beneficial, and possibly essential, to the fatty acid's ability to induce tau polymerization.Since it was evident that the FA oxidation that occurred under normal polymerization conditions was beneficial, we investigated the effects of increasing the oxidative components of the reaction. Simply increasing the amount of incubation time that AA was in the reaction buffer before the addition of tau, or by creating an oxidative environment consisting of iron, ADP, and ascorbate, produced a large increase in the amount of AA-induced filament formation. However, incubating the AA in Fe/ADP/ascorbate for one hour before the addition of tau resulted in only small amounts of filament formation. This result is perhaps not surprising if considered in the context of the DHA-induction of polymerization under the same conditions. DHA, which is more readily oxidized than AA, exhibited greater filament formation efficacy under more gentle conditions. When the oxidative environment was increased, it did not perform as well as AA. Therefore, we conclude that while oxidation of the FAs stimulates tau polymerization, they can be "over-oxidized" to a point where they are no longer functional as inducers. This "over-oxidation" of the FAs could occur by several different mechanisms. The simplest mechanism would be a concentration effect. Upon addition of FA into the aqueous polymerization reaction, oxidative products would begin to accumulate. As a "critical concentration" of oxidative product was attained, the induction process would begin.However, if the concentration of the oxidative product became too high, the amount of filament formation could be reduced. This phenomenon has been observed previously for the AA induction of tau polymerization (King, Biochemistry 38: 14851 (1999)). Other mechanisms, of course, may be responsible for the "over-oxidation" results. For example, a particular biochemically active oxidative product might actually lose its activity if it were further oxidized. This could be due to the addition of polar groups (such as hydroxyls) or a chemical breakdown into smaller oxidative products such as malondialdehyde (MDA) or 4-hdroxynonenal (HNE). EXAMPLE 7. Theoretical considerations regarding biological implications of the disclosed oxidative effects on FA-induced tau polymerization. Although not wishing to be limited by theory and based on the studies described in the preceding examples, we propose a new mechanism for the oxidative stress-induction of neurodegeneration in AD (FIG. 9). Free radicals, which may be generated by exogenous polymers of Aβ in senile plaques, would attack the plasma membrane, releasing FAs or their oxidation products into the cytoplasm. Elevated amounts of fibriliar Aβ deposits could result from the normal aging process, mutations in the amyloid precursor protein, or mutations in presenilins. This oxidative insult may also be increased in ApoE4 homozygotic individuals that appear to be less capable of clearing Aβ peptides from interstitial spaces. The increased intracellular concentrations of FAs and FA metabolites could then induce tau protein to form filamentous structures. It is possible that the increased phosphorylation of tau protein that is observed early in AD is reflective of increased concentrations of tau protein that are not bound to microtubules and are therefore free to interact with FA oxidation products. These tau filaments may lead to a disruption of neuronal transport by interfering with microtubule based transport or by simply accumulating until the neuron is filled. Either mechanism would surely result in neuronal dysfunction followed by eventual cell death.Although the actual mechanism for neurodegeneration in AD is almost certainly more complicated than the scheme presented above, this model may provide possible avenues of pursuit for the study of this process. This model attempts to combine the oxidative stress theory with the genetic risk factors of AD along with the dominant pathological structures. In addition, the model is consistent with the normal aging process being the most important risk factor in AD, since the aging process is believed to be a major contributor to increased oxidative stress. Any source of oxidative stress could potentially lead to FA oxidation and release into the cytoplasm. This could in turn lead to tau polymerization. Therefore, it is not necessarily surprising that tau pathology is seen in neurodegenerative disorders such as FTDP-17 in the relative absence of senile plaques. Future studies will be based on identifying the FA metabolite(s) that could be responsible for the induction of filament formation. We feel that such knowledge could provide important avenues of pursuit for locating potential targets for preventative or possibly curative strategies for AD.Throughout this application, various patents, publications, books, and nucleic acid and amino acid sequences have been cited. The entireties of each of these patents, publications, books, and sequences are hereby incorporated by reference into this application. SEQUENCE LISTING<110> Gamblin, T. Chris Binder, Lester I.<120> Improved Methods for IdentifyingReagents which Inhibit Tau Polymerization<130> 69450<160> 3<170> FastSEQ for Windows Version 3.0<210> 1<211> 39<212> DNA<213> Artificial Sequence<220> <223> Oligonucleotide primer<400> 1 agcaacgtcc agtccaaggc tggctcaaag gataatatc 39<210> 2 <211> 35 <212> DNA<213> Artificial Sequence<220><223> Oligonucleotide primer<400> 2 gcaaggtgac ctccaaggct ggctcattag gcaac 35<210> 3 <211> 441 <212> PRT <213> Artificial Sequence <220><223> Cysless mutant Tau protein <400> 3 Met Ala Glu Pro Arg Gin Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gin Gly Gly Tyr Thr Met His 20 25 30Gin Asp Gin Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu35 40 45Gin Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val65 70 75 80Asp Glu Gly Ala Pro Gly Lys Gin Ala Ala Ala Gin Pro His Thr Glu85 90 95He Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly He Gly Asp Thr Pro 100 105 110Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gin Ala Arg Met Val115 120 125Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly130 135 140 Ala Asp Gly Lys Thr Lys He Ala Thr Pro Arg Gly Ala Ala Pro Pro145 150 155 160Gly Gin Lys Gly Gin Ala Asn Ala Thr Arg He Pro Ala Lys Thr Pro165 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 Ser195 200 205Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys225 230 235 240Ser Arg Leu Gin Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val245 250 255Lys Ser Lys He Gly Ser Thr Glu Asn Leu Lys His Gin Pro Gly Gly 260 265 270Gly Lys Val Gin He He Asn Lys Lys Leu Asp Leu Ser Asn Val Gin275 280 285Ser Lys Ala Gly Ser Lys Asp Asn He Lys His Val Pro Gly Gly Gly290 295 ' 300 Ser Val Gin He Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser305 310 315 320Lys Ala Gly Ser Leu Gly Asn He His His Lys Pro Gly Gly Gly Gin325 330 335Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gin Ser 340 345 350Lys He Gly Ser Leu Asp Asn He Thr His Val Pro Gly Gly Gly Asn355 360 365Lys Lys He Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala 370 375 380 Lys Thr Asp His Gly Ala Glu He Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser405 410 415He Asp Met Val Asp Ser Pro Gin Leu Ala Thr Leu Ala Asp Glu Val420 425 430Ser Ala Ser Leu Ala Lys Gin Gly Leu WHAT IS CLAIMED IS:

1. A method for identifying reagents that effect tau polymerization, said method comprising:

(a) providing a polymerization buffer;

(d) adding the reagent to the tau polymerization reaction mixture to form a reagent/tau polymerization reaction mixture;

(e) incubating the reagent/tau polymerization reaction mixture to permit the formation of tau filaments wherein the free fatty acid is effectively oxidized during or before the incubating; and

(f) detecting the tau filaments in the reagent/tau polymerization reaction mixture, thereby screening the reagent for the ability to effect tau polymerization.

2. The method of claim 1 , further comprising:

(g) analyzing the tau filaments in the test reagent/tau polymerization reaction mixture.

3. The method of claim 1 , wherein steps (e), (f), and (g) are repeated over time to provide a determination of a rate of tau filament formation.

4. The method of claim 1 , wherein the substantially purified tau protein is selected from the group consisting of mammalian brain tau protein, recombinant wild type tau protein, recombinant tau protein 2N4R, and a tau mutant having no cysteine residues.

5. The method of claim 1 , wherein after adding an effective amount of fatty acid, the fatty acid-containing buffer is incubated for at least 5 minutes before adding the substantially purified tau protein.

6. The method of claim 5, wherein the fatty acid-containing buffer is incubated for about 60 minutes before adding the substantially purified tau protein.

7. The method of claim 1 , further comprising: adding an effective amount of an oxidizer to the fatty acid- containing polymerization buffer before the incubating step.

8. The method of claim 7, wherein the oxidizer is selected from the group consisting of a solution of FeCI₃, ADP, and ascorbic acid, a solution of iron citrate, a solution of lipoxygenases, a solution of cyclooxygenases, a solution of lipoxygenases and cyclooxygenases, and ultraviolet irradiation.

9. The method of claim 8, wherein the oxidizer is about 50 μM FeCI₃, about 20 mM ADP, and about 10 mM ascorbic acid.

10. The method of claim 1 , wherein the free fatty acid is selected from the group consisting of arachidonic acid and docosahexaenoic acid.

11. The method of claim 1 , wherein the method further comprises: adding an effective amount of a reducing agent to the fatty acid-containing polymerization buffer before the incubating step.

12. The method of claim 11 , wherein the reducing agent is selected from the group consisting of DTT, 2-mercaptoethanol, and glutathione

13. The method of claim 12, wherein the reducing agent is DTT at a concentration of between about 0.5 mM and about 50 mM.

14. The method of claim 12, wherein the reducing agent is DTT at a concentration of about 5 mM.

15. A method of regulating the assembly of the protein tau in the brain of a mammal in need of such treatment, said method comprising administering to the mammal a pharmacologically effective amount of an inhibitor of fatty acid oxidation.

16. The method of claim 1 , wherein the mammal is a human.

17. The method of claim 15, wherein the inhibitor of fatty acid oxidation is selected from the group consisting of vitamins with anti-oxidative properties, and non-steroidal anti-inflammatory drugs.

18. The method of claim 3, wherein the inhibitor of fatty acid oxidation is ibuprofen.

19. The method of claim 16, wherein the inhibitor is administered repeatedly over a period of at least one month.

20. The method of claim 16, wherein the inhibitor is administered at least once weekly for at least one month.