WO2005058374A1 - Methods and compositions for modulating tau in vivo - Google Patents

Abstract

Methods and compositions for modulating kinases and for inhibiting phosphorylation of tau in vivo are provided herein.

Description

METHODS AND COMPOSITIONS FOR MODULATING TAU IN VIVO

RELATED APPLICATIONS This application claims the benefit of U.S. provisional application serial nos. 60/529,469, filed December 15, 2003 and 60/615,738, filed October 4, 2004, each of which is incorporated herein by reference in its entirety. TECHNICAL FIELD This invention relates to kinases and methods for modulating kinases in vivo.

BACKGROUND Alzheimer's disease (AD) is one of a number of neurodegenerative diseases characterized by the presence of abnormal structures called paired helical filaments (PHFs) in the brain. PHFs are composed primarily of hyperphosphorylated forms of tau proteins (Grundke-lqbal et al, Proc. Natl. Acad. Sci., USA 83: 4913-4917, 1986). Thus, disorders such as AD in which PHFs are present are called tauopathies. The function of tau proteins is to bind and stabilize microtubules. Abnormally hyperphosphorylated tau proteins inhibit microtubule assembly, which may promote neuronal degeneration by disrupting normal cytoskeletal functions (Alonso et ah, Proc. Natl. Acad. Sci. USA 91:

5562-5566, 1994). Understanding regulation of tau phosphorylation can facilitate discovery of agents to prevent, delay, or treat AD and other tauopathies. Non-AD tauopathies include supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), argyrophilic grain disease, and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Each of these diseases is associated with dementia.

SUMMARY Methods and compositions for modulating kinases in vivo and identifying kinase modulators are provided herein. In one aspect, the invention features a method of identifying an agent that inhibits phosphorylation of a tau polypeptide in vivo. The method includes, for example: a) providing a test compound identified as a protein kinase A (PKA) inhibitor or a glycogen synthase kinase-3 (GSK-3) inhibitor in vitro; b) administering the test compound to a subject; and c) determining whether phosphorylation of the tau polypeptide in the subject is decreased, relative to a control, following administration of the test compound, wherein a decrease in phosphorylation of the tau polypeptide following administration of the test compound is an indication that the test compound is an agent that inhibits phosphorylation of the tau polypeptide in vivo.

The method can further include the step of administering a PKA agonist to the subject prior to or simultaneously with step b). In one embodiment, the agonist is injected into the subject's brain. In one embodiment, the PKA agonist is forskolin. The subject can be a mammal, e.g., a rodent, rabbit, cat or dog. In one embodiment, the subject is a rat. In one embodiment, the subject is a human. ₍ The determining step of the method can include evaluating phosphorylation of the tau polypeptide at one or more serine residues selected from the serine residues corresponding to serine 198, serine 199, serine 202, serine 396, and serine 404 of SEQ ID NO:l (shown in Table 1, below). The determining step can be carried out on neuronal tissue of the subject. The determining step can include preparing a Western blot. In one embodiment, the test compound of step a) of the method is identified as a PKA inhibitor (e.g., prior to step a)). The method can further include determining whether phosphorylation of the tau polypeptide in the subject is increased or decreased relative to an animal that has been administered a known PKA inhibitor that is other than the test compound. In one embodiment, the known PKA inhibitor is Rp-Adenosine 3', 5- cyclic monophosphorothioate triethyl ammonium salt (Rp-cAMPs). In one embodiment, the test compound of step a) is identified as a GSK-3 inhibitor. The method can further include, prior to step a), identifying the test compound as an inhibitor of PKA or GSK-3 by an in vitro method including: i) contacting an in vitro tau polypeptide with (1) PKA or GSK-3 or both PKA and GSK-3 and (2) the test compound; and ii) determining whether phosphorylation of the tau polypeptide is decreased in the presence of the test compound, relative to a control, wherein, if phosphorylation of the in vitro tau polypeptide is decreased in the presence of the test compound, the test compound is identified as being a PKA or GSK-3 inhibitor in vitro. In another aspect, the invention features a method for identifying an agent that inhibits a tau-related decrease in memory skill. The method can include, for example: a) providing a test compound that inhibits PKA or GSK-3; b) providing an animal under conditions in which memory skill is impaired in the animal; c) administering the test compound to the animal; and d) evaluating the animal's memory skill, wherein a decrease in the impairment of memory skill in the presence of the test compound is an indication that the test compound is an agent that inhibits a tau-related decrease in memory skill. In one embodiment, a memory skill (e.g., spatial memory skill) of the animal is impaired by administering to the animal an agent that impairs the memory skill. The memory skill impairment can be due to a genetic manipulation of the animal or an ancestor of the animal. The memory skill can be impaired in step b) by administering an agonist of PKA. In one embodiment, the animal has been trained to perform a spatial task, and the evaluating step comprises assessing the animal's performance of the task. For example the animal can be trained to identify a location in a maze (e.g., a water maze), and the evaluating step can include determining how long the animal spends in the location. The animal can be a rodent (e.g. , a rat). In one embodiment of the method for identifying an agent that inhibits a tau- related decrease in memory skill, the test compound is selected prior to step a) by an in vitro method, the method including: i) contacting an in vitro tau polypeptide with a PKA polypeptide and the test compound; ii) determining whether phosphorylation of the in vitro tau polypeptide is decreased in the presence of the test compound, relative to a control; and iii) if phosphorylation of the in vitro tau polypeptide is decreased in the presence of the test compound, selecting the test compound for use in step a). The method can include, prior to step ii), contacting the in vitro tau polypeptide with a GSK-3 polypeptide simultaneously with or subsequent to step i). In another aspect, the invention features a method for treating a tauopathy in a subject byadministering to the subject an amount of a PKA inhibitor or a GSK-3 inhibitor effective to inhibit the tauopathy, thereby treating the tauopathy in the subject. The subject can be a mammal, e.g., a rodent or a human. The tauopathy can be Alzheimer's disease (AD), supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), argyrophilic grain disease, or frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). In another aspect, the invention features a method for treating a tauopathy in a subject by administering to the subject an amount of a PKA inhibitor and a GSK-3 inhibitor effective to inhibit the tauopathy, thereby treating the tauopathy in the subject. The subject can be a mammal, e.g., a rodent or a human. The tauopathy can be AD, PSP, CBD, PiD, argyrophilic grain disease, or FTDP-17. The invention also features a method for treating a tauopathy in a subject who suffers from the tauopathy or is at risk from the tauopathy, but who is not diagnosed with bipolar depression, a blood disorder, cluster headaches, premenstrual tension, bulimia, alcoholism, a syndrome of inappropriate secretion of ADH, tardive dyskinesia, hyperthyroidism, postpartum affective psychosis, or corticosteroid-induced psychosis. The method includes, for example, administering to the subject an amount of lithium chloride (LiCl) effective to inhibit the tauopathy in the subject, thereby treating the tauopathy in the subject. The subject can be a mammal, e.g., a rodent or a human. In various embodiments, the tauopathy is PSP, CBD, PiD, argyrophilic grain disease, or FTDP-17. In one embodiment, the tauopathy is AD. In another aspect, the invention features a method for reducing loss of memory skill in a subject. The method includes identifying a subject as being susceptible to a condition involving tau-related loss of memory skill, and administering to the subject an amount of a PKA inhibitor or GSK-3 inhibitor effective to reduce loss of memory skill in the subject. The subject's memory skill can be evaluated prior to the administering step, e.g., both prior to and subsequent to the administering step. In various embodiments, the subject is administered the PKA inhibitor or GSK-3 inhibitor in at least four doses over at least four weeks, and/or the subject is administered the PKA inhibitor or GSK-3 inhibitor in at least eight doses over at least eight weeks. The invention also features a kit for identifying an in vivo inhibitor of tau phosphorylation which includes a reference compound that inhibits PKA or GSK-3 in vivo; and a reagent for detecting phosphorylation of a tau polypeptide. The reagent can be an antibody. The kit can further include instructions for comparing phosphorylation of the tau polypeptide in a first subject that has been administered a test compound to phosphorylation of the tau polypeptide in a second subject that has been administered the reference compound. Also featured is a kit for identifying an in vivo inhibitor of tau phosphorylation including: a compound that agonizes PKA in vivo; and a reagent (e.g., an antibody) for detecting phosphorylation of a tau polypeptide. The kit can further include instructions for determining whether phosphorylation of the tau polypeptide is decreased in a subject that has been administered the compound that agonizes PKA, relative to a control. An amino acid that is "corresponding" to an amino acid in a reference sequence occupies a site that is homologous to the site in the reference sequence. Corresponding amino acids can be identified by alignment of related sequences. The term "administering", as used herein, includes self-administering. The term "inhibiting", as used herein encompasses partial and complete inhibition, wherein partial inhibition is a detectable level of inhibition. A "PKA polypeptide", as used herein, refers to a catalytic subunit of protein kinase A (also known as cAMP-dependent protein kinase and A-kinase). The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. '

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 A- ID are graphs and pictures of Western blots depicting the effect of forskolin on phosphorylation of tau in rat hippocampus. The phosphorylation state and level of tau in homogenates of rat hippocampi obtained 24 hours after injection with various concentrations of forskolin were analyzed by Western blots. Phosphorylation- dependent and site-specific tau antibodies PS214 (HG. 1 A), PHF-1 (HG. IB), and Tau-1 (HG. 1C) were used to detect tau phosphorylation at Ser-214, Ser-396/404, and Ser- 198/199/202, respectively. Phosphorylation-independent tau antibody Tau-5 (HG. ID) was used to measure the total tau level. The immunoreactivity (IR) in Western blots was quantitated and expressed in each graph as mean ± SD (n = 8). *, p < 0.05; **, p < 0.01. FIGS. 2A-2F are pictures of tissue sections of rat brains which show the distribution of phosphorylated tau induced by forskolin. Coronal sections of rat brains obtained 24 hours after injection with 40 μl of 80 μM forskolin (HGS. 2B, 2D, 2F) or aCSF (HGS. 2A, 2C, 2E) were immunostained with phosphorylation-dependent and site- specific tau antibodies PS214 (HGS. 2A and 2B), PHF-1 (HGS. 2C and 2D), and Tau-1

(HGS. 2E and 2F), respectively. The insert in the upper right corner of each panel is a picture of the CA3 sector of the hippocampus at higher magnification. Bar = 400 μm, and Bar in insert = 40 μm. FIGS. 3A-3H are graphs depicting the effect of forskolin on the activities of PKA, GSK-3, cdc2, cdk-5 and MAPK in rat hippocampus and the correlation between PKA activity and tau phosphorylation. The activities of PKA (HG. 3A), GSK-3 (HG. 3B), cdc2 (HG. 3C), cdk-5 (FIG. 3D) and MAPK (HG. 3E) in hippocampal extracts from rats infused with various concentrations of forskolin for 24 hours were determined using specific peptide substrates. Bivariate correlation analysis of PKA activity and tau immunoreactivity (IR) with antibodies PS214 (HG. 3F), Tau-1 (FIG. 3G) and PHF-1

(HG. 3H) is shown. The data are presented in each graph as means ± SD of 8 experiments, ** p < 0.01. FIGS. 4A-4L are graphs and pictures of Western blots depicting the effect of LiCl and Rp-cAMPS on the activities of GSK-3 and PKA and phosphorylation of tau in rat brain hippocampus. Rats were injected into lateral ventricle with aCSF as vehicle,

80 μM forskolin alone, or forskolin combined with either 100 mM LiCl or 100 μM Rp- cAMPS. The activities of GSK-3 and PKA and the phosphorylation state of tau at various sites in the hippocampus collected 24 hours after injection were determined. The activities of GSK-3 (HGS. 4A and 4H) and PKA (HGS. 4B and 4G) of the hippocampal extracts were measured using respective specific peptide substrates. The phosphorylation levels of tau at various sites were determined by Western blots using phosphorylation- dependent and site-specific tau antibodies PS214 (for Ser-214, HGS. 4C and 41), PHF-1 (for Ser-396/404, HGS. 4D and 4J), and Tau-1 (for Ser- 198/ 199/202, HGS. 4E and 4K). The total tau level was determined by Western blots using a phosphorylation-independent tau antibody Tau-5 (FIGS. 4F and 4L). The immunoreactivities (IR) of the tau staining were quantitated. All data in the graphs are expressed as mean + SD of 8 experiments. ** p < 0.01 as compared to control injection; ## p < 0.01 as compared to injection with forskolin alone. FIGS. 5A-5F are graphs depicting the effects of Rp-cAMPS and LiCl on cdc2, cdk-5 and MAPK activities. Rats were injected into lateral ventricle with aCSF as vehicle, 80 μM forskolin alone, or forskolin combined with either 100 mM LiCl or

100 μM Rp-cAMPS. The activities of cdc2 (FIGS. 5A and 5B), cdk-5 (HGS. 5C and 5D) and MAPK (HGS. 5E and 5F) were determined using specific peptide substrates. Immunoprecipitation with an antibody specific for cdk-5 was employed to assay the cdk5 activity in the tissue extract. All data in the graphs are expressed as mean ± SD of 8 experiments. FIGS. 6A-6H are graphs and pictures of Western blots depicting the effects of cdc2/cdk5 inhibitor PNU 112455 A on GSK-3, PKA, cdc2 and cdk-5 and phosphorylation of tau in rat hippocampus. Rats were injected into lateral ventricle with aCSF as vehicle, 80 μM forskolin alone, or forskolin combined with 200 μM PNU 112455 A. The activities of GSK-3 (HG. 6A), PKA (HG. 6B), cdc2 (HG. 6C) and cdk-5 (FIG. 6D) were measured using specific peptide substrates. The phosphorylation levels of tau at various sites were determined by Western blots using phosphorylation-dependent and site- specific tau antibodies PHF-1 (for Ser-396/404, HG. 6E), Tau-1 (for Ser-198/199/202, FIG. 6F), and PS214 (for Ser-214, HG. 6G). The total tau level was determined by Western blots using a phosphorylation-independent tau antibody Tau-5 (HG. 6H). The immunoreactivities (IR) of the tau staining were quantitated. All data are expressed as mean ± SD of 8 experiments. ** p < 0.01 as compared to control injection; ## p < 0.01 as compared to injection with forskolin alone. FIGS. 7A-7N are graphs and pictures of Western blots depicting the effects of PD 98059 and SB 203580 (inhibitors of MAPK) on PKA, GSK-3 and MAPK activities and phosphorylation of tau in rat hippocampus. Rats were injected into the lateral ventricle with aCSF as vehicle, 80 μM forskolin alone, or forskolin combined with either 200 μM PD 98059 or 100 μM SB 203580. The activities of PKA (HGS. 7A and 7B) GSK-3 (FIGS. 7C and 7D), and MAPK (HGS. 7E and 7F) were assayed using specific peptide substrates. The phosphorylation levels of tau at various sites were determined by

Western blots using phosphorylation-dependent and site-specific tau antibodies PHF-1 (for Ser-396/404, HGS. 7G and 7H), Tau-1 (for Ser-198/199/202, FIGS. 71 and 7J), and PS214 (for Ser-214, FIGS. 7K and 7L). The total tau level was determined by Western blots using a phosphorylation-independent tau antibody Tau-5 (HGS. 7M and 7N). The immunoreactivities (IR) of the tau staining were quantitated. All data are expressed as mean ± SD of 8 experiments. ** p < 0.01 as compared to control injection; ##p < 0.01 as compared to injection with forskolin alone. FIGS. 8A-8C are graphs depicting the effects of forskolin, LiCl and Rp-cAMPS on spatial memory. In order to acquire spatial memory, rats were trained for 20 trials (4 trials/day) successively in a Morris water maze over a period of 5 days. On day 6, the rats were injected (into their lateral ventricles) with aCSF as vehicle, 80 μM forskolin alone, or forskolin combined with either 100 mM LiCl or 100 μM Rp-cAMPS for 24 hours. The effects of these treatments on spatial memory were measured by a Morris water maze, in which the platform hidden in milky water was removed and the quadrant time and pathway of swimming were recorded for 60 seconds by a computer. The spatial memory of rats was expressed as quadrant time ( ), i.e., the longer the quadrant time, the better the spatial memory. Forskolin impaired the spatial memory of rats (HG. 8 A), and Rp-cAMPS completely abolished this impairment (HG. 8B). LiCl only partial abolished the impairment of spatial memory induced by forskolin (HG. 8C). All data are expressed as mean ± SD of 12 animals. ** p < 0.01 as compared to control injection; ## p < 0.01 as compared to injection with forskolin alone. Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION Abnormal hyperphosphorylation of tau proteins contributes to the formation of neurofibrillary tangles in the brains of AD patients and patients with other neuronal degenerative diseases. Elucidation of the factors that contribute to phosphorylation of tau in vivo is critical for understanding these diseases and identifying agents to treat them (e.g., by preventing the formation of neurofibrillary tangles, which are primarily composed of tau-containing PHFs). The present invention is based, in part, on the discovery that cAMP-dependent protein kinase (PKA) and glycogen synthase kinase-3 (GSK-3) influence the phosphorylation of tau protein in vivo, and that inhibition of these kinases can inhibit adverse effects of tau phosphorylation in vivo, including effects on higher neurological processes such as memory. As shown herein, phosphorylation of tau in vivo is stimulated by PKA activation, which in turn primes tau for phosphorylation by GSK-3.

Tau Tau, also referred to as microtubule-associated protein tau, neurofibrillary tangle protein, and paired helical filament-tau (PHF-tau), is a major neuronal microtubule- associated protein. Its normal function is to promote microtubule assembly from tubulin subunits and to stabilize microtubules. The biological activity of tau is regulated by phosphorylation (Bramblett et al., Neuron 10: 1089-1099, 1993; Lindwall and Cole, FEBS. Lett. 349: 104-108, 1994; Drechsel et al, Mol. Biol. Cell. 3: 1141-1154, 1992; Yoshida and Ihara, J. Neurochem. 61; 1183-1186, 1993; Biernat et al., Neuron IV. 153- 163, 1993; Alonso et al, Proc. Natl. Acad. Sci. USA 91: 5562-5566, 1994; Alonso et al, Nat. Med. 2: 783-787, 1996). In normal adult human brain, six isoforms of tau are expressed by alternative mRNA splicing from a single gene (Goedert et al, EMBO J. 8: 393-399, 1989; Goedert et al, Neuron 3: 519-526, 1989; Goedert and Jakes, EMBO J. 9: 4225-4320, 1990). The isoforms differ by the presence or absence of 29- or 58-amino acid inserts in the N- terminal half of the protein and an additional 31 -amino acid microtubule binding sequence in the C-terminal half of the protein. Similar levels of each isoform are observed in normal brains and PHFs from AD patients. However, all six isoforms of tau from AD patients are hyperphosphorylated. In normal brain, 2-3 moles of phosphate per mole of tau are present. Tau from AD brains exhibits 3-4 fold more phosphorylation than observed in normal brains (Grundke-Iqbal et al, Proc. Natl. Acad. Sci. USA 83:

4913-4917, 1986; Iqbal et al, Lancet 2: 421-426, 1986; Kopke et al, J. Biol. Chem. 268: 24374-24384, 1993). Hyperphosphorylated tau is incapable of promoting microtubule assembly and aggregates into tangles of PHFs (Grundke-Iqbal et al, Proc. Natl. Acad. Sci. USA 83: 4913-4917, 1986; Iqbal et al, Lancet 2: 421-426, 1986). At least 29 phosphorylation sites have been identified in tau found in PHFs

(Morishima-Kawashima et al, Neurobiol. Aging 16: 365-71, 1995; Lovestone and Reynolds, Neurosc. 78: 309-324, 1997; Johnson and Hartigan, J. Alzheimer's Dis. h 329- 351, 1999; Hanger et al, J. Neurochem. 71: 2465-2476, 1998). More than ten protein kinases have been shown to phosphorylate tau in vitro. These include GSK-3, cyclin- dependent kinase 5 (cdk5), cell division cycle 2 kinase (cdc2), mitogen-activated protein kinases (MAP kinases), CaM kinase II, protein kinase-C, and PKA (Morishima- Kawashima et al, Neurobiol. Aging 16: 365-71, 1995; Lovestone and Reynolds, Neurosc. 78: 309-324, 1997; Johnson and Hartigan, I. Alzheimer's Dis. 1: 329-351, 1999; Hanger et al, J. Neurochem. 71: 2465-2476, 1998). The amino acid sequence of a human tau polypeptide is shown in Table 1. This sequence is also available in GenBank^® under GL2144820, Accession QRHUT1.

Table 1. Amino acid sequence of a long splice form of human tau.

MΆEPRQEFEVMΞDHAGTYGLGDRKDQGGYTMHQDQEGDTDAG KESP QTPTEDGSEEPGSETSDAKSTP TAΞDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDK KAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRS RTPSLPTPPTREPKKVAWRTPPKSPSSAKSRLQTAPVP PD KNVKSKIGSTENLKHQPGGGKVQIIN LDLS VQSKCGSKDNIKHVPGGGSVQIVYKPVD SKVTSKCGSLGNIHHKPGGGQVEVKSEK DFKDRV QSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPWSGDTSPRH SNVSSTGSIDMV DSPQLATLADEVSASLAKQGL ( SEQ ID NO : L )

Identification of Kinase Inhibitors Screening assays can be used to identify agents that inhibit PKA or GSK-3. A potential PKA or GSK-3 inhibitor can be essentially any physiologically acceptable substance. For example, an inhibitor can be a protein, peptide, or polypeptide (all of these terms refer to linear polymers of amino acid residues, regardless of glycosylation or other post-translational modification; the term "protein" being commonly used to refer to polypeptides possessing the structure of full-length, naturally occurring proteins and the term "peptide" being commonly used to refer to polypeptides 2 to about 50 amino acid residues in length). The kinase inhibitor can also be a peptidomimetic, a peptoid, another small molecule (e.g., a small synthetic molecule), a nucleic acid, or any other type of molecule. While potential inhibitors are not limited to agents that act by any particular mechanism, some of these agents (e.g., dominant negative mutants or active site inhibitors such as substrate analogs) may inhibit the activity of the kinase, while others (e.g., an antisense oligonucleotide, siRNA, or transcription factor) can alter kinase expression. Likewise, an inhibitor can affect the expression or activity of a molecule that acts on and thereby up- or down-regulates the kinase. Agents identified as inhibitors of PKA or GSK-3 can be used in in vivo methods described herein, e.g., in methods to inhibit tau phosphorylation in vivo. Exemplary PKA inhibitors are listed in Table 2. These agents are available from Calbiochem^® Biochemicals (San Diego, CA). The catalog number for each agent is provided in Table 2. Exemplary GSK-3 inhibitors are listed in Table 3. These are also available from Calbiochem^® Biochemicals and catalog numbers are provided. GSK-3 is also inhibited by lithium chloride and valproic acid. In some embodiments, the screening assays described herein employ a PKA activator (i.e., an agonist) and/or a GSK-3 activator. Exemplary PKA activators (also available from Calbiochem Biochemicals) are listed in Table 4. GSK-3, which is constituitively active in vivo, can be further activated indirectly through inhibition of PI3- kinase. Inhibition of PI3-kinase with wortmannin results in inhibition of protein kinase B (PKB) activity. Active PKB phosphorylates GSK-3β/3 at Ser-9/39 and causes inhibition of GSK-3. Thus, inhibition of PI3-kinase can relieve inhibition of GSK-3 and heighten GSK-3 activation.

Table 2. PICA inhibitors available from Calbiochem Biochemicals

Table 3. GSK-3 inhibitors available from Calbiochem Biochemicals

Table 4. PKA activators available from Calbiochem Biochemicals

Assays used to identify additional PKA or GSK-3 kinase inhibitors in vitro can be carried out, e.g., in cell culture or in a cell -free system, and they can be designed to reveal the presence or absence of the kinase, or the presence or absence of phosphorylation of a substrate (e.g., tau) (i.e., the assays can be qualitative) or the level of its expression or activity (i.e., they can be quantitative). Moreover, the assays can be conducted in a heterogeneous format (where a kinase or a kinase substrate is anchored to a solid phase) or a homogeneous format (where the entire reaction is carried out in a liquid phase). In either approach, the order in which the reactants are added can be varied to obtain different information about the agents being tested. For example, exposing the kinase to the test agent and a binding partner at the same time identifies agents that interfere with binding (by, e.g., competition), whereas adding the test agent after binding has occurred identifies agents capable of disrupting preformed complexes. The in vitro methods can employ purified components (e.g., purified kinase and purified substrate) or more complex biological samples (e.g., a sample obtained from a test subject that includes a tissue, cell or biological fluid in which PKA and/or GSK-3 and/or tau are normally expressed). The sample can be tested for kinase expression (e.g., mRNA or protein expression), structural integrity (e.g., full-length or C-terminally truncated) or kinase activity. In vitro techniques for detecting kinases and kinase substrates include enzyme linked immunosorbent assays (ELISAs), immuno- precipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques can be carried out with labeled probes, such as anti-tau or anti-kinase antibodies, which can be detected by standard imaging techniques. Labeled agents include agents that are directly labeled by being linked or coupled (i.e., physically linked) to a detectable substance as well as agents that are indirectly labeled by virtue of being capable of reacting with a detectable substance or participating in a reaction that gives rise to a detectable signal. To determine the activity of a PKA or GSK-3 kinase, any standard assay for protein phosphorylation can be carried out. One can use a natural kinase substrate (e.g., tau) or another protein or peptide that the kinase phosphorylates. Assays for kinase activity can also be carried out with biologically active fragments of the kinase (e.g., a fragment that retains catalytic activity). More specifically, a screen (e.g., a high throughput screen) for PKA or GSK-3 inhibitors can be carried out by: (a) binding one or more types of substrate proteins or peptides (e.g. , tau proteins or fragments of tau proteins) to a solid support (e.g., the wells of microtiter plates); (b) exposing the support to a blocking agent (e.g., a standard blocking agent to prevent reaction components from non-specifically adhering to the support); and (c) exposing the substrate to PKA and/or GSK-3, a source of phosphate (e.g., ATP with a radioactively labeled gamma-phosphate), and a test compound (i.e., a potential kinase inhibitor). The components of the reaction (e.g., the kinase, phosphate source, and test compound) are typically supplied in a buffered solution and the reaction is allowed to proceed at a temperature (the temperature can vary from, for example, room temperature (about 23°C) to a physiological temperature (about 37°C) or higher) and for a period of time that is in the linear range of the assay. The reaction can be terminated in a number of ways (by, for example, rinsing the support several times with a buffered solution), and the amount of phosphate incorporated into the bound substrate can be determined (standard techniques are available to measure, for example, radioactive tags; or phosphorylation of particular residues in a substrate may be detected by antibodies specific for the phosphorylated form of the residue). Inhibitors are identified as the agents that reduce the extent to which the kinase was able to phosphorylate the substrate, or the rate of phosphorylation. Appropriate controls can be carried out in connection with any of the methods of screening potential inhibitors. For example, the method described above (and others aimed at identifying kinase inhibitors) can be carried out in the presence and absence of a test compound (representing experimental and control paradigms, respectively). Alternatively, test compounds and placebos (e.g., biologically inactive test compounds, such as denatured or mutant proteins or nucleic acids that lack biological activity) can be used. Positive controls (employing compounds known to possess the desired activity) may also be useful. The agents tested for inhibitory activity can be those within a library, and the screen can be carried out using any of the numerous approaches used with combinatorial libraries. One can use, for example, libraries of biological molecules, or alternatively peptoid libraries that contain molecules having the functionalities of peptides, but with novel, non-peptide backbones that are resistant to enzymatic degradation but that nevertheless are bioactive (see, e.g., Zuckermann et al, J. Med. Chem. 37:2678-85, 1994). One can also use spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one- compound" library method; and synthetic library methods using affinity chromatography selection. Molecular libraries can be synthesized according to methods known in the art (see, e.g., DeWitt et al, Proc. Natl. Acad. Sci. USA 90:6909, 1993; Erb et al, Proc. Natl. Acad. Sci. USA 91:11422, 1994; Zuckermann et al, I. Med. Chem. 37:2678, 1994; Cho et al, Science 261:1303, 1993; Carrell et al, Angew. Chem. Int. Ed. Engl. 33:2059, 1994;

Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al, J. Med. Chem. 37:1233, 1994). Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Patent No. 5,223,409), spores

(Ladner, U.S. Patent No. 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Cwirla et al, Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301-310. 1991; Ladner, supra.). Regardless of the precise mode of presentation, the agents in the libraries are exposed to a PKA or GSK-3 and a substrate; here, as above, agents within the libraries can be identified as inhibitors by virtue of their ability to prevent, to any extent, the ability of the kinase to phosphorylate its substrate. PKA and GSK-3 activity can also be assayed in cell-based systems. These methods can be carried out by, for example, contacting a cell that expresses a PKA and/or GSK-3 protein, or a biologically active portion thereof, with a test agent and assessing the ability of the test agent to inhibit kinase activity (any assay to examine kinase activity can be carried out with a biologically active portion of the whole kinase). The inhibitor can affect the kinase directly or indirectly (by inhibiting or activating a molecule that acts on, or that is acted on by, the kinase). Cell-based systems can also be used to identify agents that inhibit PKA or GSK-3 by inhibiting its expression (in that event, it is expected that the test agents will be nucleic acids (e.g., siRNA or antisense oligonucleotides) or molecules that regulate the activity of transcription factors). The cell can be any biological cell that expresses the kinase of interest, whether naturally or as a result of genetic engineering. For example, the cell can be a mammalian cell, such as a murine, canine, feline, bovine, ovine, porcine, or human cell. The cell can also be non- mammalian (e.g., a Drosophϊla, Xenopus, or yeast cell). The cell can be compared to a cell that expresses a small-interfering RNA (siRNA) that inhibits kinase expression, as a control. In addition to, or as an alternative to, assessing kinase activity, the assays can reveal whether a test agent interferes with the ability of PKA and/or GSK-3 to simply bind to, or otherwise associate with, another molecule (e.g., tau). These methods can be carried out by, for example, labeling either the kinase or its binding partner with a marker, such as a radioisotope or enzymatic label, so that the kinase-containing moieties can be detected. Suitable labels are known in the art and include, for example, ¹²^I, ³⁵S, ¹⁴C, or ³H (which are detectable by direct counting of radioemmissions or by scintillation counting). Enzymatic labels include horseradish peroxidase, alkaline phosphatase, and luciferase, which are detected by determining whether an appropriate substrate of the labeling enzyme has been converted to product. Fluorescent labels can also be used. Another way to detect interaction (between any two molecules (e.g., GSK-3 and tau)) using a fluorophore is by fluorescence energy transfer (FET) (see, e.g., Lakowicz et al, U.S. Patent No. 5,631,169 and Stavrianopoulos et al, U.S. Patent No. 4,868,103. Binding can also be detected without using a labeled binding partner. For example, a microphysiometer can be used to detect the interaction of a kinase with a substrate without the labeling the kinase or substrate (McConnell et al, Science 257:1906-1912. 1992). Another label-free option is to assess interaction between a kinase and a target molecule (be it a kinase substrate or other binding protein) with realtime Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, Anal. Chem. 63:2338-2345, 1991 and Szabo et al, Curr. Opin. Struct. Biol. 5:699-705, 1995). BIA detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that indicates real-time reactions between biological molecules. As noted above, kinase inhibitors can be detected in assays where a substrate is bound to a solid support. More generally, wherever kinase-related binding is assessed, one of the binding partners can be anchored to a solid phase (e.g. , a microtiter plate, a test tube (e.g., a microcentrifuge tube) or a column). The non-anchored binding partner can be labeled, either directly or indirectly, with a detectable label (including any of those discussed herein), and binding can be assessed by detecting the label. If desired, the kinase (or a biologically active fragment thereof) can be fused to a protein that binds a matrix. For example, one can identify a kinase inhibitor by fusing a kinase to glutathione-S-transferase; absorbing the fusion protein to a support (e.g., glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-derivatized microtiter plates); exposing the immobilized fusion protein to a potential binding partner (e.g., an agent that inhibits the activity of the kinase; i.e., a test compound); washing away unbound material; and detecting bound material. The exposure should take place under conditions conducive to complex formation (e.g., a physiologically acceptable condition).

Alternatively, the complexes can be dissociated from the matrix, and the level of kinase binding or activity can be determined using standard techniques. PKA and GSK-3 kinases or molecules with which they interact or with which they may interact can also be immobilized on matrices using biotin and avidin or streptavidin. For example, biotinylated kinases or molecules to which they bind can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., using the biotinylation kit sold by Pierce Chemicals, Rockford, IL), and immobilized in the wells of avidin- or streptavidin-coated 96 well plates (Pierce Chemical). Regardless of the precise way in which a kinase is immobilized, the kinase is exposed to a potential binding partner, any unreacted components are removed (e.g., by washing under conditions that retain any complexes); and the remaining complexes are detected (e.g., by virtue of a label or with an antibody, e.g., an antibody that specifically binds the kinase used in the assay). The step of detecting the kinase or substrate can also be carried out by enzyme-linked assays, which rely on detecting an enzymatic activity associated with the kinase or its substrate. Where the binding assay is carried out in a liquid phase, the reaction products (e.g., tau-containing complexes) can be separated from unreactive components by, for example: differential centrifugation (see, e.g., Rivas and Minion, Trends Biochem. Sci. 18:284-287, 1997); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al, Eds. Current Protocols in Molecular Biology 1999, J. Wiley & Sons, New York.); and immunoprecipitation (as described, for example, in Ausubel, supra). Where kinase expression is assessed, a cell or cell-free mixture is contacted with a candidate compound and the expression of kinase mRNA or protein is evaluated (the level can be compared to that of the kinase mRNA or protein in the absence of the candidate compound or in the presence of another control substance (e.g., where the candidate compound is an antisense oligonucleotide, the "control" can include a "sense" oligonucleotide)). Clearly, where mRNA or protein expression is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is an inhibitor of the kinase mRNA or protein expression. The level of kinase mRNA or protein expression can be readily determined using methods well known in the art (e.g., Northern blot analysis, Western blot analysis or other immunoassay, polymerase chain reaction analyses (e.g., rtPCR; see U.S. Patent No. 4,683,202), probe arrays, or serial analysis of gene expression (SAGE) (see U.S. Patent No. 5,695,937)). The methods described above can be carried out in concert with other methods. For example, a PKA or GSK-3 inhibitor can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of the kinase can be confirmed in vivo (e.g., in an animal such as a mouse or a non-human primate).

Screening Kinase Inhibitors In Vivo Test agents or agents identified as inhibitors of PKA or GSK-3 can be administered to an animal and the effects of the agent on the animal can be evaluated. For example, agents identified as inhibitors of PKA or GSK-3 in vitro can be examined for inhibition of these enzymes in vivo, e.g., by obtaining a sample of a tissue, such as a neuronal tissue, from an animal that has been treated with the agent, and determining whether the kinase activity is altered relative to a control (e.g., a sample from an animal that has not been treated with the agent). Alternatively, or in addition, the effects of an agent on a tau protein can be determined by treating an animal with the agent and isolating a sample from a tissue of the animal that expresses the tau protein (e.g., neuronal tissue) and comparing the tau protein to tau protein isolated from a control sample (e.g., neuronal tissue isolated from an animal that has not been treated with the agent). Effects on tau which can be examined include, for example, effects on tau's phosphorylation, aggregation, subcellular localization, or association with tubulin, and can be assayed by methods known in the art, such as immunohistochemistry and Western blotting. Methods in which a sample is removed from an animal and analyzed ex vivo can be performed, e.g., according to the in vitro methods described in the section above. In addition, or alternative to biochemical abnormalities, the effects of agents on one or more neurological functions can be examined. In particular, functions that are impaired in AD patients (or patients suffering from another tauopathy) can be examined. For example, AD patients lose recent memories, have difficulty retaining new information, and have difficulty with visual/spatial information. Methods for evaluating memory and learning in animals (e.g., rodents) include, e.g., experiments in which animals are trained to perform a task, and performance of the task is evaluated (e.g., under conditions in which the animal is treated with a candidate PKA and/or GSK-3 inhibitor). Animals may be tested in methods that include the use of a Morris water maze, a Barnes (non-swimming) maze, a radial maze, a T-maze, and other devices. Methods that measure conditioning (e.g., eyeblink conditioning, conditioned taste aversion, cued and contextual fear conditioning) may also be used. See, e.g., Current Protocols in Neuroscience (John Wiley & Sons, Inc., 2000) for methods for testing memory and other cognitive functions in animals. Animal models for AD or another tauopathy may be used, or animals may be treated such that they exhibit a feature of AD or another tauopathy prior to treatment. Animal models for AD or AD-related conditions include cholesterol-fed rabbits (Sparks et al, Proc. Natl. Acad. Sci. USA 100(19): 11065-9, 2003) and various transgenic mouse strains, e.g., expressing human and/or mutant amyloid precursor proteins (see, e.g.,

Dodart et al, Genes Brain Behav. l_(3):142-55, 2002), or mutant tau proteins (see, e.g., Tatebayashi et al, Proc. Natl. Acad. Sci. USA 99(21):13896-901, 2002). Animals may be administered a test compound by any suitable means, such as by infusion into the brain, parenteral injection, oral administration, inhalation administration, transdermal administration, use of a slow release device or implant, etc. Methods for evaluating the effects of agents on kinase activity, tau proteins, and spatial memory are described further in the Examples, below.

Pharmaceutical Compositions Compositions useful for inhibiting phosphorylation of tau in vivo (e.g., PKA and

GSK-3 inhibitors, whether previously known or identified by the screening assays described herein), can be incorporated into pharmaceutical compositions and administered to patients who have, or who are at risk of developing, a tauopathy (e.g., AD, surpanuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), argyrophilic grain disease, and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17)). Such compositions will include one or more inhibitors and a pharmaceutically acceptable carrier (e.g., a solvent, dispersion medium, coating, buffer, absorption delaying agent, and the like, that are substantially non-toxic). Supplementary active compounds can also be incorporated into the compositions. In general, agents that inhibit phosphorylation of tau in vivo will either cross the blood/brain baπier or will be administered so as to be accessible to brain tissue (e.g., into the brain, e.g., into a compartment continuous with cerebrospinal fluid surrounding the brain, e.g., into a ventricle of the brain). Pharmaceutical compositions are formulated to be compatible with their intended route of administration, whether oral or parenteral (e.g., intravenous, intradermal, subcutaneous, transmucosal (e.g., nasal sprays are formulated for inhalation), or transdermal (e.g., topical ointments, salves, gels, patches or creams as generally known in the art). The compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents; antibacterial or antifungal agents such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and isotonic agents such as sugars (e.g., dextrose), polyalcohols (e.g., manitol or sorbitol), or salts (e.g., sodium chloride). Liposomal suspensions (including liposomes targeted to affected cells with monoclonal antibodies specific for neuronal antigens) can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). Inhibitor preparations can be formulated and enclosed in ampules, disposable syringes or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating such as lecithin, or a surfactant. Absorption of the active ingredient can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.). Where oral administration is intended, the inhibitor can be included in pills, capsules, troches and the like and can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Compositions containing inhibitors can be formulated for oral or parenteral administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage). Toxicity and therapeutic efficacy of compounds, including any potential kinase inhibitor, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. One can, for example, determine the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population), the therapeutic index being the ratio of LD₅₀:ED₅₀. Inhibitors that exhibit high therapeutic indices are preferred. Where an inhibitor exhibits an undesirable side effect, care should be taken to target that agent to the site of the affected tissue (the aim being to minimize potential damage to unaffected cells and, thereby, reduce side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures. Data obtained from the cell culture assays and animal studies can be used in formulating an appropriate dosage of any given kinase inhibitor for use in humans. A therapeutically effective amount of a kinase inhibitor will be an amount that delays progression of a tauopathy, or improves one or more symptoms of the tauopathy, whether evident by improvement in an objective sign or subjective symptom of the disease. Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present). Kinase inhibitors identified and administered according to the methods of the invention can be small molecules (e.g., peptides, peptidomimetics (e.g., peptoids), amino acid residues (or analogs thereof), polynucleotides (or analogs thereof), nucleotides (or analogs thereof), or organic or inorganic compounds (e.g., heteroorganic or organometallic compounds). Typically, such molecules will have a molecular weight less than about 10,000 grams per mole (e.g., less than about 7,500, 5,000, 2,500, 1,000, or 500 grams per mole). Salts, esters, and other pharmaceutically acceptable forms of any of these compounds can be assayed and, if kinase-inhibitory activity is detected, administered according to the therapeutic methods described herein. Exemplary doses include milligram or micro gram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 μg - 500 mg/kg; about 100 μg - 500 mg/kg; about 100 μg - 50 mg/kg; 10 μg - 5 mg/kg; 10 μg - 0.5 mg/kg; or 1 μg - 50 μg/kg). While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including small molecules, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending physician or veterinarian (in the case of therapeutic application) or a researcher (when still working at the clinical development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. EXAMPLES

Example 1. Forskolin Induces Tau Phosphorylation at Ser-214. Ser- 198/ 199/202 and Ser-396/404 In Vivo To study the effect of PKA activation on tau phosphorylation in vivo, different doses of forskolin, a specific PKA activator (Adashi and Resnick, I. Cell. Biochem. 31: 217-228, 1986; Laurenza et al, Trends. Pharmacol. Sci. 10: 442-447, 1989), were injected the left lateral ventricle of rats, and the rats were sacrificed 24 hours after injection. Sprague-Dawley rats (Grade II, male, weight 200-250g, 4 months old) were supplied by Experimental Animal Central of Tongji Medical College. Rats were first anesthetized by chloral hydrate (30 mg/kg, i.p) and placed on a stereotactic instrument with the incisor bar set 2 mm below the ear bars (i.e., flat skull). After the scalp was incised and retracted, a 50 μl syringe (Hamilton) was stereotactically placed into the lateral ventricle of cerebrum at the co-ordinates from bregma and dura of AP-0.8, L-l .5 and V-4 (in mm). Forskolin (dissolved at different dosages as described below in artificial cerebrospinal fluid (aCSF) composed of 140 mM NaCl, 3.0 mM KC1, 2.5 mM CaCl₂, 1.0 mM MgCl₂, 1.2 mM Na₂HPO₄, pH 7.4) was injected (40 μl) into the left ventricle of the brain. The same volume of aCSF without forskolin was infused into the left ventricle of control animals. All surgical procedures were completed under sterile conditions and penicillin (200,000 U, i.m) was injected to prevent infection (Hauss-

Wegrzyniak et al, Brain Res. 780: 294-303, 1998; Bennecib et al, FEBS. Lett. 485: 87- 93, 2000; Bennecib et al, Alzheimer's Reports 3: 295-303, 2000). To prepare rat hippocampal extracts, rats were killed 24 hours after injection. The left hippocampus was immediately removed and homogenized at 4°C using a Teflon glass homogenizer in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM NaF, 1 mM

Na₃VO , 10 mM β-mercaptoethanol, 5 mM EDTA, 2 mM benzamidine, 1.0 mM phenylmethyl sulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, and 2 μg/ml pepstatin. The tissue homogenates were then divided into two portions. One portion of each homogenate was centrifuged at 12,000 g for 20 minutes at 4°C, and the resulting supernatant was stored at -80°C for assaying activities of protein kinases. The other portion was mixed in 2:1 (v/v) ratio with lysis buffer containing 200 mM Tris-HCl, pH7.6, 8% SDS, 40% glycerol, boiled for 10 minutes in a water bath, then centrifuged at 12,000 g for 30 minutes, and the supernatant was stored at -80°C for Western blot analysis. The concentration of protein in the hippocampal extracts was measured by BCA kit (Bennecib et al, FEBS. Lett. 485: 87-93, 2000; Bennecib et al, Alzheimer's Reports 3: 295-303, 2000) according to manufacturer's instructions (Pierce Chemical

Company, Rockford, IL, USA). Changes in the phosphorylation of tau at several sites were then examined by Western blots using phosphorylation-dependent and site-specific tau antibodies as described previously (Bennecib et al, FEBS. Lett. 485: 87-93, 2000). Blots were developed with antibodies specific for PS214 (1 : 1 ,000), Tau-1 (1 :30,000), PHF-1

(1:500), or Tau-5 (1:200), and visualized using an enhanced chemiluminescent substrate kit (Pierce, Rockford, IL, USA) and exposure to CL-Xposure^® film (Pierce, Rockford, IL, USA). PS214 is a rabbit polyclonal antibody specific for a tau protein phosphorylated at Ser-214 (Biosource International, Camarillo, CA, USA). Tau-1 is a monoclonal antibody specific for a tau protein that is not phosphorylated at Ser-198/199/202. PHF-1 is a monoclonal antibody that is specific for a tau protein phosphorylated at Ser-396/404. Tau-5 is a monoclonal antibody that reacts with all forms of the tau protein, whether or not phosphorylated (Lab Vision Corporation, Fremont, CA, USA). The immunoreactivity of tau bands was quantitatively analyzed by Kodak Digital Science ID software (Eastman Kodak Company, New Haven, CT, USA) and expressed as sum optical density. The levels of total tau and tau phosphorylated at various sites were expressed as relative level of the sum optical density against control. Phosphorylation of tau at Ser-214 in rat hippocampus increased to approximately 4, 7, and 15-fold of the control level 24 hours after injection of 20 μM, 40 μM and 80 μM forskolin, respectively

(FIG. 1 A). Furthermore, under the same conditions, phosphorylation of tau at Ser- 396/404 (PHF-1 site) increased 1.4-, 2-, and 3-fold (HG. IB), respectively, and the level of non-phosphorylated tau at Ser 199/202 (Tau-1 sites) was decreased to 77%, 50% and 38% of the control level (FIG IC), respectively. The total level of tau measured by Tau-5 was not changed significantly by forskolin treatment (FIG. ID). These data show that forskolin increased the phosphorylation of tau in rat hippocampus in a dose-dependent manner, and the effects are not due to changes in the level of tau protein. The immunohistochemical distribution of tau phosphorylation induced by forskolin infusion in the lateral ventricle was also determined as follows. Twenty four hours after injection, rats were fixed in situ by perfusion for 20 minutes at 4°C by Zamboni's solution containing 2% paraformaldehyde, 15% picric acid, and 24 mM NaH₂PO - 126 mM Na₂HPO₄ (pH 7.2). The brain was removed from the skull of the fixed animals and sliced coronally into blocks that contained hippocampus. These tissue blocks were further fixed in Zamboni's solution for another 12 hours at 4°C, paraffin embedded, and cut into 5 μm-thick sections. The immunocytochemical staining was performed as described previously (Pei et al, J. Neuropathol. Exp. Neurol. 56: 70-78, 1997; Pei et al, J. Neuropathol. Exp. Neurol. 58: 1010-1019, 1999). Briefly, the tissue sections were first treated with 100 mM NaOH at room temperature for 30 minutes, followed by incubation at 4°C for 48 hours with one of the following primary antibodies: PS214 (1:500), Tau-1 (1:30,000), PHF-1 (1:500). The bound primary antibodies were detected using

Histostain™-SP kits (Zymed Laboratories Inc., South San Francisco, CA, USA), and visualized with diaminobenzidine (DAB). All sections were counterstained lightly with hematoxylin to show cell nuclei. A marked increase in the immunostaining of hippocampi of forskolin-treated animals as compared with that of vehicle-injected controls was observed with antibodies

PS214 (HGS. 2A, 2B) and PHF-1 (FIGS. 2C, 2D). The most dramatic increase in the staining was seen in the mossy fibers of CA3 sector. When stained with Tau-1 (which stained tau unphosphorylated at Ser-198/199/202), decreased staining compared to control was observed in sections from forskolin-treated animals (FIG. 2E, 2F). The topology of the change of staining with Tau-1 was similar to those of staining with PS214 and PHF-1. These results indicated that the infusion of forskolin induced phosphorylation of tau at various sites on tau in the same region of the brain. The tau phosphorylation occurred most dramatically in the mossy fibers of CA3 sector of hippocampus. Example 2. Forskolin Increases PKA But Not GSK-3. Cdc2. Cdk-5 and MAPK Activities in Rat Hippocampus To confirm the activation of PKA in situ in the hippocampus by injection of forskolin into the lateral ventricle, PKA activity in the hippocampal extracts were measured. The PKA activity was measured using Kemptide (Leu-Arg-Arg-Ala-Ser-Leu- Gly; SEQ ID NO:8) as a substrate, as described previously (Kemp et al, J. Biol. Chem. 252: 4888-4894, 1977; Casnellie, Meth. Enzymol. 200: 115-20, 1991). Briefly, samples of tissue extract containing 7.5 μg of protein were incubated for 10 minutes at 30°C with 100 μM Kemptide, 5 μM cAMP and 100 μM [γ-³²P]-ATP (2,000 cpm/ pmol ATP) in 40 mM Tris-HCl (pH7.4), 20 mM MgCl₂ and 0.1 mg/ml BSA. The reaction was stopped and the kinase activity of each sample was determined. The reaction mixture was applied in triplicate to phosphocellulose filters (Pierce, Rockford, IL, USA). The filters were washed 3 times with 75 mM O-phosphoric acid, dried and counted by liquid scintillation counter. PKA activity was expressed as pmol phosphate incorporated/mg of protein min at 30°C. The level of protein kinase activity is expressed in FIGS. 3A-3H as relative to the enzymatic activity in control animals. Forskolin activated PKA in a dose-dependent manner (FIG. 3A). Up to 6-fold activation of PKA was observed following injection with 80 μM forskolin. A positive correlation between PKA activity and immunoreactivities of PS214 (FIG. 3F) and PHF-1 (FIG. 3H), both of which recognize only phosphorylated tau, and a negative correlation between PKA activity and immunoreactivity of Tau-1, which recognizes only unphosphorylated tau (FIG. 3G), were observed. However, among the tau phosphorylation sites studied, only Ser-214 is a PKA site. Several studies have demonstrated that Tau-1 sites (Ser-198/199/202) and the PHF-1 site (Ser-396/404) cannot be phosphorylated by PKA (Scott et al, J. Biol. Chem. 268: 1168-1173, 1993; Litersky et al, Biochem. J. 316: 655-660, 1996; Wang et al, FEBS. Lett. 436: 28-34, 1998). Rather, these sites can be phosphorylated by each of GSK-3, cdc2, cdk-5 and MAPK in vitro (Mandelkow et al, FEBS. Lett. 314: 315-321, 1992; Song and Yang, J. Protein. Chem. 14: 95-105, 1995; Godemann et al, FEBS. Lett. 454: 157-164, 1999; Reynolds et al, J. Neurochem. 74: 1587-1595, 2000; Liu and Wang, Ada Pharmacol. Sin. 22: 183-187,

2002). Hence, we studied whether infusion of forskolin could indirectly activate GSK-3, cdc2, cdk-5 or MAP kinase (MAPK) activity in the brain, which might have induced phosphorylation of tau at Tau-1 and PHF-1 sites. The GSK-3 activity in rat hippocampal extracts was measured using phospho-GS peptide 2 as described previously (Pei et al, J. Neuropathol. Exp. Neurol. 56: 70-78, 1997; Tanaka et al, FEBS. Lett. 426: 248-254, 1998; Tsujio et al, FEBS. Lett. 469: 111-

117, 2000). Briefly, a sample of tissue extract containing 7.5 μg of protein was incubated for 30 minutes at 30°C with 20 μM peptide substrate and 200 μM [γ-³²P]-ATP (1,500 cpm/ pmol ATP) in 30 mM Tris, pH 7.4, 10 mM MgCl₂, 10 mM NaF, 1 mM Na₃VO₄, 2 mM EGTA, and 10 mM β-mercaptoethanol in a total volume of 25 μl. The reaction was stopped by addition of 25 μl of 300 mM O-phosphoric acid. The reaction mixture was applied in triplicate to phosphocellulose filters (Pierce, Rockford, IL, USA). The filters were washed 3 times with 75 mM O-phosphoric acid, dried and counted by liquid scintillation counter. The GSK-3 activity was expressed as pmol phosphate incorporated/mg of protein/min at 30°C. The cdc2 kinase activity was measured using as a substrate a synthetic peptide

(PKTPKKAKKL; SEQ ID NO:9) corresponding to amino acids 9-18 of histone HI (Lew et al, I. Biol. Chem. 267: 13383-90, 1992). Briefly, a sample of tissue extract containing 10 μg protein was incubated for 10 minutes at 30°C with 50 μM peptide substrate and 200 μM [γ-³²P]-ATP (2,000 cpm/ pmol ATP) in 30 mM Tris, pH 7.4, 10 mM MgCl₂, 10 mM NaF, 1 mM Na₃VO , 2 mM EGTA, and 10 mM β-mercaptoethanol in a total volume of 25 μl. The reaction was stopped and the kinase activity was determined as described above for the GSK-3 activity assay. The MAP kinase activity was measured using as a substrate a synthetic peptide (APRTPGGRR; SEQ ID NO: 10) corresponding to a fragment of bovine myelin basic protein (Boulton et al, Science 249: 64-7, 1990; Blumer and Johnson, Trends. Biochem. Sci. 19: 236-40, 1994; Lewis et al, I. Biol Chem. 266: 15180-4, 1991). Briefly, a sample of tissue extract containing 10 μg of protein was incubated for 10 minutes at 30°C with 250 μM peptide substrate in assay buffer: 20mM MOPS, pH 7.2, 25mM β-glycerol phosphate, 5mM EGTA,1 mM Na₃VO , ImM dithiothreitol. The reaction was stopped and the kinase activity was determined as described above for the GSK-3 activity assay. The cdk5 activity was measured using immunoprecipitation of cdk5 (Bennecib et al, Alzheimer's Reports 3: 295-303, 2000). Briefly, 50 μg protein was mixed with 2 μg of antibody specific for cdk5 (rabbit anti-cdk5 polyclonal antibody, Santa Cruz, CA, USA). After incubation at 4°C overnight, 30 μl immobilized protein G suspension was added to the reaction mixture and the mixture was constantly mixed for 2-4 hours. The sedimented beads were suspended in 25 μl assay buffer solution: 30 mM Tris, pH 7.4, 10 mM MgCl₂, 10 mM NaF, 1 mM Na₃VO₄, 2 mM EGTA, and 10 mM β- mercaptoethanol, and reacted with 200 μM [γ-³²P]-ATP for 10 minutes at 30°C. The reaction was stopped and the kinase activity was determined as described above for the GSK-3 activity assay. The level of each protein kinase activity was expressed as relative to the enzymatic activity in control animals (HG. 3D). Determination of GSK-3, cdc2, cdk-5 and MAPK activities of the hippocampal extracts indicated there was no increase in the activities of these protein kinases upon forskolin treatment (HGS. 3B, C, D and E, respectively). These results suggest that the increased phosphorylation of tau at Tau-1 and PHF-1 sites was not due to an increase of

GSK-3, cdc2, cdk-5 or MAPK activity in forskolin-injected rat brain.

Example 3. Phosphorylation of Tau at Ser-198/199/202 and Ser-396/404 by GSK-3 Basal Activity When It Is Prephosphorylated by PKA in Rat Hippocampus It was examined whether phosphorylation of tau by PKA at Ser-214 enhanced phosphorylation at PHF-1 and Tau-1 sites by the basal activity of GSK-3 in the forskolin- injected brain. LiCl, which is commonly used to inhibit GSK-3 activity (Phiel et al, Nature 423:435-439, 2003), was injected into rat brains together with forskolin, as described in Example 1. Injection with 100 mM LiCl induced approximately a 40% reduction in GSK-3 activity in rat brain hippocampus (HG. 4A), but it had no effect on PKA activity (HG. 4B). This inhibition of GSK-3 did not affect forskolin-induced tau phosphorylation at Ser-214 (HG. 4C), but dramatically decreased forskolin-induced tau phosphorylation at PHF-1 (FIG. 4D) and Tau-1 sites (HG.4E). These changes in tau immunoreactivities were not due to any changes in total tau level, because neither forskolin alone nor forskolin plus LiCl altered total tau level in the rat hippocampus (HG. 4F). These results suggested that forskolin-induced phosphorylation of tau at PHF- 1 and Tau-1 sites is catalyzed by the basal activity of GSK-3. To confirm whether the GSK-3-catalyzed phosphorylation of tau at PHF-1 and Tau- 1 sites is dependent on PKA-catalyzed tau phosphorylation in rat hippocampus, Rp- Adenosine 3', 5 '-cyclic monophosphorothioate triethyl ammonium salt (Rp-cAMPS;

Sigma Chemical Co., St. Louis, MO, USA), a specific PKA inhibitor (Dostmann et al, J. Biol. Chem. 265: 10484-10491, 1990; Eckstein, Annu. Rev. Biochem. 54: 367-402, 1985), was injected together with forskolin. This combination blocks forskolin-induced PKA activation but not other potential forskolin-induced effects. Kinase assays confirmed the blockage of forskolin-induced PKA activation by Rp-cAMPS (HG. 4G). Injection of Rp-cAMPS did not affect GSK-3 activity (HG. 4H). Blockage of PKA activation also blocked the forskolin-induced phosphorylation of tau at Ser-214 (HG. 41), PHF-1 (HG. 4J), and Tau-1 (HG. 4K) sites. These changes in tau immunoreactivities were not due to any changes in total tau level, because neither forskolin alone nor forskolin plus Rp-cAMPS altered total tau level in the rat hippocampus (HG. 4L). These results indicated that the increase in the phosphorylation of tau at PHF-1 and Tau-1 sites by GSK-3 may result from the prephosphorylation of tau at other sites by activation of PKA in rat brain. Example 4. Tau Is Not Phosphorylated at Ser-198/199/202 and Ser-396/404 by

Cdc2, Cdk-5 or MAPK When It Is Prephosphorylated by PKA in Rat Hippocampus Tau is known to be phosphorylated in vitro at Ser-198/199/202 and Ser-396/404 by cdc2, cdk-5 or MAPK as proline directed protein kinases (PDPK) (Mandelkow et al, FEBS. Lett. 314: 315-321, 1992; Drewes et al, EMBO. J. 11: 2131-8, 1992; Baumann et al, FEBS. Lett. 336: 417-24, 1993; Reynolds et al, J. Neurochem. 74: 1587-1595, 2000).

To investigate possible activation of these kinases by forskolin and inhibition of these kinases by LiCl and or Rp-cAMPs in the present study, the activities of these protein kinases in rats injected with these compounds were measured as described in Example 2. Forskolin did not increase the activities of cdc2, cdk5 or MAPK, and 100 mM LiCl and 80 μM Rp-cAMPS did not affect the activities of cdc2, cdk-5 or MAPK (FIG. 5). To determine whether basal activities of cdc2, cdk5 or MAPK could have phosphorylated tau at Ser-198/199/202 and Ser-396/404 in hippocampus of rat when tau was prephosphorylated by PKA, we injected 200 μM PNU 112455 A (an inhibitor of cdc2 and cdk-5, also known as N⁴-(6-Aminopyrimidin-4-yl)-sulfanilamide) (Clare et al, J. Biol. Chem. 51 : 48292-9, 2001), 200 μM PD 98059 (inhibitor of MAP kinase kinase, also known as 2'-Amino-3'-methoxyflavone) (Kultz et al, J. Biol. Chem. 273:13645-51, 1998) or 100 μM SB 203580 (inhibitor of p38 MAP kinase, also known as 4-(4- fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) lH-imidazole) (LoGrasso et al, Biochemistry 36: 10422-7, 1997) combined with 80 μM forskolin into the lateral ventricle of rats for 24 hours. It was found that 200 μM PNU 112455 A neither affected GSK-3 activity (FIG. 6A) nor altered the forskolin-induced elevation in PKA activity (HG. 6B). As expected, 200 μM PNU 112455A significantly decreased cdc2 and cdk-5 activities to approximately 46% and approximately 60% of the controls, respectively (HGS. 6C and 6D). Under these conditions, no significant change was observed in forskolin-induced tau hyperphosphorylation at Ser-396/404 (FIG. 6E), Ser-198/199/202

(HG. 6F), and Ser-214 (FIG. 6G) or in total tau levels (HG. 6H). Likewise, 200 μM PD 98059 and 100 μM SB 203580, which decreased MAPK activity to approximately 54% and approximately 43% of the controls (FIGS. 7E and 7F), respectively, did not significantly affect PKA (HGS. 7A and 7B) or GSK-3 (FIGS. 7C and 7D) activity. Neither of these two MAPK inhibitors had any significant effect on the forskolin-induced hyperphosphorylation of tau at Ser-396/404 (FIGS. 7G and 7H), Ser- 198/199/202 (FIGS. 71 and 7J) and Ser-214 (FIGS. 7K and 7L). These results further confirmed that cdc2, cdk-5 and MAPK were not responsible for the increase in phosphorylation of tau at Ser-198/199/202, Ser-396/404 and Ser-214 when PKA was activated by forskolin.

Example 5. Induction of an Impairment of Spatial Memory by Forskolin and Inhibition of this Effect by Rp-cAMPS and by LiCl The impairment of spatial memory is a typical symptom of AD in early stages. Therefore, the influence of forskolin on spatial memory in rats was examined using a

Morris water maze. Sprague-Dawley rats were allowed free access to food and water, and maintained at constant temperature (25°C). Spatial memory was measured using a Morris water maze, according to the classical Morris protocol, which consists of two stages: acquiring spatial memory and assaying spatial memory retention (Morris, J. Neurosci. Methods. 11: 47-60, 1984). For spatial learning, rats were trained by 20 successive trials (4 trials/day) in a water pool to find a platform hidden in milky water, using spatial clues outside the water pool. Through these training sessions, rats acquired spatial memory about location of the platform. These rats were then injected with test drug(s) or vehicle (aCSF) only. Twenty-four hours after injection, the animals were retested for spatial memory retention in the Morris water maze. The platform was removed from the water pool and rats were allowed to freely swim in the water pool for 60 seconds to assay the influence of various drug treatments on spatial memory. The time rats stayed in the previous platform quadrant (quadrant time) and the pathway of rats swimming were recorded by a video camera fixed to the ceiling of the room, 1.5 meters from the water surface. The camera was connected to a digital-tracking device attached to an IBM computer equipped with software to track the movements of the rats. The longer a rat stayed in the previous platform-located quadrant, the better it scored spatial memory. Spatial memory of rats is expressed as quadrant time (%). Data were analyzed using SPSS 10.0 statistical software. The One- Way ANOVA procedure followed by LSD's post hoc tests was used to determine the statistical significance of differences of the means. To analyze the correlations among variables, Pearson correlations were computed with bivariate correlations procedure. The control rats treated with aCSF alone spent approximately 55% of total swimming time (quadrant time %) in the previous platform-located quadrant. In contrast, treatment with 20μM, 40 μM or 80 μM forskolin significantly decreased this quadrant time in a dose dependent manner (HG. 8A). The forskolin-induced decrease in the quadrant time was abolished by 100 μM Rp-cAMPS (HG. 8B), a concentration that also essentially completely inhibited hyperphosphorylation of tau at Ser-198/199/202, Ser- 396/404 and Ser-214. (see HGS. 4G, 4H, 41, 4J, 4K and 4L). On the other hand, 100 mM LiCl only partially abolished the decrease in quadrant time induced by 80 μM forskolin (FIG. 8C). Co-injection of 80 μM forskolin and 100 mM LiCl completely abolished hyperphosphorylation of tau at Ser-198/199/202 and Ser-396/404 but not at Ser-214 (see FIGS. 4A, 4B, 4C, 4D, 4E and 4F). These results revealed that hyperphosphorylation of tau both by GSK-3 and PKA impairs spatial memory and that the phosphorylation of tau by the two kinases has additive effect on the impairment of this type of memory. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying an agent that inhibits phosphorylation of a tau polypeptide in vivo, the method comprising:

5 a) providing a test compound identified as a protein kinase A (PKA) inhibitor or a glycogen synthase kinase-3 (GSK-3) inhibitor in vitro; b) administering the test compound to a subject; and c) determining whether phosphorylation of the tau polypeptide in the subject is decreased, relative to a control, following administration of the test o compound, wherein a decrease in phosphorylation of the tau polypeptide following administration of the test compound is an indication that the test compound is an agent that inhibits phosphorylation of the tau polypeptide in vivo.

2. The method of claim 1, further comprising administering a PKA agonist to the 5 subject prior to or simultaneously with step b).

3. The method of claim 2, wherein the agonist is injected into the subject's brain.

4. The method of claim 2, wherein the PKA agonist is forskolin.0

5. The method of claim 1, wherein the subject is a mammal.

6. The method of claim 5, wherein the subject is a rodent, rabbit, cat or dog. 5

7. The method of claim 6, wherein the subject is a rat.

8. The method of claim 5, wherein the subject is a human.

9. The method of claim 1, wherein the determining step comprises evaluating0 phosphorylation of the tau polypeptide at one or more serine residues selected from the serine residues corresponding to serine 198, serine 199, serine 202, serine 396, and serine 404 of SEQ ID NO: 1.

10. The method of claim 1, wherein the determining step is carried out on neuronal tissue of the subject.

11. The method of claim 10, wherein the determining step comprises preparing a Western blot.

12. The method of claim 1, wherein the test compound of step a) is identified as a

PKA inhibitor.

13. The method of claim 1, further comprising determining whether phosphorylation of the tau polypeptide in the subject is increased or decreased relative to an animal that has been administered a known PKA inhibitor that is other than the test compound.

14. The method of claim 13, wherein the known PKA inhibitor is Rp-Adenosine 3', 5-cyclic monophosphorothioate triethyl ammonium salt (Rp-cAMPs).

15. The method of claim 1, wherein the test compound of step a) is identified as a GSK-3 inhibitor.

16. The method of claim 1, further comprising, prior to step a), identifying the test compound as an inhibitor of PKA or GSK-3 by an in vitro method comprising: i) contacting an in vitro tau polypeptide with (1) PKA or GSK-3 or both PKA and GSK-3 and (2) the test compound; and ii) determining whether phosphorylation of the tau polypeptide is decreased in the presence of the test compound, relative to a control, wherein, if phosphorylation of the in vitro tau polypeptide is decreased in the presence of the test compound, the test compound is identified as being a PKA or GSK-3 inhibitor in vitro.

17. A method for identifying an agent that inhibits a tau-related decrease in memory skill, the method comprising: a) providing a test compound that inhibits PKA or GSK-3; b) providing an animal under conditions in which memory skill is impaired in the animal; c) administering the test compound to the animal; and d) evaluating the animal's memory skill, wherein a decrease in the impairment of memory skill in the presence of the test compound is an indication that the test compound is an agent that inhibits a tau-related decrease in memory skill.

18. The method of claim 17, wherein memory skill of the animal is impaired by administering to the animal an agent that impairs memory skill.

19. The method of claim 17, wherein the memory skill impairment is due to a genetic manipulation of the animal or an ancestor of the animal.

20. The method of claim 17, wherein the memory skill is spatial memory skill.

21. The method of claim 17, wherein the memory skill is impaired in step b) by administering an agonist of PKA.

22. The method of claim 20, wherein the animal has been trained to perform a spatial task, and the evaluating step comprises assessing the animal's performance of the task.

23. The method of claim 22, wherein the animal has been trained to identify a location in a maze, and the evaluating step comprises determining how long the animal spends in the location.

24. The method of claim 23, wherein the maze is a water maze.

25. The method of claim 17, wherein the animal is a rodent.

26. The method of claim 25, wherein the rodent is a rat.

27. The method of claim 17, wherein the test compound is selected prior to step a) by an in vitro method comprising: i) contacting an in vitro tau polypeptide with a PKA polypeptide and the test compound; ii) determining whether phosphorylation of the in vitro tau polypeptide is decreased in the presence of the test compound, relative to a control; and iii) if phosphorylation of the in vitro tau polypeptide is decreased in the presence of the test compound, selecting the test compound for use in step a).

28. The method of claim 27, wherein the method includes, prior to step ii), contacting the in vitro tau polypeptide with a GSK-3 polypeptide simultaneously with or subsequent to step i).

29. A method for treating a tauopathy in a subject, the method comprising administering to the subject an amount of a PKA inhibitor or a GSK-3 inhibitor effective to inhibit the tauopathy, thereby treating the tauopathy in the subject.

30. The method of claim 29, wherein the subject is a mammal.

31. The method of claim 30, wherein the subject is a rodent or a human.

32. The method of claim 29, wherein the tauopathy is Alzheimer's disease (AD), supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), argyrophilic grain disease, or frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).

33. A method for treating a tauopathy in a subject, the method comprising administering to the subject an amount of a PKA inhibitor and a GSK-3 inhibitor effective to inhibit the tauopathy, thereby treating the tauopathy in the subject.

34. The method of claim 33, wherein the subject is a mammal.

35. The method of claim 34, wherein the subject is a rodent or a human.

36. The method of claim 33, wherein the tauopathy is AD, PSP, CBD, PiD, argyrophilic grain disease, or FTDP-17.

37. A method for treating a tauopathy in a subject who suffers from the tauopathy or is at risk from the tauopathy, but who is not diagnosed with bipolar depression, a blood disorder, cluster headaches, premenstrual tension, bulimia, alcoholism, a syndrome of inappropriate secretion of ADH, tardive dyskinesia, hyperthyroidism, postpartum affective psychosis, or corticosteroid-induced psychosis, the method comprising administering to the subject an amount of lithium chloride (LiCl) effective to inhibit the tauopathy in the subject, thereby treating the tauopathy in the subject.

38. The method of claim 37, wherein the subject is a mammal.

39. The method of claim 38, wherein the subject is a rodent or a human.

40. The method of claim 37, wherein the tauopathy is AD, PSP, CBD, PiD, argyrophilic grain disease, or FTDP-17.

41. A method for reducing loss of memory skill in a subject, the method comprising: identifying a subject as being susceptible to a condition involving tau- related loss of memory skill, and administering to the subject an amount of a PKA inhibitor or GSK-3 inhibitor effective to reduce loss of memory skill in the subject.

42. The method of claim 41, wherein the subject's memory skill is evaluated prior to the administering step.

43. The method of claim 42, wherein the subject's memory skill is evaluated both prior to and subsequent to the administering step.

44. The method of claim 41, wherein the subject is administered the PKA inhibitor or GSK-3 inhibitor in at least four doses over at least four weeks.

45. The method of claim 41, wherein the subject is administered the PKA inhibitor or GSK-3 inhibitor in at least eight doses over at least eight weeks.

46. A kit for identifying an in vivo inhibitor of tau phosphorylation comprising: a reference compound that inhibits PKA or GSK-3 in vivo; and a reagent for detecting phosphorylation of a tau polypeptide.

47. The kit of claim 46, wherein the reagent is an antibody.

48. The kit of claim 46, wherein the kit further comprises instructions for comparing phosphorylation of the tau polypeptide in a first subject that has been administered a test compound to phosphorylation of the tau polypeptide in a second subject that has been administered the reference compound.

49. A kit for identifying an in vivo inhibitor of tau phophorylation comprising: a compound that agonizes PKA in vivo; a reagent for detecting phosphorylation of a tau polypeptide.

50. The kit of claim 49, wherein the reagent is an antibody.

51. The kit of claim 49, wherein the kit further comprises instructions for determining whether phosphorylation of the tau polypeptide is decreased in a subject that has been administered the compound that agonizes PKA, relative to a control.