P4-424 Identifying inhibitors of tau polymerization

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Poster Session P4: Therapeutics and Therapeutic Strategies - Therapeutic Strategies, NeurofibrilIary Pathology-based

Poster Session P4: Therapeutics and Therapeutic Strategies - Therapeutic Strategies, Neurofibrillary Pathology-based

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INHIBITORY ACTION O F ( - ) C L A U S E N A M I D E ON TAU P R O T E I N H Y P E R P H O S P H O R Y L A T I O N IN E X P E R I M E N T A L DIABETIC M I C E

Cheng Yong*, Jun-tian Zbang. Institute of Material Medica, Peking Union Medical College, Beijing, China. Contact e-mail: [email protected]

Background: Neurofibrillary tangles (NFTs) are histopathological lesions in Alzheimer's disease. The core component of paired helical filaments, which make up NFTs, is result from abnormal hyperphosphorylation of microtubule-associated protein tau. The extent of tan phosphorylation depends on the balance of phosphorylated enzymes and dephosphorylated enzymes. PP-land PP-2A are dephosphorylated enzymes, and CDK-5, GSK-3 are important enzymes for hyperphosphorylation of tau protein. The diabetes mouse induced by streptozotocin (STZ, 200 mg/kg body weight, i.p.), the insulin-secreting [3-cell killer, appeared tau protein hyperphosphorylation at Ser199/Thr202 sits and the microtubules prominent sighs of fragmentation and dissolution in hippocampal neurons. (-)Clausenamide, a new chiral compound isolated from traditional chinese herb - Clausena lausium (lour) Skeels, which showed neuroprotective effects in central nervous system. Objectlve(s): To detect the effects of (-)clausenamide on inhibiting tau protein hyperphosphorylation in these experimental diabetic mice. Methods: (-)Clausenamide was oral administration at doses of 7.5,15,30mg/kg. The behavior assays were performed 7 weeks after STZ treated, then mice were fixed, and microtubules in neurons of CA1 of hippocampus were detected by electron microscope and immunohistochemistry was performed with AT-8, tau-1, GSK-3, CDK-5 and PP-1 and tubulin antibodies. Results: (-)Clausenamide could improve the ability of learning and memory in diabetes mice and inhibit hyperphosphorylation of tan protein which leads to stabilization of microtubules through increasing PP-1 and decreasing the GSK-3 and CDK-Ys expression. In addition, (-)clausenamide increased protein kinase C (PKC) activity with or without diacylglycerol. Condusions: (-)Clausenamide could inhibit hyperphosphorylation of tau protein, which leads to stabilization of microtubules. PKC plays an important role in inhibiting tan hyperphosphorylation through inhibiting GSK-3 and elevating PP-1 expression. These may indicate the mechanism underlying the inhibitory effect of (-)clausenamide on tan protein hyperphosphorylation through increasing PKC activity.

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MECHANISM OF NEUROFIBRILLARY D E G E N E R A T I O N IN A M O U S E M O D E L OF TAUOPATHY AND P R O G R E S S TOWARDS IDENTIFICATION O F A T H E R A P E U T I C TARGET

Mike Hutton*, Heather Melrose, Cindy Zehr, Eilecn McGowan, Dennis W. Dickson, Shu-Hui Yen, Katherine Kehoe, Julie Taylor, Jason Eriksen, Leonardo Petrucelli, Jada Lewis. Mayo Clinic College of Medicine,

for neurofilament-H, luxol fast blue and with electron microscopy. Elevation of Hsp70 in JNPL3 mice and primary cell cultures was achieved by treatment with the antibiotic Geldanamycin. Results: Analysis of the pathology in JNPL3 mice by unbiased stereology showed significant neuron and volume loss in several regions (including spinal cord, pontine grey and trigeminal motor nucleus). Neuronal number was inversely correlated with the number of neurons containing neurofibrillary lesions. Tan accumulation and aggregation initially occurred in axons and was accompanied by axonal degeneration, in spinal cord, that preceded secondary myelin changes. Induction of Hsp70 in neuronal cell cultures and in initial studies in JNPL3 mice resulted in reduced total and pathological tan levels. Conclusions: These studies demonstrate that the development of neurofibrillary pathology in JNPL3 mice is accompanied by progressive neuronal degeneration. Early accumulation of aggregated tau in axons and associated axonal destruction suggests that one mechanism of neuronal loss, in this model, involves a "dying back" process. Investigation of multiple therapeutic targets to halt this neurodegenerative process is ongoing with induction of Hsp70 showing evidence of pathology clearance in initial studies suggesting that prevention and even reversal of neurofibrillary lesion formation maybe feasible in JNPL3 mice.

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P H A R M A C O L O G I C A L MODULATION O F TAU FIBRILLIZATION IN VITRO

Mihaela Necula*, Carmen N. Chirita, Jeff Kuret. Ohio St. University,

Columbus, OH, USA. Contact e-mail: [email protected]

Background: In vitro, tau fibrillizes through a kinetic pathway that includes the presence of intermediates with significant folded structure, suggesting that it may be feasible to modulate fibrillization with small molecule inhibitors. Objective(s): To test this hypothesis, a library of random small molecules was screened for the ability to block the formation or detection of thioflavin S-positive structures from full-length, unphosphorylated recombinant tau protein incubated in the presence of fibrillization inducers such as arachidonic acid. Methods: Active compounds identified by the screen were characterized biochemically using quantitative electron microscopy, static light scattering, and fluorescence spectroscopic assays. Results: Three distinct classes of antagonist were identified. Class I consisted of agents that competed with thioflavin S for binding to tau structures, but did not inhibit fibrillization. Class II agents competed with thioflvain S binding but also inhibited fibrillization as measured by both total filament length and number. These agents were also able to destabilize mature synthetic filaments through an endwise depolymerization mechanism. Class IH agents competed with thioflavin S binding and inhibited tau fibrillization as measured by total filament number but not by total filament length. Conclusions: These data suggest that pharmacological modulation of the tau fibrillization pathway is possible, and that small molecule antagonists may facilitate further dissection of the tan fibrillization pathway in vitro and in biological model systems.

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IDENTIFYING INHIBITORS OF TAU POLYMERIZATION

Jacksonville, FL, USA. Contact e-mail: [email protected]

David M. Wilson*, Jake Ni, Li-An Yeh, Ross Stein. Brigham and Women's

Background: Neurofibrillary lesions composed of MAPt (tau) protein are a

Hospital, Boston, MA, USA. Contact e-mail: dwilson @caregroup.harvard.edu

defining pathological feature of several neurodegenerative diseases, termed tauopathies, including Alzheimer's disease. The importance of tau in the pathogenic cascade in these diseases was confirmed by the identification of mutations in the tau gene, in families with autosomal dominant forms of tauopathy, which demonstrated that tau dysfunction is sufficient to cause neurodegeneration. The recent generation of transgenic models that recapitulate many neuropathological features of the human tauopathies now allows us to examine the mechanism(s) of tau-associated neurofibrillary degeneration and to identify therapeutic targets for treatment of these conditions. Objective(s): To determine the mechanism(s) of neuronal loss in a mouse model of tauopathy (JNPL3) expressing mutant (P301L) tau and m identify a therapeutic target that will prevent this process. Methods: Neuronal loss and the progression of tan pathology in JNPL3 mice were quantified by unbiased stereology. Axonal and myelin degeneration was demonstrated by staining

Background: Given a proposed causative role for tau polymerization in the etiology of Alzheimer's disease and other tauopathies, we have designed and implemented a high-throughput assay for identifying appropriate inhibitors. Objective(s): Our goals in designing the assay were to, 1) target an early step in assembly thereby avoiding problems associated with inter-filament aggregation, 2) conduct the assay under relatively physiologic conditions and in a timeframe suitable for HTS, and 3) conduct the assay in the absence of cofactors (i.e., polyanions or fatty acids) that could contribute to false positives and negatives. Methods: All experiments employed human 4R tau with a C322A mutation. Protein from E.Coli was purified by standard methods. Results: We have identified relatively physiologic buffer conditions that promote rates of filament formation comparable to the fastest rates achieved with fatty acid cofactors. These "permissive"

Poster Session P4: Therapeutics and Therapeutic Strategies- Therapeutic Strategies, Neurofibrillary Pathology-based buffers also promote non-covalent dimerization and subsequent disulfide formation, with the former proceeding 106 times faster than the latter. Since folding events associated with dimerization were expected to bring disulfideforming cysteine residues into close apposition, these residues were labeled with pyrene-maleimide to generate a fluorescent readout of dimerization. Closely apposed pyrene molecules produce excimer fluorescence at a longer wavelength (470 nm) than the monomer emission (375 nm). Various experiments have validated the utility of these conjugates. Notably, excimer fluorescence increases in permissive buffers, and is a function of fatty acid concentration in non-permissive buffers. Excimer fluorescence is also closely paralleled by the thioflavin fluorescence associated with tan folding. [Thioflavin was not deemed a suitable reporter, however, as compounds could potentially block dimerization without affecting folding (false negatives) or simply displace thioflavin without affecting dimerization (false positives).] An assay based on inhibition of excimer fluorescence was successfully adapted to a 384 well format. The assay is robust, with typical z' factors of 0.5-0.8 using tan at a concentration of 1 IxM. To date we have screened 30,000 compounds and several classes of inhibitors have been identified. Inhibition of polymerization has been confirmed by centrifugal separations and electron microscopy. Conclusions: Standard HTS methodologies can be used to identify inhibitors of tan polymerization.

~ H I G H THROUGHPUT SCREEN ASSAYS OF INHIBITION OF PP-2A CATALYZED DEPHOSPHORYLATION OF ABNORMALLY HYPERPHOSPHORYLATED TAU BY I1PP2AAND I2 PP2A

Sabiha Khatoon*, Inge Grundke-Iqbal, Khalid Iqbai. NYS Institute for Basic Research, Staten Island, Ny, USA. Contact e-mail: iqbalk@ worldnet.att.net

Background: The microtubule associated protein tau is abnormally hyperphosphorylated and in this state is polymerized into filaments in Alzheimer disease (AD) and other tanopathies. The activity of protein pbosphatase-2A (PP-2A), a major regulator of the phosphorylation of tau, is compromised in AD brain and is believed to be a cause of the abnormal hyperpbosphorylation of tan. The activity of PP-2A is regulated by two small endogenous inhibitor proteins, I1 vP2A and I2PP2A. Objective(s): To develop a mlcrotiter plate-based high throughput screening assay in which potential drugs that restore the PP-2A activity by inhibiting the activities of Il PP2A and or I2PP2A can be screened. Methods: We have combined in a microtiter plate-based assay, biochemical dephosphorylation of abnormally hyperphosphorylated tau/tau peptide by PP-2A and its inhibition by Ii vP2A or 12vv2A, with ELISA that measures the level of dephosphorylated polypeptide by monoclonal antibody Tan-1. This monoclonal antibody recognizes tan unphosphorylated at Ser-195/198/199/202. Results: PP-2A dephosphorylated tan at Tau-1 site which could be assayed by the high throughput screening assay. Both Ii PP2A and I2 PP2A inhibited the PP-2A catalyzed dephosphorylation of the abnormally hyperpbosphorylated tau at the Tan-1 site. Conclusions: A ]high throughput screening assay has been developed by which drugs that disinhibit PP-2A activity through the inhibition of I1PP2A and or I2PP2A call be generated.(Supported in part by the New York State Office of Mental Retardation and Developmental Disabilities, NIH grant AG19158 and a grant from the Institute for the Study of Aging, New York, NY.)

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EFFECT OF CHRONIC LITHIUM TREATMENT ON AN ANIMAL M O D E L O F TAUOPATHIES

Hanae Nakashima* 1, Takeshi Ishihara 1, Seishi Terada 1, Osamu Yokota 1, Aki Kugo 1, Takashi Hamamura 1, John Q. Trojanowski 2, Virginia M. Lee 2, Shigetoshi Kuroda I . 10kayarna Univ Grad Seh Med & Dent,

Okayarna, Japan; 2Center for Neurodegenerative Disease Research, University of Pennsylvania, Philadelphia, PA, USA. Contact e-rnail: [email protected] Background: Previous studies have shown that transgenic (Tg) mice overexpressing human tau protein develop filamentous tan aggregates in

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the CNS. We overexpressed the smallest human tan isoform (T44, also known as fetal tan) in the CNS of Tg mice to model tauopathies. These tau (T44) Tg mice acquire age-dependent CNS pathologies including insoluble, hyperphosphorylated tan and argyrophilic intraneuronal inclusions formed by tau-immunoreactive filaments. Therefore, these Tg mice are model that can be exploited for drug discovery in studies that target amelioration of tan-induced neurodegeneration as well as for elucidating mechanisms of filamentous tau pathology in various neurodegenerative tauopathies. Lithium is the major drug for bipolar disorder, and is also a direct inhibitor of GSK-3beta, a major tan kinase, so it is expected to have a therapeutic effect for tauopathies by suppressing hyperphosphorylation of tau. Objective(s): To elucidate the effect of lithium, as an inhibitor of GSK3beta, on an animal model of tauopathies. Methods: T44 Tg mice were fed with long-term lithium supplementation. The effect of lithium on T44 Tg mice was evaluated pathologically, biochemically and behaviorally using immunohistochemistry, Western blot and tail suspension test, respectively. Results: The long-term administration of lithium has a significant effect on the consequences of tau overexpreesion. For example, the number of tau-positive spheroids in the spinal cord of lithium-treated T44 Tg mice increased at 6 months of age compared with non-treated Tg mice, but the number decreased dramatically at 9 months. Biochemically, lithium treatment reduced accumulation of insoluble tau protein in the CNS of Tg mice at 6 months in spite of the fact that tan-positive spheroids in the spinal cord increased at the same age. Conclusions: The long-term administration of lithium, by inhibiting GSK-3beta activity, has a protective effect on tan-induced pathologies possibly via attenuation of tan-induced toxicity on neuronal cells thereby suggesting that lithium may be neuroprotective in various neurodegenerative tanopathies.

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E T H A N O L E X T R A C T OF ALPINIA OXYPHYLLA FRUCTUS SHOWS INHIBITION OF TAU PROTEIN PHOSPHORYLATION IN C E L L C U L T U R E

K.K. Wong*, C.C. Wan, P.C. Shaw. Department of Biochemistry, The

Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region of China. Contact e-mail: kelvinrnike@hotmail, corn Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to dementia in the ageing population. One of the pathological markers of AD is the accumulation of neurofibrillary tangels (NFT). NFT composes of damaged microtubule and paired helical filaments (PHF), which mainly consist of hyperphosphorylated tau protein (this kind of hyperphosphorylated tan is called PHF-tau). In AD, hyperpbosphorylation of tau into PHF-tau would cause it to lose its normal physiological function. PHF-tau is disintegrated from microtubules and polymerized with straight filament to form PHF. Hyperphosphorylation of tau has been proven to be an early event in the NFT pathology. Therefore, blockade of this hyperphosphorylation step may be a prime target to interrupt the pathogenic cascade. One of the main tau kinases responsible for the hyperphosphorylation of tan has been identified as glycogen synthase kinase 313 (GSK-3[3). We have established a cell model overexpresses recombinant GSK-3~, mimicking the hyperphosphorlation of Tan by GSK-3[3 in-vitro. This cell model has been established using COS-7 cells transiently co-transfected with the smallest tan isoform and GSK-3[3 cDNAs. The expressed Tan protein is hyperphosphorylated by the GSK-3[3 which could be monitored by Western blot analysis. Through this screening assay, ethanol extract of Alpinia oxyphylla Fructus shows strong GSK-3[~-dependant inhibition of tau phosphorylation at sites T231, T202 and $396. It inhibition also shown in differentiated and non-differentiated neuroblastoma SHSY5Y cell line.