Spatial learning deficit in transgenic mice that ... - Wiley Online Library

Journal of Neurochemistry, 2002, 83, 1529–1533

RAPID COMMUNICATION

Spatial learning deficit in transgenic mice that conditionally over-express GSK-3b in the brain but do not form tau filaments Fe´lix Herna´ndez,*,1 Jose´ Borrell, ,1 Carmen Guaza,  Jesu´s Avila* and Jose´ J. Lucas* *Centro de Biologı´a Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Cientı´ficas (CSIC)/Universidad Auto´noma de Madrid (UAM), Madrid, Spain  Instituto Cajal, CSIC Avda, Madrid, Spain

Abstract Deregulation of glycogen synthase kinase-3 (GSK-3) activity in neurones has been postulated as a key feature in Alzheimer’s disease (AD) pathogenesis. This was further supported by our recent characterization of transgenic mice that conditionally overexpress GSK-3b in hippocampal and cortical neurones. These mice, designated Tet/GSK-3b, showed many of the biochemical and cellular aspects of AD neuropathology such as tau hyperphosphorylation and somatodendritic localization, decreased nuclear b-catenin, neuronal death and reactive gliosis. Tet/GSK-3b mice, however, did not show tau filament formation up to the latest tested age of 3 months at least. Here we report spatial learning deficits of Tet/GSK-3b mice in the Morris water maze. In parallel, we also measured the increase in GSK-3 activity while further exploring the possibility of tau filament formation in aged mice. We

Deregulation of glycogen synthase kinase-3 (GSK-3) activity in neurones has been postulated as a key feature in Alzheimer’s disease (AD) pathogenesis. This is based on the interaction of GSK-3 (and more precisely its b isoform, GSK-3b) with many of the cellular components related to the neuropathology of AD, such as the amyloid precursor protein, the Ab peptide, the metabolic pathway leading to acetylcholine synthesis, the presenilins, which are mutated in many cases of familial AD, and tau protein, which is the primary constituent of neurofibrillary tangles (NFT) (see Grimes and Jope 2001 for a review). More precisely, GSK-3b has been proposed as the link between the two neuropathological hallmarks of AD, the extracellular Ab deposits and the intraneuronal fibrillary tangles made of hyperphosphorylated tau. Now it seems clear that tau changes in AD are secondary to Ab deposition (Gotz et al. 2001; Lewis et al. 2001), and that tau is essential for Ab-induced toxicity (Rapoport et al. 2002). However, the intracellular connection between Ab and tau changes in AD is still a matter for debate, and GSK-3b has emerged as one of the possible mediators. GSK3b has been shown to phosphorylate tau in most sites hyperphosphorylated in NFT both in transfected cells (Lovestone et al. 1994) and in vivo (Hong et al. 1997; Munoz-Montano et al. 1997). Increased levels of GSK-3b have been found in AD brains and GSK-3b accumulates in the cytoplasm of pre-tangle neurones and can be found associated to NFT (Yamaguchi et al. 1996; Imahori and Uchida 1997; Pei et al. 1999). On the other hand, exposure of cortical and hippocampal primary neuronal cultures to Ab has been shown to induce activation of GSK-3b (Takashima et al. 1996), and blockade of either GSK-3b expression or

found a significant increase in GSK-3 activity in the hippocampus of Tet/GSK-3b mice whereas no tau fibrils could be found even in very old mice. These data reinforce the hypothesis of GSK-3 deregulation in AD pathogenesis, and suggest that Tet/GSK-3b mice can be used as an AD model and, most remarkably, can be used to test the therapeutic potential of the selective GSK-3 inhibitors that are currently under development. Additionally, these experiments suggest that destabilization of microtubules and alteration of intracellular metabolic pathways contribute to AD pathogenesis independent of toxicity triggered by the aberrant tau deposits. Keywords: Alzheimer’s disease, GSK-3, spatial learning, tau, transgenic mice. J. Neurochem. (2002) 83, 1529–1533.

activity, either by antisense oligonucleotides or by lithium, prevents Abinduced neurodegeneration of cortical and hippocampal primary cultures (Takashima et al. 1993; Alvarez et al. 1999). This has led to the postulation of the therapeutic potential of selective GSK-3 inhibitors for the treatment of AD (Eldar-Finkelman 2002). We have recently generated transgenic mice that conditionally overexpress GSK-3b in hippocampal and cortical neurones (Lucas et al. 2001). These mice, designated Tet/GSK-3b, showed many of the biochemical and cellular aspects of AD neuropathology such as tau hyperphosphorylation and somatodendritic localization, decreased nuclear b-catenin, neuronal death and reactive gliosis; thus, further supporting a critical role of GSK-3 in AD pathogenesis. However, Tet/GSK-3b mice did not show tau filament formation despite the somatodendritic accumulation of hyperphosphorylated tau (at least up to the latest tested age of 3 months). Here we continue characterizing Tet/GSK-3b mice to explore whether, apart from mimicking neuropathological aspects of AD, an

Received September 16, 2002; revised manuscript received October 1, 2002; accepted October 19, 2002. Address correspondence and reprint requests to Jose´ J. Lucas, Centro de Biologı´a Molecular ‘Severo OchoaCSIC/UAM, Fac. Ciencias, Universidad Auto´noma de Madrid., Cantoblanco, 28049 Madrid, Spain. E-mails: [email protected] 1 These authors contributed equally to this work. Abbreviations used: AD, Alzheimer’s disease; GSK-3, glycogen synthase kinase-3; NFT, neurofibrillary tangles.


2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1529–1533

1529

1530 F. Herna´ndez et al.

increase in GSK-3 activity also results in learning deficits. We also measure the level of GSK-3 hyperactivity and analyse whether tau fibrils appear in old Tet/GSK-3b mice in order to test their relevance to 5 potential learning deficits.

Materials and methods Animals Tet/GSK-3b mice were generated as previously described (Lucas et al. 2001). Briefly, Tet/GSK-3b mice result from the breeding of TetO mice (carrying the bi-directional tet-responsive promoter followed by GSK-3b and b-galactosidase cDNAs, one in each direction) with CamKIIa-tTA mice. The double transgenic mice are designated Tet/GSK-3b and overexpress GSK-3b in cortical and hippocampal neurones in a conditional manner repressible by tetracycline administration. GSK-3 activity assay Brain tissue was homogenated in 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM NaF, 1 mM VO4Na, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, and a cocktail of peptidase inhibitors (Roche). Homogenates were centifugated at 14 000 g for 15 min and supernatants collected for GSK-3 activity assays. The GS1 peptide (YRRAAVPPSPSLSRHSSPHQS*EDEE) containing ser21 in phosphorylated form was used as substrate (Stambolic and Woodgett 1994). Supernatants were incubated at 37C with 30 lM of GS1 peptide in the presence of 50 lM [c-32P]ATP (NEN–Dupont) in 25 mM Tris pH 7.5, 1 mM DTT (Dithiothreitol) 10 mM MgCl2 and either 10 mM NaCl or 10 mM LiCl. The assays were stopped by spotting aliquots on P81 phosphocellulose paper. Filters were processed as described previously (Moreno et al. 1996). GSK-3 activity was calculated as the difference between the activity in the presence of 10 mM NaCl and the activity in the presence of 10 mM LiCl. Results were expressed as a percentage with respect to activity levels in wild-type extracts. The wild-type activities were 607 ± 77, 584 ± 27 and 1224 ± 122 pmol/min mg of protein for cortex, cerebellum and hippocampus, respectively. Behavioural testing Only male mice were used for this study and they were tested at the age of 4 months. The distribution of mice in the different genotypes was as follows: wild type (n ¼ 15), tTA (n ¼ 14), TetO (n ¼ 14) and Tet/GSK3b (n ¼ 27). Morris water maze Apparatus The water maze apparatus consisted of a circular pool (1.2 m diameter and 40 cm high) made of white plastic. It was located in the centre of a testing room (3 · 4 m). The pool was filled to the depth of 20 cm with water (24–25C), and divided into four quadrants of equal size. An invisible escape platform (10 cm diameter), made of transparent plastic was placed in the middle of one of the quadrants (0.5 cm below the water level). During a probe trial the platform was removed from the pool. In the cued learning test, the platform position was marked with a grey plastic cube (9 cm). The position of a mouse in the tank was recorded by a video camera suspended 2.5 m above the centre of the tank and connected to a video tracking system (Ethovision1.50; Noldus IT) and a PC running HVS software. Procedure Four different starting positions were equally spaced around the perimeter of the pool. On each day, all four start positions were used once in a random sequence. During the acquisition or training phase, each mouse was given 20 trials in blocks of four trials for five

consecutive days. During training, the time taken to locate the escape 7 platform (escape latency) was recorded. If the mouse failed to escape within 100 s it was placed onto the platform by the experimenter. After trial 20, each animal was given a 100-s probe trial when quadrant search times and platform crossings were measured. The day after the probe test, mice were trained to locate a visible-cued platform in a block of four trials. On each trial of the visible platform test, the platform was randomly located in one of the four quadrants. Spontaneous locomotor activity A digiscan Animal Activity Monitor System (activity cage), model RXYZCM TAO (Omnitech Electronics Inc.) was used to assess the activity of the animals. Mice were tested for a 10-min session and activity recorded at 5-min intervals. Data for the horizontal activity, vertical activity, number of stereotypic movements and time spent in the centre or margins of the cage were collected by an IBM-compatible computer system. Immunohistochemical detection of phosphorylated tau and isolation of sarkosyl-insoluble filaments Immunohistochemistry with PHF-1 antibody was performed as previously described (Lucas et al. 1999). Preparation of sarkosylinsoluble extracts from mouse brain and electron microscopy of filaments was performed as previously described (Perez et al. 1998). Briefly, each mouse brain was homogenized in 10 vol. of buffer (10 mM Tris-HCl pH 7.4, 0.8 M NaCl, 1 mM EGTA and 10% sucrose) 8 and centrifuged at 20 000 g for 20 min. Then, N-lauroyl sarcosine (Sigma) was added to the supernatant to a final concentration of 1%. After 1 h at 37C, the sample was centrifuged at 20 000 g for 1 h. The pellet was resuspended in 50 mM Tris-HCl pH 7.4. To test for the presence of isolated filaments, samples were placed on a carbon-coated grid for 2 min and then stained with 2% (wt/vol) uranyl acetate for 1 min. Transmission electron microscopy was performed in a JEOL Model 1200EX electron microscope operated at 100 kV. Electron micrographs were obtained at a magnification of 40 000 on Kodak SO-163 film.

Results In Tet/GSK-3b mice, transgene expression is driven to the forebrain by the CamK-IIa promoter and it is conditional in a tetracycline-regulated manner (Lucas et al. 2001). To achieve the conditional transgene expression, a double transgenic approach was used in which Tet/GSK-3b mice occur as a result of breeding tTA mice (CamK-IIa promoter driving expression of the tetracycline sensitive transcription factor tTA, also termed Tet-Off) with TetO mice (inducible TetO promoter driving expression of GSK-3b). The initial characterization of Tet/GSK-3b mice revealed increased GSK-3b levels in hippocampal and, to a lesser extent, cortical neurones. This resulted in changes in the GSK-3 substrates tau and b-catenin, thus indicating an increase in kinase activity in these neurones (Lucas et al. 2001). To measure the level of GSK-3 hyperactivity in the brain of Tet/GSK-3b mice, we performed GSK-3 kinase activity measurements in extracts from different brain regions (Fig. 1). Hippocampal extracts from Tet/GSK-3b mice showed a significant (p < 0.02) 24.5% increase in GSK-3 activity when compared with wildtype mice. However, despite the increase in GSK-3b levels in cortical extracts of Tet/GSK-3b mice, no concomitant increase in GSK-3 activity was found. This is probably a result of the limited number of cortical neurones that show transgenic expression of GSK-3b (see Lucas et al. 2001). In cerebellum, no differences were found, as expected, because the CamkIIa promoter that drives the expression of the transgene is not active in this brain region.


2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1529–1533

1

Spatial learning deficit in Tet GSK-3b mice

1531

Fig. 1 GSK-3 activity is increased in the hippocampus of Tet/GSK-3b mice. Histogram showing comparison of GSK-3 activity in extracts from different brain regions of Tet/GSK-3b and wild-type mice (*p < 0.02).

To address the question of whether the increase in GSK-3 activity results in altered learning capabilities, we performed the Morris water maze test with Tet/GSK-3b mice. To rule out unspecific effects of the transgenes caused by their sites of insertion, single transgenic mice tTA and TetO (that do not over-express GSK-3b) were tested in parallel with Tet/GSK-3b (that do over-express GSK-3b) and wild-type mice. In the hidden platform test (Fig. 2a), the three control groups (wild-type, tTA and TetO) behaved similarly and learnt to locate the platform during the 9 training sessions (F2,39 ¼ 0.8794, p ¼ not significant). Tet/GSK-3b mice, however, performed significantly worse both in terms of their 10 latency to find the platform (F3,65 ¼ 2.7828; p < 0.05) and the distance that they swam to reach the platform (F3,65 ¼ 5.6056; p < 0.005). The deficit found in the Tet/GSK-3b mice was even more significant when wild-type, tTA and TetO mice were grouped as a single control group (p < 0.001, both for latency and distance). No significant difference was found in the swimming speed for any of the genotypes (not shown). In the probe test, Tet/GSK-3b mice spent significantly less time in the target quadrant than did control mice (p < 0.05) (Fig. 2b). Similarly, the frequency of entries into the virtual platform area was also significantly lower in Tet/GSK-3b mice than in control mice (p < 0.05). At the end of the water maze test, mice were assayed in the activity cage for 10 min in two consecutive days. No differences were found in terms of total horizontal and vertical activities, time spent in movement, time spent in centre vs. periphery, or stereotypic movements for any of the four genotypes (data not shown). Thus further ruling out that a motor abnormality could account for the impaired performance of Tet/GSK-3b mice in the water maze test. To address whether tau filament formation was required for the impaired spatial learning observed in Tet/GSK-3b mice, we performed the biochemical purification procedure that is used to obtain tau filaments from AD brains. We ran in parallel samples from 5-month-old Tet/GSK3b mice (killed one week after the behavioural testing), 16-month-old Tet/GSK-3b mice, and 5-month-old TauVLW mice (transgenic mice that express human tau with a triple FTDP-17 mutation and that we have previously shown to form tau filaments; Lim et al. 2001 and Fig. 3d). Despite the somatodendritic accumulation of hyperphosphorylated tau evident in hippocampal neurones of 5-month-old Tet/GSK-3b mice (Fig. 3b), no filaments were purified from the brain of these mice (not shown), and not even from aged (16 month old) mice (Fig. 3c).

Discussion By behaviourally testing Tet/GSK-3b mice, we showed that an increase in GSK-3 activity in the hippocampus is enough to elicit a deficit in spatial learning in the Morris water maze. These results, together with the previous characterization of these mice, support the theory that a

Fig. 2 Morris water maze performance of Tet/GSK-3b transgenic mice. (a) Time spent and the distance swam to find the hidden platform over the five days of the training phase. Analysis by ANOVA revealed that Tet/GSK-3b mice performed significantly different with respect to the other three genotypes (+F1,41 ¼ 51.4070, p < 0.001 for latency; ++F1,41 ¼ 80.3571, p < 0.001 for distance). (b) Probe test after the acquisition phase: the time spent in the quadrant where the platform was formerly located and the number of crosses over the platform location were significantly lower (*p < 0.05) in Tet/GSK-3b mice vs. control mice (wild-type, tTA and TetO mice grouped together).

deregulation of GSK-3 activity may be key in the pathogenesis of AD. Furthermore, we find that tau filament formation is not required for the behavioural deficit, therefore suggesting that toxicity elicited by aberrant tau deposits may not be determinant in the cognitive symptomatolgy of AD. The spatial learning deficit exhibited by Tet/GSK-3b mice reinforces their validity as an AD animal model. As GSK-3 has been suggested as a 11 key link between Ab toxicity and tau pathology, Tet/GSK-3b mice are valuable in helping us to understand the intracellular determinants of neuronal dysfunction in AD. Furthermore, the fact that transgenic expression in Tet/GSK-3b mice is conditional, and that it can be shut down in adult mice once they have developed both the neuropathological changes and the behavioural deficit, makes these mice even more interesting. This will allow us to explore if both neuropathology and behaviour are susceptible to revert and to analyse if some aspects of the


2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1529–1533

1532 F. Herna´ndez et al.

(a)

(c)

(b)

Fig. 3 Absence of sarcosyl-insoluble filaments in the brain of Tet/GSK-3b mice despite somatodendritic accumulation of hyperphosphorylated tau. (a) and (b) Immunohistochemistry with PHF-1 antibody revealed somatodendritic accumulation of phosphorylated tau in hippocampal neurones of a 5-month-old Tet/GSK-3b mouse (b) but not in a wild-type mouse (a). (c) No sarcosyl-insoluble filaments were obtained from the brains of aged (16-month-old) Tet/GSK-3b mice. (d) Filaments (5-nm diameter) were obtained from the brains of 5-month-old TauVLW mice as previously reported (Lim et al. 2001).

(d)

12 neuropathology revert in parallel to the behaviour while others do not. This will help to clarify which aspects of the neuropathology are responsible for the behavioural deficit, in a similar way as we did previously in the conditional mouse model of Huntington’s disease (Yamamoto et al. 2000; Martin-Aparicio et al. 2001). The ability to silence transgene expression in adult symptomatic mice can also be very useful when testing the new and selective GSK-3 inhibitors that pharmaceutical companies are currently developing in view of the increasing evidence supporting the GSK-3 deregulation hypothesis of AD pathogenesis (Cross et al. 2001; Eldar-Finkelman 2002; Martinez et al. 2002a,b). In this regard, the efficacy of different concentrations of GSK-3 inhibitors in Tet/GSK-3b mice can be compared with the restoration of normal levels of GSK-3 achieved by giving tetracycline to the mice. Aberrant intraneuronal protein deposits is a common feature for many neurodegenerative diseases such as tauopathies, Huntington’s disease and other disorders caused by expanded poly glutamines, and Parkinson’s disease. This has led to the hypothesis that toxicity elicited by these protein aggregates may be a common pathogenic mechanism for all these diseases (Price et al. 1998; Tobin and Signer 2000). However, we were neither able to find tau filaments in Tet/GSK-3b mice, despite the somatodendritic accumulation of hyperphorylated tau, nor in the initial characterization of the mice at the age of three months (Lucas et al. 2001), nor in the behaviourally impaired mice at the age of 5 months as reported here and not even in very old mice (up to 16 months old). Thus ruling out that the behavioural deficit of Tet/GSK-3b mice could be a result of the toxicity triggered by the aberrant tau deposits, and therefore suggesting that destabilization of microtubules and alteration of intracellular metabolic pathways, rather than tau filament elicited toxicity, contribute to the aetiology of AD. In summary, the data presented here strongly support the conclusion that deregulation of GSK-3 activity is a key feature in AD pathogenesis and suggest that Tet/GSK-3b mice can be used as an AD animal model that will enable the exploration of the intracellular effectors of neuronal dysfunction, and to test whether the behavioural deficit and the

neuropathology are reversible, to dissect which aspects of the neuropathology account for the behavioural deficit, and to test the potential as AD therapeutic agents of new GSK-3 inhibitors.

Acknowledgements We thank Drs Mar Pe´rez and Filip Lim for helpful discussion. We are also grateful to Helena Garuti for her assistance in running the behavioural tests, and to Raquel Cuadros and Elena Langa for laboratory technical assistance. This work was supported by Neuropharma, and by grants from Fundacio´n La Caixa, Spanish CICYT, Comunidad de Madrid, Fundacio´n Lilly, and by an institutional grant from Fundacio´n Ramo´n Areces.

References Alvarez G., Munoz-Montano J. R., Satrustegui J., Avila J., Bogonez E. and DiazNido J. (1999) Lithium protects cultured neurons against beta-amyloidinduced neurodegeneration. FEBS Lett. 453, 260–264. Cross D. A., Culbert A. A., Chalmers K. A., Facci L., Skaper S. D. and Reith A. D. (2001) Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J. Neurochem. 77, 94–102. Eldar-Finkelman H. (2002) Glycogen synthase kinase 3: an emerging therapeutic target. Trends Mol. Med. 8, 126–132. Gotz J., Chen F., van Dorpe J. and Nitsch R. M. (2001) Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils. Science 293, 1491–1495. Grimes C. A. and Jope R. S. (2001) The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog. Neurobiol. 65, 391–426. Hong M., Chen D. C., Klein P. S. and Lee V. M. (1997) Lithium reduces tau phosphorylation by inhibition of glycogen synthase kinase-3. J. Biol. Chem. 272, 25326–25332. Imahori K. and Uchida T. (1997) Physiology and pathology of tau protein kinases in relation to Alzheimer’s disease. J. Biochem. (Tokyo) 121, 179–188. Lewis J., Dickson D. W., Lin W. L., Chisholm L., Corral A., Jones G., Yen S. H., Sahara N., Skipper L., Yager D., Eckman C., Hardy J., Hutton M. and McGowan E. (2001) Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP. Science 293, 1487–1491.


2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1529–1533

1

Spatial learning deficit in Tet GSK-3b mice

Lim F., Hernandez F., Lucas J. J., Gomez-Ramos P., Moran M. A. and Avila J. (2001) FTDP-17 mutations in tau transgenic mice provoke lysosomal abnormalities and Tau filaments in forebrain. Mol. Cell Neurosci. 18, 702–714. Lovestone S., Reynolds C. H., Latimer D., Davis D. R., Anderton B. H., Gallo J. M., Hanger D., Mulot S., Marquardt B., Stabel S., Woodgett J. R. and 13 Miller C. C. J. (1994) Alzheimer’s disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Curr. Biol. 4, 1077–1086. Lucas J. J., Hernandez F. and Avila J. (1999) Nuclear localization of beta-catenin in adult mouse thalamus correlates with low levels of GSK-3beta. Neuroreport 10, 2699–2703. Lucas J. J., Hernandez F., Gomez-Ramos P., Moran M. A., Hen R. and Avila J. (2001) Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice. EMBO J. 20, 27–39. Martin-Aparicio E., Yamamoto A., Hernandez F., Hen R., Avila J. and Lucas J. J. (2001) Proteasomal-dependent aggregate reversal and absence of cell death in a conditional mouse model of Huntington’s disease. J. Neurosci. 21, 8772–8781. Martinez A., Alonso M., Castro A., Perez C. and Moreno F. J. (2002a) First nonATP competitive glycogen synthase kinase 3 beta (GSK-3beta) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer’s disease. J. Med. Chem. 45, 1292–1299. Martinez A., Castro A., Dorronsoro I. and Alonso M. (2002b) Glycogen synthase kinase 3 (GSK-3) inhibitors as new promising drugs for diabetes, neurodegeneration, cancer, and inflammation. Med. Res. Rev. 22, 373–384. Moreno F. J., Munoz-Montano J. R. and Avila J. (1996) Glycogen synthase kinase 3 phosphorylation of different residues in the presence of different factors: analysis on tau protein. Mol. Cell. Biochem. 165, 47–54. Munoz-Montano J. R., Moreno F. J., Avila J. and Diaz-Nido J. (1997) Lithium inhibits Alzheimer’s disease-like tau protein phosphorylation in neurons. FEBS Lett. 411, 183–188. Pei J. J., Braak E., Braak H., Grundke-Iqbal I., Iqbal K., Winblad B. and Cowburn R. F. (1999) Distribution of active glycogen synthase kinase-3 beta

1533

(GSK-3beta) in brains staged for Alzheimer disease neurofibrillary changes. J. Neuropathol. Exp. Neurol. 58, 1010–1019. Perez M., Valpuesta J. M., de Garcini E. M., Quintana C., Arrasate M., Lopez Carrascosa J. L., Rabano A., Garcia de Yebenes J. and Avila J. (1998) Ferritin is associated with the aberrant tau filaments present in progressive supranuclear palsy. Am. J. Pathol 152, 1531–1539. Price D. L., Sisodia S. S. and Borchelt D. R. (1998) Genetic neurodegenerative diseases: the human illness and transgenic models. Science 282, 1079–1083. Rapoport M., Dawson H. N., Binder L. I., Vitek M. P. and Ferreira A. (2002) Tau is essential to beta-amyloid-induced neurotoxicity. Proc. Natl Acad. Sci. USA 99, 6364–6369. Stambolic V. and Woodgett J. R. (1994) Mitogen inactivation of glycogen synthase kinase-3 beta in intact cells via serine 9 phosphorylation. Biochem. J. 303, 701–704. Takashima A., Noguchi K., Sato K., Hoshino T. and Imahori K. (1993) Tau protein kinase I is essential for amyloid beta-protein-induced neurotoxicity. Proc. Natl Acad. Sci. USA 90, 7789–7793. Takashima A., Noguchi K., Michel G., Mercken M., Hoshi M., Ishiguro K. and Imahori K. (1996) Exposure of rat hippocampal neurons to amyloid beta peptide (25–35) induces the inactivation of phosphatidyl inositol-3 kinase and the activation of tau protein kinase I/glycogen synthase kinase-3 beta. Neurosci. Lett. 203, 33–36. Tobin A. J. and Signer E. R. (2000) Huntington’s disease: the challenge for cell biologists. Trends Cell Biol. 10, 531–536. Yamaguchi H., Ishiguro K., Uchida T., Takashima A., Lemere C. A. and Imahori K. (1996) Preferential labeling of Alzheimer neurofibrillary tangles with antisera for tau protein kinase (TPK) I/glycogen synthase kinase-3 beta and cyclin-dependent kinase 5, a component of TPK II. Acta Neuropathol. (Berl) 92, 232–241. Yamamoto A., Lucas J. J. and Hen R. (2000) Reversal of neuropathology and motor dysfunction in a conditional model of Huntington’s disease. Cell 101, 57–66.


2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1529–1533