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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Neurobiol Aging. Author manuscript; available in PMC Jan 1, 2010.
Published in final edited form as:
PMCID: PMC2611957
Wnt-pathway activation during the early stage of neurodegeneration in FTDP-17 mice
Martina Wiedau-Pazos,* Eugene Wong, Esther Solomon, Maricela Alarcon, and Daniel H. Geschwind*
Department of Neurology, David Geffen School of Medicine at UCLA, 635 Charles E. Young Drive South, Los Angeles, CA 90095, USA
* Corresponding authors: MWP: mwiedau/at/; phone 310-206-9933; fax 310-206-8082
Glycogen synthase kinase-3beta (GSK-3β), a key component of the Wnt signaling pathway, has been recognized as an important tau kinase with a potential pathogenic role in dementia. We have previously shown that GSK-3β induced tau-hyperphosphorylation and Wnt-activation enhance tau-induced degeneration in drosophila. Here, we demonstrate that Wnt-activation occurs prior to three months of age in the JNPL3 mouse model of frontotemporal dementia (FTD). We observed that GSK-3β becomes associated with insoluble tau, concomitant with the increase in the downstream Wnt-pathway component β-catenin. We demonstrate that this induces downstream Wnt signaling via the activation of nuclear transcription factors associated with β-catenin, suggesting that Wnt pathway activation is an early feature of the neurodegenerative process.
Keywords: dementia, transgenic animal model, Wnt-pathway
Tau pathology is considered one of the hallmarks of a group of disorders that includes Alzheimer’s disease (AD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, Pick’s disease and corticobasal degeneration, leading to the term, tauopathies, to describe these disorders [16].
Glycogen synthase kinase-3beta (GSK-3β) is a kinase that has been implicated in insulin signaling. Its role in phosphorylation of tau linked to forms of degenerative dementia is well described [4,10,14,18,25]. GSK-3β co-localizes with PHF in AD and enhances neurodegeneration via tau hyperphosphorylation and PHF formation in fly models of human tau expression [12,28]. When GSK-3β is inhibited in vivo, a reduction of tau-phosphorylation and neurodegeneration is observed demonstrating a key role for GSK-3β in tau-induced neurodegeneration [2,13,15,23,23].
In addition to its role in insulin signaling and tau-phosphorylation, GSK-3β holds a pivotal position in the canonical Wnt-pathway, where it phosphorylates β-catenin in concert with APC, Frizzled, and Axin, causing β-catenin degradation via the ubiquitin-proteasome pathway [1,3,21,29]. When the Wnt signal is activated, and GSK-3β is down regulated, un-phosphorylated/activated β-catenin accumulates and translocates to the nucleus where it interacts with cofactors to regulate the transcription of target genes that are involved in the cell cycle and cell survival [5]. Our previous data in Drosophila suggested that the role of GSK-3β in the effects of Wnt pathway signaling via β-catenin and the role of GSK3-β in the phosphorylation of tau were independent of each other [12].
Here, we extend the observations in a fly model to a mammalian system and test the hypothesis that the association of GSK-3β with mutant tau may interfere with the role of GSK-3β in the canonical Wnt-pathway. We confirm the observation that GSK-3β becomes insoluble early in the disease process, likely via its association with mutant insoluble tau. To further assess the consequence of this on Wnt signaling in vivo, we bred a P301L tau reporter mouse by crossing JNPL3 with the Fos-lacZ34Efu/J strain, which expresses lacZ in proportion to the amount of activated β-catenin. We show an increase of cytosolic and nuclear β-catenin early in the disease process, prior to the onset of significant tau-hyperphosphorylation and signs of neuronal degeneration, suggesting that Wnt pathway activation may be an initial step in the neurodegenerative process.
3.1. Transgenic animals
3.1.1. JNPL3
Hybrid (C57BL/DBA2/SW) JNPL3 transgenic mice (Taconic), expressing the longest four-repeat isoform of the human tau protein with the P301L missense mutation, were initially obtained from Michael Hutton’s laboratory [17]. Transgenic mice and non-transgenic littermates were bred by mating hemizygous JNPL3 mice with the B6 strain (Taconic). Animals were maintained on the B6 background. The tau transgene was genotyped by PCR between exons 1 and 5 of human tau cDNA according to published protocols [17].
3.1.2. LacZ mice
To generate a double transgenic mouse that allows the detection of activated β-catenin and Tcf/Lef expression, hemizygous JNPL3 mice were crossed with Tg(Fos-lacZ)34Efu (The Jackson Laboratory) [7] and bred onto a B6 background to create the JNPL3-lacZ strain. Offspring were genotyped after weaning, amplifying a 315 bp sequence of the transgene against a Tcrd control.
All procedures involving the animals and their care were approved by the UCLA Animal Research Committee.
3.2. Central nervous system (CNS) protein extracts
Brains and spinal cords were rapidly harvested after cardiac perfusion of the sacrificed animal with 25 ml of ice-cold phosphate-buffered saline (PBS). Whole spinal cords were collected, the brains were dissected into cortical, brainstem and cerebellar regions. The tissues were weighed, quickly frozen on dry ice and stored at −80°C. Each CNS piece was homogenized in ten volumes (v/w) of PBS containing protease and phosphatase inhibitors (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM sodium pyrophosphate, 30 mM β-glycerophosphate, 30 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM sodium orthovanadate, sonicated and spun 30 minutes at 13000 RPM at 4°C. For soluble protein extract electrophoresis, the supernatants were collected; for sarcosoyl-insoluble fractions, extracts were prepared as described [9,12].
3.2. Western blotting
All samples were separated on 10% sodium dodecyl sulfate–polyacrylamide (SDS PAGE) gels, electroblotted overnight onto PVDF membrane (Bio-Rad) and probed with primary and peroxidase labeled secondary antibodies (company). Blots were developed using enhanced chemiluminescent (PierceEndogen) and band intensities were quantified using a laser scanner densitometer (Molecular Dynamics).
3.3. Antibodies
Antibodies against tau protein included monoclonal T14 and T46 (Zymed), monoclonal AT100, AT8, AT270 against phosphorylated forms of tau (Zymed)–recognizing epitopes that are phosphorylated by GSK-3β, polyclonal GSK-3β and β-catenin (Chemicon), and polyclonal lacZ (Cappel). For loading controls, monoclonal anti-β-tubulin antibody TUB 2.1 (Sigma) was used.
3.4. Immunohistochemistry
Brains from 6 month-old JNPL3 transgenic mice or 3, 6 and 9 month-old JNPL3-lacZ mice and an equal number of control mice were used for immunohistochemical analysis. After euthanizing the animals, they were perfused transcardially with 4% paraformaldehyde in saline and sodium phosphate buffer (pH 7.4). Frozen specimen were cut into 50μm floating sections on a cryotome, and stained using the Vectastain kit (Vector) with the primary antibodies as described in the Western blotting section. Sections were counterstained using cresyl violet and coverslipped.
3.5. Data analysis
Densitometry results of three Western blots were analyzed by ANOVA using SAS statistics software. Post-hoc data analysis was performed using Tukey test [26].
4.1. Phosphorylation of tau and association of tau with GSK-3β in the CNS of JNPL3 mice
Cerebral cortex from six-month-old female JNPL3 mice was evaluated using immunoblotting with antibody against hyperphosphorylated tau (AT100). We confirmed, as previously reported [24], that tau protein extracted from JNPL3 cortex and spinal cord exhibits a shift toward increased amounts of hyperphosphorylated tau in the sarkosyl-insoluble protein fraction when compared to CNS extracts from controls (figure 1, panel A.). In contrast, extracts from the cerebellum, which does not degenerate, do not contain AT-100 positive PHF-tau, although similar amounts of total human tau (figure 1, panel H lanes 5–8) expression are observed in cerebellum when compared to the cortex, spinal cord and brainstem. Immunoblotting of the soluble and sarkosyl-insoluble protein fractions from cortex using antibody against GSK-3β reveals that GSK-3β is shifted toward the sarkosyl-insoluble fraction in cortex, concomitant with tau (figure 1, panel A), suggesting a possible association between hyperphosphorylated tau and GSK-3β in the cortex of JNPL3 mice.
Figure 1
Figure 1
GSK-3β co-localizes with hyperphosphorylated mutant tau in vulnerable CNS regions of the JNPL3 mouse
To further analyze the association of tau with GSK-3β, we performed double staining immunohistochemistry. Panels B-G of figure 1 show frontal cortex sections of the JNPL3 and non-transgenic animals labeled with antibody against PHF tau (AT100, red, panels B, E) and GSK-3β (green, panels C, F). Varying loads of PHF tau are observed in neurons in the P301L brain, ranging from small clusters to large protein aggregates filling out the entire cell in a tangle shape (panel B). The distribution of GSK-3β in neurons overlaps mainly with the small clusters of PHF tau (panel D, arrows), indicating that GSK-3β is primarily found overlapping with tau in a less soluble, pathological confirmation early in the process of forming hyperphosphorylated tau aggregates, months prior to overt neurodegeneration [17].
4.2. Total β-catenin levels are elevated in vulnerable CNS regions of the JNPL3 strain
We next tested the hypothesis that if enough GSK-3β were compartmentalized in the insoluble protein fraction, less would be available to phosphorylate cytosolic β-catenin, potentially resulting in an increase of total β-catenin. We first evaluated CNS β-catenin levels in JNPL3 and control mice. Figure 1 (panel H) reveals that β-catenin levels are higher in CNS areas of the JNPL3 mice that are most prone to degeneration. Cortex, spinal cord, and brain stem contain higher amounts of β-catenin than the cerebellum, a region that does not degenerate in this model. Since GSK phosphorylation of β-catenin and its subsequent degradation is a key means of suppressing canonical Wnt signaling, this increase in β-catenin supported the notion that Wnt signaling may be altered.
4.3. β-Catenin elevation results in increased tcf activity in vivo
β-catenin exists in several cellular pools, therefore it was possible that an increase in total cytosolic β-catenin may not result in increased Wnt signaling. To directly evaluate the Wnt pathway signaling activity of β-catenin, we assessed its transcriptional activating effect in an in vivo system. β-catenin transduces the Wnt signal via its interaction with Tcf/Lef transcription factors, therefore we used a well-characterized Tcf/Lef reporter mouse strain, Tg(Fos-lacZ)34Efu/J (lacZ) [6]. In this reporter strain, the β-galactosidase gene is activated in the presence of β-catenin/Tcf/Lef complex binding to its transcriptional activation site. We generated a JNPL3/tcf-lacZ reporter mouse by crossbreeding JNPL3 with the Tg(Fos-lacZ)34Efu/J strain (lacZ) [7] expressing β-galactosidase in the presence of the lymphoid enhancer binding factor 1/transcription factor 3 (LEF1/TCF3) mediated signaling pathway and activated β-catenin, thus acting as an in vivo readout of β-catenin/Tcf/Lef induced transcriptional activation.
Female JNPL3/tcf-lacZ mice, containing both the dominantly active tau transgene and the Tcf/Lef reporter, and Tcf-lacZ control littermates (without the tau transgene) were analyzed at ages 3, 6 and 9 months for PHF-tau and appearance of neurodegeneration, GSK-3β and β-catenin levels and concomitant β-galactosidase expression as an indicator of Tcf activation/signaling. First, in parallel to the findings in the JNPL3 strain, increasing levels of hyperphosphorylated tau are observed between 3 and 9 months in the JNPL3-lacZ mouse cortex (figure 2, panels A, B and C) and spinal cord (figure 3, panels A, B and C) in contrast to wild type controls (figures 2 and and3,3, panels J, K and L). The expression of GSK-3β remains elevated throughout the disease process (figures 2 and and3,3, panels D, E and F) when compared to the wild type controls (figures 2 and and3,3, panels M, N and O). At early disease stages, prior to a significant accumulation of PHF tau, β-catenin/Tcf/Lef levels are elevated in the JNPL3-lacZ cortex and spinal cord when compared to the wild type controls (figures 2 and and3,3, panels J, K and L). Wnt activation via β-catenin/Tcf/Lef diminishes with ongoing accumulation of PHF-tau during aging as demonstrated by β-galactosidase reporter expression (figures 2 and and3,3, panels G, H and I).
Figure 2
Figure 2
GSK-3β co-localizes with PHF-tau in cortical neurons
Figure 3
Figure 3
Total β-catenin expression is elevated in the vulnerable CNS regions of the JNPL3 mouse
Western blotting was used to verify these histochemical observations. Figure 4.a. confirms that β-galactosidase levels are significantly increased in soluble protein extracts from the frontal cortex and spinal cord of 3 month-old JNPL3 mice compared to the cerebellum. Figure 4.b. depicts the reduced expression of β-galactosidase in the cortex as the JNPL3-lacZ mouse ages to 9 months, whereas the β-catenin/Tcf/Lef as indicated by β-galactosidase levels remained stable in the wild type control.
Figure 4
Figure 4
Increased β-catenin/Tcf/Lef in cortex and spinal cord affected by degeneration in the presence of mutant P301L tau
In this study, we first confirmed previous reports that the expression of human P301L tau in the JNPL3 mouse CNS leads to the formation of PHF-tau aggregates and elevated levels of insoluble GSK-3β in a pattern that correlates with regional degeneration in this model. High levels of PHF-tau and GSK-3β are observed in spinal cord and cortex, whereas lower levels of GSK-3β and PHF-tau are observed in the cerebellum. In parallel with the increase of GSK-3β in affected CNS regions, we report the novel observation that total β-catenin levels are elevated in the same pattern in affected CNS regions early in the disease course, prior to overt pathology. In addition, we show that this elevation of β-catenin specifically reflects activated Wnt signaling, which induces Tcf transcriptional activity. This may seem paradoxical, since GSK-3β activity is expected to diminish β-catenin levels. In turn, a reduction of β-catenin would be expected to lead to reduced Wnt signaling/Tcf transcriptional activity. However, we also find that during this early phase of disease, GSK-3β becomes co-localized with pathologically phosphorylated tau in the insoluble protein fraction, which we hypothesized would lead to a reduced availability of GSK-3β in the cytosol, causing elevated β-catenin due to decreased phosphorylation of β-catenin by GSK-β. We observed this elevation of β-catenin at three months in an early stage of the disease prior to overt neurodegeneration.
These findings are in accordance with an earlier invertebrate study, in which we evaluated the function of β-catenin/Tcf/Lef in both over expression and loss-of-function studies in a Drosophila eye model of human tau over expression. When homologs of β-catenin and the nuclear co-transcription factor Tcf were increased in Drosophila, the neurodegeneration phenotype was enhanced [12]. In contrast to PHF formation, which was observed only in adult flies, the exacerbation of degeneration caused by GSK-3β occurred at all developmental stages, - larval, pupal and adult. Thus, the tangle formation took place later in the fly than the initiation of cell death. Feany and colleagues also observed cell death without NFT’s in drosophila overexpressing human tau wild type and several mutants [28].
Our previous fly data suggested that increased Wnt signaling via β-catenin could be an early feature in tau related neurodegeneration. Here we tested this hypothesis in a mouse model of FTD. The present study is the first in vivo report providing evidence in mammals, that total and activated β-catenin, and canonical Wnt signaling via β-catenin/Tcf/Lef complex are elevated many months prior to overt pathology with neuronal loss. Since we observed that β-catenin is elevated only early in the disease process in the JNPL3 mouse, our results suggest that β-catenin may play a role in the initial phase of degenerative changes.
These findings are particularly interesting in light of previous reports in mice with conditional over expression of GSK-3β. Aspects of tau-induced neuropathology, such as tau hyperphosphorylation, reactive astrocytosis, and neuronal death [27], and spatial learning deficit [11] were reported when GSK-3β was over expressed. When GSK-3β over expression was inhibited, normal GSK-3β activity, reduced levels of phosphorylated tau, and diminished neuronal death were shown [8], supporting a more complex role of GSK-3β in neurodegeneration than solely its role in tau-phosphorylation.
Thus, complementary to observations in the fly [12], the present evidence that β-catenin is increased early in the disease course before NFT formation peaks in the mouse supports the hypothesis that cellular processes leading to neuronal degeneration may not be initiated by tau hyperphosphorylation itself. We hypothesize that tau hyperphophorylation is a product of increased interaction between GSK-3β and tau, whereas the effect of this interaction on the Wnt pathway may be a separate consequence of the tau/GSK-3β interaction, both, leading eventually to neurodegeneration. This hypothesis is supported by previous reports that the link of tau phosphorylation and GSK-3β activity to neurodegeneration is not consistent in studies of transgenic mice [19,20,27]. Therefore, based on the present data, we conclude that tau toxicity is not solely attributable to altered microtubule binding and its aggregation, but that tau can also indirectly affect cellular pathways causing neurodegeneration. The role of the Wnt pathway, and its connection to other processes that are implicated in neurodegeneration, such as cell cycle activation, and its interaction with other pro-degenerative factors, such as β-amyloid, as well as Wnt-pathway inhibitors such as proteins of the Dickkopf family that have been implicated in Alzheimer’s disease [6], are deserving of further investigation.
Figure 5
Figure 5
β-catenin/Tcf/Lef expression is elevated in the vulnerable CNS regions of the JNPL3/tcf-lacZ mouse
Our thanks to Dr. Virginia Lee for fruitful discussions of this project. Funding provided by the John Douglas French Alzheimer’s Foundation (MWP, DHG), the UCLA Alzheimer’s disease research center (ADRC) (MWP, DHG) and NIH, K08 NS002240 (MWP).
6.1. Disclosure statement
(a) No conflicts of interest are present.
(b) The appropriate institutional approval for all animal experiments was obtained, UCLA Animal Research Committee approval number 2003-103.
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1. Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. Beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 1997;16(13):3797–804. [PubMed]
2. Alvarez G, Munoz-Montano JR, Satrustegui J, Avila J, Bogonez E, Diaz-Nido J. Regulation of tau phosphorylation and protection against beta-amyloid-induced neurodegeneration by lithium. Possible implications for Alzheimer’s disease. Bipolar Disord. 2002;4:153–165. [PubMed]
3. Behrens J, Jerchow BA, Wuertele M, Grimm J, Asbrand C, Wirtz R, Kuehl M, Wedlich D, Birchmeier W. Functional interaction of an axin homolog, conductin, with β-catenin, APC, and GSK-3β Science. 1998;280(5363):596–9. [PubMed]
4. Biernat J, Mandelkow EM, Schröter C, Lichtenberg-Kraag B, Steiner B, Berling B, Meyer H, Mercken M, Vandermeeren A, Goedert M. The switch of tau protein to an Alzheimer-like state includes the phosphorylation of two serine-proline motifs upstream of the microtubule binding region. EMBO J. 1992;11(4):1593–1597. [PubMed]
5. Clevers H, van de Wetering M. TCF/LEF factors earn their wings. Trends Genet. 1997;13:485–9. [PubMed]
6. Caricasole A, Copani A, Caraci F, Aronica E, Rozemuller AJ, Caruso A, Storto M, Gaviraghi G, Terstappen GC, Nicoletti F. Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is associated with neuronal degeneration in Alzheimer’s brain. J Neurosci. 2004;24:6021–6027. [PubMed]
7. DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development. 1999;126(20):4557–68. [PubMed]
8. Engel T, Hernandez F, Avila J, Lucas JJ. Full Reversal of Alzheimer’s Disease-Like Phenotype in a Mouse Model with Conditional Overexpression of Glycogen Synthase Kinase-3.2. Neurobiol Dis. 2002;26(19):5083–5090. [PubMed]
9. Goedert M, Spillantini MG, Cairns NJ, Crowther RA. Tau proteins of Alzheimer paired helocal filaments: Abnormal phosphorylation of all six brain isoforms. Neuron. 1997;8:159–168. [PubMed]
10. Gustke N, Steiner B, Mandelkow EM, Biernat J, Meyer HE, Goedert M, Mandelkow E. The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves Ser-Pro and Thr-Pro motifs. FEBS Lett. 1992;307:199–205. [PubMed]
11. Hernandez F, Borrell J, Guaza C, Avila J, Lucas J. Spatial learning deficit in transgenic mice that conditionally over-express GSK-3β in the brain but do not form tau-filaments. J Neurochem. 2002;83:1529–1533. [PubMed]
12. Jackson GR, Wiedau-Pazos M, Sang TK, Wagle N, Brown CA, Massachi S, Geschwind DH. Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila. Neuron. 2002 May 16;34(4):509–19. [PubMed]
13. Jin N, Kovacs AD, Sui Z, Dewhurst S, Maggirwar SB. Opposite effects of lithium and valproic acid on trophic factor deprivation-induced glycogen synthase kinase-3 activation, c-Jun expression and neuronal cell death. Neuropharmacology. 2005;48(4):576–83. [PubMed]
14. Jope RS, Johnson GV. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci. 2004;29:95–102. [PubMed]
15. Lee CW, Lau KF, Miller CC, Shaw PC. Glycogen synthase kinase-3 beta-mediated tau phosphorylation in cultured cell lines. Neuroreport. 2003 Feb 10;14(2):257–60. [PubMed]
16. Lee VM, Goedert M, Trojanowski JQ. Neurodegenerative tauopathies. Ann Rev Neurosci. 2001;24:1121–59. [PubMed]
17. Lewis J, McGowan E, Rockwood J, Melrose H, Nacharaiu P, Van Slegtenhorst M, Gwinn-Hardy K, Murphy MP, Baker M, Yu X, Duff K, Hardy J, Corral A, Lin WL, Yen SH, Dickson DW, Davies P, Hutton M. Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein. Nat Genet. 2000 Aug;25(4):402–5. [PubMed]
18. Lovestone S, Reynolds CH. The phosphorylation of tau: a critical stage in neurodevelopment and neurodegenerative processes. Neuroscience. 1997;78:309–324. [PubMed]
19. Lucas JJ, Hernandez F, Gomez-Ramos P, Moran MA, Hen R, Avila J. Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice. EMBO J. 2001;20:27–39. [PubMed]
20. Mattson MP. Neuronal death and GSK-3β: a tau-fetish? Trends Neurosci. 2001;24(5):255–6. [PubMed]
21. Moon RT, Bowerman B, Boutros M, Perrimon N. The promise and perils of Wnt signaling through beta-catenin. Science. 2002;296:1644–6. [PubMed]
22. Mudher A, Shepherd D, Newman TA, Mildren P, Jukes JP, Squire A, Mears A, Drummond JA, Berg S, MacKay D, Asuni AA, Bhat R, Lovestone S. GSK-3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila. Mol Psychiatry. 2004 May;9(5):522–30. [PubMed]
23. Noble W, Planel E, Zehr C, Olm V, Meyerson J, Suleman F, Gaynor K, Wang L, LaFrancois J, Feinstein B, Burns M, Krishnamurthy P, Wen Y, Bhat R, Lewis J, Dickson D, Duff K. Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo. Proc Natl Acad Sci U S A. 2005 May 1;102(19):6990–5. [PubMed]
24. Sahara N, Lewis J, De Ture M, McGowan E, Dickson DW, Hutton M, Yen SH. Assembly of tau in transgenic animals expressing P301L tau: alteration of phosphorylation and solubility. J Neurochem. 2002;83(6):1498–1508. [PubMed]
25. Selkoe DJ. The molecular pathology of Alzheimer’s disease. Neuron. 1991;6:487–98. [PubMed]
26. Sheskin DJ. The handbook of parametric and nonparametric statistical procedures. 3. CRC Press; Boca Raton: 2003. p. 719.
27. Spittaels K, Van Den Haut C, Van Dorpe, Geerts H, Mercken M, Bruynseels K, Lasrado R, Vandezande K, Laenen L, Boon T, Van Lint J, Vandenheede J, Moechars D, Loos R, Van Leuven F. Glycogen synthase kinase-3beta phosphorylates protein tau and rescues the axonopathy in the central nervous system of human four-repeat tau transgenic mice. J Biol Chem. 2000;275:41340–41349. [PubMed]
28. Wittmann CW, Wszolek MF, Shulman JM, Salvaterra PM, Lewis J, Hutton M, Feany MB. Tauopathy in Drosophila: Neurodegeneration Without Neurofibrillary Tangles. Science. 2001;293(5530):711–714. [PubMed]
29. Yost CM, Torres M, Miller JR, Huang E, Kimelman D, Moon RT. The axis-inducing activity, stability, and subcellular distribution of beta-catenin are regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev. 1996;10:1443–54. [PubMed]