- Email: [email protected]
Using okadaic acid as a tool for the in vivo induction of hyperphosphorylated tau
Pergamon
Neuroscience Vol. 85, No. 4, pp. 1329 1332. 1998 Copyright ;C 1998 IBRO. Publishedby ElsevierScienceLtd Printed in Great Britain.All rights reserved PII: S0306-4522(97)00695-7 0306-4522/98 $19.00+0.00
MATTERS ARISING USING OKADAIC ACID AS A TOOL FOR THE IN VIVO INDUCTION OF HYPERPHOSPHORYLATED TAU A. K. MUDHER* and V. H. PERRY CNS Inflammation Group, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OXI 3QT, U.K.
Neurofibrillary tangles (NFTs) are an established pathological hallmark of Alzheimer's disease (AD), and yet there is no animal model in which they have been induced in vivo. NFTs comprise paired helical filaments] 9 the main constituent of which is a 57,000-68,000 mol. wt microtubule-associated protein called tau ~s't6,2° found in an abnormally hyperphosphorylated state. 5'12'17 The role that hyperphosphorylated tau plays in the AD neuropathology is unclear. It would be of value to induce hyperphosphorylated tau in vivo in order to investigate its effect(s) on neurobiology. Hyperphosphorylated tau can be induced in vitro by potentiating kinase activitys'2~'23 or by inhibiting phosphatase activity, 9"13'14"26'27 Arendt et al. 1 have proposed an animal model in which they showed that the injection of the phosphatase 1/2A inhibitor okadaic acid (OA) into the rat brain resulted in the induction of hyperphosphorylated tau, with maximal induction seen at seven days post-injection. They then went on to report that the chronic intraventricular infusion of OA into the rat brain also results in the induction of hyperphosphorylated tau, and moreover the animals exhibit a memory deficit that is apparent two weeks after the infusion is begun. These reports make this an attractive model for the induction of hyperphosphorylated tau in vivo because it appears that OA can mimic the neuropathological as well as some of the clinical symptoms of AD. We were interested in investigating the effect that hyperphosphorylated tau has on neuronal function and we chose to replicate the acute model described by Arendt et al.~ to induce hyperphosphorylated tau in vivo.
Adult male Wistar rats were injected with 0.5 ~1 of saline containing 5, 50, 100, 500 or 1000 ng of OA or with 0.5 gl of saline either in the hippocampus or in the basal forebrain nucleus, using stereotaxic surgery as previously described. 1 The ammonium salt of OA *To whom correspondence should be addressed. Abbreviations: AD, Alzheimer's disease, NFTs, neurofibril-
lary tangles, OA, okadaic acid.
was used and this was obtained from two sources-Sigma (Poole, U.K.) and RBI (Natick, MA, U.S.A). The rats were killed at 2 or 24 h post-injection and we used immunohistochemistry on 50~tm-thick freefloating sections to look for hyperphosphorylated tau. The phosphorylation dependent antibody AT8 (Autogen Bioclear, Calne, U.K.) was used. We found, as reported by Arendt et al.,~3 that OA injection into the hippocampus or basal forebrain nucleus induced an upregulation of phosphorylated tau which appeared within 2 h (Fig. la,b) and disappeared by 24h post-injection (Fig. lc,d). The up-regulation of the AT8 staining was seen both in terms of neuropil and somatodendritic staining around the injection site, over and above the constitutive level of staining seen on the contralateral side (data not shown). This effect was dose-dependent. However we found that OA is very neurotoxic at this dose required to induce hyperphosphorylated tau. At 2 h post-injection in both the hippocampus and basal forebrain nucleus, a small lesion began to appear around the injection site which became quite widespread by 24 h (Fig. la-d). No such lesion was seen in the saline-injected animals (Fig. le,f). The main features of the lesion were extensive parenchymal disintegration, thinning of the granule cell layers in the hippocampus and appearance of neutrophils and pyknotic cells (data not shown). The neurotoxicity was dose-dependent. Similar neurotoxicity was seen in rats injected with OA obtained from either source (RBI or Sigma). Our results show that a single 50 ng dose of OA in the hippocampus, or a 500 ng dose in the basal forebrain, results in complete neuropil disintegration by 24 h post-injection; surprisingly Arendt el al. report no such lesion in their acute model even though they injected up to 4000 ng of OA into the basal forebrain. ~ Similarly no neurotoxicity was reported in the chronic model despite the fact that up to 140 ng of OA a day was being infused into the lateral ventricles of the the rats for eight weeks. 2 In the light of our results, it would seem that the memory deficit seen in the animals infused with large doses of OA for
1329
1330
A . K . Mudher and V. H. Perry
Fig. 1. AT8 staining in the rat hippocampus or basal forebrain nucleus following a single injection of OA or saline. OA was injected into the hippocampus (a and c) or basal forebrain nucleus (b and d) and induction of hyperphosphorylated tau was looked for, using the phosphorylation dependant anti tau antibody AT8, at 2 h (a and b) or 24 h (c and d) post-injection. The hippocampal injections were 50 ng of OA (in a 0.5 ~1 volume); the basal forebrain injections were 500 ng of OA (in a 0.5 gl volume). Control injections were made using 0.5 p_l of saline (e and f). An inert blue dye (shown by the + in a, d, e and f) indicates the site of injection. At 2 h post-injection, up-regulation of AT8 staining is seen in both brain regions (a and b) that is not seen in the saline-injected controls (data not shown). A neurotoxic lesion also begins to appear at 2 h post-injection of OA in both brain regions (arrows in a and b) and this is extensive by 24 h (c and d). No such lesion is seen in the animals injected with saline at 2 h (data not shown), or 24 h post-injection (e and f).
The in vivo toxicity of okadaic acid
1331
prolonged periods of time, may have been the result of large OA-mediated lesions in various parts of the brain, and may have little to do with any hyperphosphorylated tau induced in that model. The neurotoxicity that we have seen is in agreement with tissue culture experiments that demonstrate OA's potent toxicity even at doses 2000-fold
after brief exposures. 24 Some of these actions may be attributed to OA's ability to inhibit phosphatase 1/2A, since these phosphatases have a broad spectrum of substrate specificities.
lower than those that were used in our stud y."4 7 1.0 1.8 . .2 4.2 5 Moreover O A has a plethora of
because of its potent toxicity, but also because of its ability to affect a number of biological processes.
other effects, e.g., it is pro-inflammatory, 6'22 has tumour-promoting ability, ~ and can inhibit protein, D N A and RNA synthesis at pM concentrations
Acknowledgements~This work was supported by a grant from Bristol Myers Squibb.
One can then conclude that although OA can indeed up-regulate hyperphosphorylated tau #~ v i v o , it is of limited use as a tool in this context not only
REFERENCES 1. Arendt T., Hanisch F., Holzer M. and Bruckner M. K. (1994) In vivo phosphorylation in the rat basal nucleus induces PHF-like and APP immunoreactivity. NeuroReporl 5, 1397 1400. 2. Arendt T., Holzer M., Fruth R., Bruckner M. K. and Gartner U. (1995) Paired helical filament-like phosphorylation of tau, deposition of beta/A4 amyloid and memory impairment in rat induced by chronic inhibition of phosphatase 1 and 2A. Neuroscience 69, 691-698. 3. Arendt T., Holzer M., Fruth R., Bruckner M. and Gartner U. (1997) Chronic inhibition of phosphatases 1 and 2A in rat brain: a nontransgenic model of Alzheimers disease. In Alzheimer's Disease: Biology, Diagnosis and Therapeutics (eds Iqbal K., Winblad B., Nishimura T., Takeda T. and Wisneiwski H.), pp. 587 594, John Wiley and Sons Ltd, New York. 4. Arias C., Sharma N., Davies P. and Shafit-Zagardo B. (1993) Okadaic acid induces early changes in microtubuleassociated protein 2 and tau phosphorylation prior to neurodegeneration in cultured cortical neurons. J. Neurochem. 61,673 682. 5. Bancher C., Grundke lqbal I., Iqbal K., Fried V. A., Smith H. T. and Wisniewski H. M. (1991) Abnormal phosphorylation of tau precedes ubiquitination in neurofibrillary pathology of Alzheimer disease. Brain Res 539, 11 18. 6. Berkow R. L. and Dodson R. W. (1993) Regulation ofneutrophil respiratory burst by protein phosphatases. Lf/b Sci. 52, 1727 1732. 7. Candeo P., Favaron M., Lengyel I., Manev R. M., Rimland J. M. and Manev H. (1992) Pathological phosphory[ation causes neuronal death: effect of okadaic acid in primary culture of cerebellar granule cells. Z Ak, urochem. 59, 1558 1561. 8. Drewes G., Lichtenberg Kraag B., Doring F., Mandelkow E. M., Biernat J., Goris J., Doree M. and Mandelkow E. (1992) Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. Eur. molec. Biol. Org. J. l l , 2131-2138. 9. Drewes G., Mandelkow E. M., Baumann K., Goris J., Merlevede W. and Mandelkow E. (1993) Dephosphorylation o1" tau protein and Alzheimer paired helical filaments by calcineurin and phosphatase-2A. Fedn Eur. biocehem. Sots Lett. 336, 425~32. 10. Fernandez M. T., Zitko V., Gascon S. and Novelli A. (1991) The marine toxin okadaic acid is a potent neurotoxin for cultured cerebellar neurons. Lij~, Sci. 49, L157-LI62. 11. Fujiki H., Suganuma M., Sugur H., Yoshizawa S., Ojika M., Wakatsa K. and Sugimura T. (1987) Induction of ornithine decarboxylase activity in mouse skin by a possible tumor promoter, okadaic acid. Proc. Jap. Acad. Ser. B. 63,51 53. 12. Goedert M., Cohen E. S., Jakes R. and Cohen P. (1992) p42 MAP kinase phosphorylation sites in microtubuleassociated protein tau are dephosphorylated by protein phosphatase 2A. Implications for Alzheimer's disease. Fedn Eur. biochem. Socs Lett. 312, 95 99. 13. Gong C. X., Grundke Iqbal l., Damuni Z. and lqbal K. (1994) Dephosphorylation of microtubule-associated protein tau by protein phosphatase-1 and -2C and its implication in Alzheimer disease. Fedn Eur. biochem. Sots Lett. 341, 94 -98. 14. Gong C. X., Singh T. J., Grundke Iqbal I. and lqbal K. (1994) Alzheimer's disease abnormally phosphorylated tau is dephosphorylated by protein phosphatase-213 (calcineurin). J. Neurochem. 62, 803 806. 15. Grundke lqbal I., Iqbal K., Tung Y. C., Quinlan M., Wisniewski H. M. and Binder L. I. (1986) Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc. hath. Acad. Sci. U.S.A. 83, 4913-4917. 16. Ihara Y., Nukina N., Miura R. and Ogawara M. (1986) Phosphorylated tau protein is integrated into paired helical filaments in lzkheimer's disease. J. Biochem., Tokyo 99, 1807 1810. 17. lqbal K., Grundke Iqbal 1., Smith A. J., George L., Tung Y. C. and Zaidi T. (1989) Identification and localization of a tau peptide to paired helical filaments of Alzheimer disease. Proc. ham. Acad. Sci. U.S.A. 86, 5646 5650. 18. Ishida Y., Furukawa Y., Decaprio J. A., Saito M. and Griffin J. D. (1992) Treatment of myeloid leukemic cells with the phosphatase inhibitor okadaic acid induces cell cycle arrest at either G1/S or G2/M depending on dose. J. ( W l Physiol. 150, 484-492. 19. Kosik K. and Greenberg S. (1994) Tau protein and Alzheimer's disease. In Alzheimer's Disease (eds Terry R. D., Katzman R. and Bick K. L.), pp. 335 344. Raven, New York. 20. Lee V. M., Balin B. J., Otvos L. Jr and Trojanowski J. Q. (1991) A68: a major subunit of paired helical filaments and derivatized forms of normal tau. Science 251,675-678. 21. Lovestone S. and Reynolds C. (1997) The phosphorylation of Tau: a critical stage in neurodevelopment and neurodegenerative processes. Neuroscieme 78, 309 324.
1332 22. 23. 24. 25. 26. 27.
A . K . Mudher and V. H. Perry Lu D. J., Takai A., Leto T. L. and Grinstein S. (1992) Modulation of neutrophil activation by okadaic acid, a protein phosphatase inhibitor. Am. J. Physiol. 262, C39-C49. Mandelkow E. M., Drewes G., Biernat J., Gustke N., Van Lint J., Vandenheede J. R. and Mandelkow E. (1992) Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau. Fedn Eur. biochem. Socs Lett. 314, 315 321. Matias W. G., Bonini M. and Creppy E. E. (1996) Inhibition of protein synthesis in a cell-free system and Vero cells by okadaic acid, a diarrhetic shellfish toxin. Z Toxicol. environ. Health 48, 309 317. Tubaro A., Florio C., Luxich E., Sosa S., Della Loggia R. and Yasumoto T. (1996) A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels. Toxicon 34, 743-752. Wang J. Z., Gong C. X., Zaidi T., Grundke Iqbal I. and Iqbal K. (1995) Dephosphorylation of Alzheimer paired helical filaments by protein phosphatase-2A and -2B. Z biol. Chem. 270, 48544860. Wang J. Z., Grundke Iqbal I. and Iqbal K. (1996) Restoration of biological activity of Alzheimer abnormally phosphorylated tau by dephosphorylation with phosphatase -2A, 2B and 1. Molec. Brain Res. 38, 200 208. (Accepted 15 December 1997)
Neuroscience Vol. 85, No. 4, pp. 1329 1332. 1998 Copyright ;C 1998 IBRO. Publishedby ElsevierScienceLtd Printed in Great Britain.All rights reserved PII: S0306-4522(97)00695-7 0306-4522/98 $19.00+0.00
MATTERS ARISING USING OKADAIC ACID AS A TOOL FOR THE IN VIVO INDUCTION OF HYPERPHOSPHORYLATED TAU A. K. MUDHER* and V. H. PERRY CNS Inflammation Group, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OXI 3QT, U.K.
Neurofibrillary tangles (NFTs) are an established pathological hallmark of Alzheimer's disease (AD), and yet there is no animal model in which they have been induced in vivo. NFTs comprise paired helical filaments] 9 the main constituent of which is a 57,000-68,000 mol. wt microtubule-associated protein called tau ~s't6,2° found in an abnormally hyperphosphorylated state. 5'12'17 The role that hyperphosphorylated tau plays in the AD neuropathology is unclear. It would be of value to induce hyperphosphorylated tau in vivo in order to investigate its effect(s) on neurobiology. Hyperphosphorylated tau can be induced in vitro by potentiating kinase activitys'2~'23 or by inhibiting phosphatase activity, 9"13'14"26'27 Arendt et al. 1 have proposed an animal model in which they showed that the injection of the phosphatase 1/2A inhibitor okadaic acid (OA) into the rat brain resulted in the induction of hyperphosphorylated tau, with maximal induction seen at seven days post-injection. They then went on to report that the chronic intraventricular infusion of OA into the rat brain also results in the induction of hyperphosphorylated tau, and moreover the animals exhibit a memory deficit that is apparent two weeks after the infusion is begun. These reports make this an attractive model for the induction of hyperphosphorylated tau in vivo because it appears that OA can mimic the neuropathological as well as some of the clinical symptoms of AD. We were interested in investigating the effect that hyperphosphorylated tau has on neuronal function and we chose to replicate the acute model described by Arendt et al.~ to induce hyperphosphorylated tau in vivo.
Adult male Wistar rats were injected with 0.5 ~1 of saline containing 5, 50, 100, 500 or 1000 ng of OA or with 0.5 gl of saline either in the hippocampus or in the basal forebrain nucleus, using stereotaxic surgery as previously described. 1 The ammonium salt of OA *To whom correspondence should be addressed. Abbreviations: AD, Alzheimer's disease, NFTs, neurofibril-
lary tangles, OA, okadaic acid.
was used and this was obtained from two sources-Sigma (Poole, U.K.) and RBI (Natick, MA, U.S.A). The rats were killed at 2 or 24 h post-injection and we used immunohistochemistry on 50~tm-thick freefloating sections to look for hyperphosphorylated tau. The phosphorylation dependent antibody AT8 (Autogen Bioclear, Calne, U.K.) was used. We found, as reported by Arendt et al.,~3 that OA injection into the hippocampus or basal forebrain nucleus induced an upregulation of phosphorylated tau which appeared within 2 h (Fig. la,b) and disappeared by 24h post-injection (Fig. lc,d). The up-regulation of the AT8 staining was seen both in terms of neuropil and somatodendritic staining around the injection site, over and above the constitutive level of staining seen on the contralateral side (data not shown). This effect was dose-dependent. However we found that OA is very neurotoxic at this dose required to induce hyperphosphorylated tau. At 2 h post-injection in both the hippocampus and basal forebrain nucleus, a small lesion began to appear around the injection site which became quite widespread by 24 h (Fig. la-d). No such lesion was seen in the saline-injected animals (Fig. le,f). The main features of the lesion were extensive parenchymal disintegration, thinning of the granule cell layers in the hippocampus and appearance of neutrophils and pyknotic cells (data not shown). The neurotoxicity was dose-dependent. Similar neurotoxicity was seen in rats injected with OA obtained from either source (RBI or Sigma). Our results show that a single 50 ng dose of OA in the hippocampus, or a 500 ng dose in the basal forebrain, results in complete neuropil disintegration by 24 h post-injection; surprisingly Arendt el al. report no such lesion in their acute model even though they injected up to 4000 ng of OA into the basal forebrain. ~ Similarly no neurotoxicity was reported in the chronic model despite the fact that up to 140 ng of OA a day was being infused into the lateral ventricles of the the rats for eight weeks. 2 In the light of our results, it would seem that the memory deficit seen in the animals infused with large doses of OA for
1329
1330
A . K . Mudher and V. H. Perry
Fig. 1. AT8 staining in the rat hippocampus or basal forebrain nucleus following a single injection of OA or saline. OA was injected into the hippocampus (a and c) or basal forebrain nucleus (b and d) and induction of hyperphosphorylated tau was looked for, using the phosphorylation dependant anti tau antibody AT8, at 2 h (a and b) or 24 h (c and d) post-injection. The hippocampal injections were 50 ng of OA (in a 0.5 ~1 volume); the basal forebrain injections were 500 ng of OA (in a 0.5 gl volume). Control injections were made using 0.5 p_l of saline (e and f). An inert blue dye (shown by the + in a, d, e and f) indicates the site of injection. At 2 h post-injection, up-regulation of AT8 staining is seen in both brain regions (a and b) that is not seen in the saline-injected controls (data not shown). A neurotoxic lesion also begins to appear at 2 h post-injection of OA in both brain regions (arrows in a and b) and this is extensive by 24 h (c and d). No such lesion is seen in the animals injected with saline at 2 h (data not shown), or 24 h post-injection (e and f).
The in vivo toxicity of okadaic acid
1331
prolonged periods of time, may have been the result of large OA-mediated lesions in various parts of the brain, and may have little to do with any hyperphosphorylated tau induced in that model. The neurotoxicity that we have seen is in agreement with tissue culture experiments that demonstrate OA's potent toxicity even at doses 2000-fold
after brief exposures. 24 Some of these actions may be attributed to OA's ability to inhibit phosphatase 1/2A, since these phosphatases have a broad spectrum of substrate specificities.
lower than those that were used in our stud y."4 7 1.0 1.8 . .2 4.2 5 Moreover O A has a plethora of
because of its potent toxicity, but also because of its ability to affect a number of biological processes.
other effects, e.g., it is pro-inflammatory, 6'22 has tumour-promoting ability, ~ and can inhibit protein, D N A and RNA synthesis at pM concentrations
Acknowledgements~This work was supported by a grant from Bristol Myers Squibb.
One can then conclude that although OA can indeed up-regulate hyperphosphorylated tau #~ v i v o , it is of limited use as a tool in this context not only
REFERENCES 1. Arendt T., Hanisch F., Holzer M. and Bruckner M. K. (1994) In vivo phosphorylation in the rat basal nucleus induces PHF-like and APP immunoreactivity. NeuroReporl 5, 1397 1400. 2. Arendt T., Holzer M., Fruth R., Bruckner M. K. and Gartner U. (1995) Paired helical filament-like phosphorylation of tau, deposition of beta/A4 amyloid and memory impairment in rat induced by chronic inhibition of phosphatase 1 and 2A. Neuroscience 69, 691-698. 3. Arendt T., Holzer M., Fruth R., Bruckner M. and Gartner U. (1997) Chronic inhibition of phosphatases 1 and 2A in rat brain: a nontransgenic model of Alzheimers disease. In Alzheimer's Disease: Biology, Diagnosis and Therapeutics (eds Iqbal K., Winblad B., Nishimura T., Takeda T. and Wisneiwski H.), pp. 587 594, John Wiley and Sons Ltd, New York. 4. Arias C., Sharma N., Davies P. and Shafit-Zagardo B. (1993) Okadaic acid induces early changes in microtubuleassociated protein 2 and tau phosphorylation prior to neurodegeneration in cultured cortical neurons. J. Neurochem. 61,673 682. 5. Bancher C., Grundke lqbal I., Iqbal K., Fried V. A., Smith H. T. and Wisniewski H. M. (1991) Abnormal phosphorylation of tau precedes ubiquitination in neurofibrillary pathology of Alzheimer disease. Brain Res 539, 11 18. 6. Berkow R. L. and Dodson R. W. (1993) Regulation ofneutrophil respiratory burst by protein phosphatases. Lf/b Sci. 52, 1727 1732. 7. Candeo P., Favaron M., Lengyel I., Manev R. M., Rimland J. M. and Manev H. (1992) Pathological phosphory[ation causes neuronal death: effect of okadaic acid in primary culture of cerebellar granule cells. Z Ak, urochem. 59, 1558 1561. 8. Drewes G., Lichtenberg Kraag B., Doring F., Mandelkow E. M., Biernat J., Goris J., Doree M. and Mandelkow E. (1992) Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. Eur. molec. Biol. Org. J. l l , 2131-2138. 9. Drewes G., Mandelkow E. M., Baumann K., Goris J., Merlevede W. and Mandelkow E. (1993) Dephosphorylation o1" tau protein and Alzheimer paired helical filaments by calcineurin and phosphatase-2A. Fedn Eur. biocehem. Sots Lett. 336, 425~32. 10. Fernandez M. T., Zitko V., Gascon S. and Novelli A. (1991) The marine toxin okadaic acid is a potent neurotoxin for cultured cerebellar neurons. Lij~, Sci. 49, L157-LI62. 11. Fujiki H., Suganuma M., Sugur H., Yoshizawa S., Ojika M., Wakatsa K. and Sugimura T. (1987) Induction of ornithine decarboxylase activity in mouse skin by a possible tumor promoter, okadaic acid. Proc. Jap. Acad. Ser. B. 63,51 53. 12. Goedert M., Cohen E. S., Jakes R. and Cohen P. (1992) p42 MAP kinase phosphorylation sites in microtubuleassociated protein tau are dephosphorylated by protein phosphatase 2A. Implications for Alzheimer's disease. Fedn Eur. biochem. Socs Lett. 312, 95 99. 13. Gong C. X., Grundke Iqbal l., Damuni Z. and lqbal K. (1994) Dephosphorylation of microtubule-associated protein tau by protein phosphatase-1 and -2C and its implication in Alzheimer disease. Fedn Eur. biochem. Sots Lett. 341, 94 -98. 14. Gong C. X., Singh T. J., Grundke Iqbal I. and lqbal K. (1994) Alzheimer's disease abnormally phosphorylated tau is dephosphorylated by protein phosphatase-213 (calcineurin). J. Neurochem. 62, 803 806. 15. Grundke lqbal I., Iqbal K., Tung Y. C., Quinlan M., Wisniewski H. M. and Binder L. I. (1986) Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc. hath. Acad. Sci. U.S.A. 83, 4913-4917. 16. Ihara Y., Nukina N., Miura R. and Ogawara M. (1986) Phosphorylated tau protein is integrated into paired helical filaments in lzkheimer's disease. J. Biochem., Tokyo 99, 1807 1810. 17. lqbal K., Grundke Iqbal 1., Smith A. J., George L., Tung Y. C. and Zaidi T. (1989) Identification and localization of a tau peptide to paired helical filaments of Alzheimer disease. Proc. ham. Acad. Sci. U.S.A. 86, 5646 5650. 18. Ishida Y., Furukawa Y., Decaprio J. A., Saito M. and Griffin J. D. (1992) Treatment of myeloid leukemic cells with the phosphatase inhibitor okadaic acid induces cell cycle arrest at either G1/S or G2/M depending on dose. J. ( W l Physiol. 150, 484-492. 19. Kosik K. and Greenberg S. (1994) Tau protein and Alzheimer's disease. In Alzheimer's Disease (eds Terry R. D., Katzman R. and Bick K. L.), pp. 335 344. Raven, New York. 20. Lee V. M., Balin B. J., Otvos L. Jr and Trojanowski J. Q. (1991) A68: a major subunit of paired helical filaments and derivatized forms of normal tau. Science 251,675-678. 21. Lovestone S. and Reynolds C. (1997) The phosphorylation of Tau: a critical stage in neurodevelopment and neurodegenerative processes. Neuroscieme 78, 309 324.
1332 22. 23. 24. 25. 26. 27.
A . K . Mudher and V. H. Perry Lu D. J., Takai A., Leto T. L. and Grinstein S. (1992) Modulation of neutrophil activation by okadaic acid, a protein phosphatase inhibitor. Am. J. Physiol. 262, C39-C49. Mandelkow E. M., Drewes G., Biernat J., Gustke N., Van Lint J., Vandenheede J. R. and Mandelkow E. (1992) Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau. Fedn Eur. biochem. Socs Lett. 314, 315 321. Matias W. G., Bonini M. and Creppy E. E. (1996) Inhibition of protein synthesis in a cell-free system and Vero cells by okadaic acid, a diarrhetic shellfish toxin. Z Toxicol. environ. Health 48, 309 317. Tubaro A., Florio C., Luxich E., Sosa S., Della Loggia R. and Yasumoto T. (1996) A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels. Toxicon 34, 743-752. Wang J. Z., Gong C. X., Zaidi T., Grundke Iqbal I. and Iqbal K. (1995) Dephosphorylation of Alzheimer paired helical filaments by protein phosphatase-2A and -2B. Z biol. Chem. 270, 48544860. Wang J. Z., Grundke Iqbal I. and Iqbal K. (1996) Restoration of biological activity of Alzheimer abnormally phosphorylated tau by dephosphorylation with phosphatase -2A, 2B and 1. Molec. Brain Res. 38, 200 208. (Accepted 15 December 1997)