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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 14, 2006 as doi:10.1096/fj.05-4362fje. |
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* Laboratory for Alzheimer’s Disease, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan;
Department of Mood Disorder Research and Suicide Prevention, Tokyo Institute of Psychiatry, Tokyo, Japan;
New York University, Center for Dementia Research, Nathan S. Kline Institute, New York, USA;
Department of Pathology and Microbiology, University of Nebraska Medical Center, Nebraska, USA;
¶ Department of Neuropathology, Faculty of Medicine, University of Tokyo, Tokyo, Japan;
|| Division of Cell Cycle Regulation, Cancer Research Institute, Kanazawa, Japan;
# The Netherlands Brain Bank, Amsterdam, The Netherlands; and
** Neuroscience Research Institute, Peking, China
1Correspondence: Laboratory for Alzheimer’s Disease, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. E-mail: kenneth{at}brain.riken.go.jp
SPECIFIC AIM
In tauopathies such as Alzheimer disease (AD) and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), tau is abnormally hyperphosphorylated at multiple Ser/Thr sites; however, the mechanisms and significance underlying this phenomenon are unclear due to its complexity. The specific aim of this study was to investigate the mechanisms of PHF-1 (phospho-Ser396 and phospho-Ser404) hyperphosphorylation of tau with a FTDP17 mutation R406W that causes AD-like dementia and tauopathy in humans but has a unique ability to reduce tau phosphorylation at the PHF-1 site in vitro and in cultured cells.
PRINCIPAL FINDINGS
1. Glycogen synthase kinase (GSK)-3ß requires priming phosphorylation at Ser404 to further phosphorylate tau at the PHF-1 site (mostly at Ser396)
The mechanisms of tau phosphorylation at the PHF-1 site by GSK-3ß, the major physiological tau kinase at the PHF-1 site, was investigated. Since the PHF-1 epitopes (phospho-Ser404 and 396) are located within the amino acid sequence that may serve as primed substrates for GSK-3ß phosphorylation (Fig. 1
), we studied the effects of a Ser-to-Ala (S404A) substitution on tau phosphorylation at the PHF-1 site by GSK-3ß. While GSK-3ß significantly enhanced the phosphorylation of WT tau at Ser400, 396, and the PHF-1 site, it failed to phosphorylate S404A tau at these sites. This finding suggests that GSK-3ß requires Ser404 to be primed before it can phosphorylate tau at Ser400, 396, and the PHF-1 site.
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2. The R406W mutation inhibits tau phosphorylation at Ser404, thereby reducing GSK-3ß-mediated tau phosphorylation at the PHF-1 site
We then studied the phosphorylation state of Ser404 in R406W tau using S404D and R406W/S404D tau that mimic WT and R406W tau phosphorylated at Ser404, respectively. While R406W tau migrated as a single major band whose position was almost aligned with those of recombinant tau, unphosphorylated WT tau, and S404A tau, S404D as well as R406W/S404D tau migrated significantly slower than did S404A or R406W tau, suggesting that reduced phosphorylation at Ser404 accounts for the faster migration patterns similar to those of R406W tau, S404A tau, and recombinant tau. These data demonstrate that the R406W mutation probably reduces tau phosphorylation at Ser404. Taken together, our data suggest a mechanism by which the R406W mutation reduces GSK-3ß-mediated tau phosphorylation at the PHF-1 site (Ser404 and Ser396): by primarily reducing phosphorylation at Ser404, GSK-3ß-mediated tau phosphorylation at the PHF-1 site is further inhibited (Fig. 1)
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3. JNK is involved in the phosphorylation of R406W tau at the PHF-1 site
In cultured hippocampal neurons or SY5Y cells expressing either WT or R406W human tau, both endogenous and exogenous tau was phosphorylated at Ser396 and the PHF-1 site via the mechanism schematically shown in Fig. 1
. Lithium inhibited phosphorylation of WT and R406W tau at Ser396, Ser400, and the PHF-1 site, supporting that endogenous GSK-3ß is the major physiological tau kinase that targets these sites in these cells. In contrast, when tau is expressed in rapidly proliferating cells such as COS7 cells, R406W tau was found to be more phosphorylated at the PHF-1 site than WT tau. Furthermore, this physiological tau phosphorylation could not be inhibited by the GSK-3ß inhibitor lithium (20 mM), but diminished mostly by a JNK inhibitor SP600125 (50 µM), suggesting an involvement of JNKs in the PHF-1 phosphorylation of R406W tau in COS7 cells. In fact, overexpression of active JNK in COS7 cells enhanced the phosphorylation of R406W tau at Ser396 and the PHF-1 site more effectively than that of WT tau. Furthermore, overexpression of active JNK induced phosphorylation of S404A tau at Ser396 and the PHF-1 site where GSK-3ß was unable to do so.
4. Endogenous JNK is activated in the cytoplasm and hyperphosphorylates R406W tau at the PHF-1 site during mitotsis
Immunocytochemical studies using anti-active JNK antibodies revealed that these antibodies only faintly stained the nucleus of the majority of COS7 cells. In 
5% of the mitotic cells, however, anti-active JNK antibodies stained the cytoplasm. In SY5Y cells, similarly anti-active JNK antibodies immunostained the cytoplasm of the mitotic cells (1%). The presence of active JNK in the cytoplasm was always associated with the presence of the PHF-1 epitope in mitotic SY5Y cells. These data further suggest that JNKs are generally activated in the cytoplasm of mitotic cells and may mediate the mitotic tau phosphorylation at the PHF-1 site.
5. Microtubule-interfering agents increase tau phosphorylation at Ser396 and the PHF-1 site in SY5Y cells that can be inhibited by the JNK inhibitor SP600125
We therefore examined the effects of the microtubule interfering agents nocodazole and taxol, on tau phosphorylation at Ser396 and the PHF-1 site in SY5Y cells transiently expressing WT tau or R406W tau. During the initial 2 h of treatment, these agents dephosphorylated tau as demonstrated by increased electrophoretic mobility of tau bands and by decreased immunolabeling with the anti-PS396 antibody. After 3 h elapsed, however, 72 kDa exogenous (WT, R406W) and 60 kDa endogenous (SY5Y) tau bands began to appear. The staining intensities of these bands, which increased in a time-dependent manner, were associated with the increased proportion of cells arrested in the G2/M phase.
The PS396 (Fig. 2
) and the PHF-1 antibodies reacted with all of these slowly migrating species of both exogenous (72 kDa) and endogenous (60 kDa) tau. Phosphorylation of R406W tau at these sites was first inhibited at 0 h, then increased, and became almost the same as that of WT tau both in taxol-treated (6 h) and nocodazole-treated (12 h) SY5Y cells.
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To verify that JNK is primarily responsible for phosphorylating tau at the PHF-1 site during mitosis, we treated these cells with one of four different kinase inhibitors (LiCl for GSK-3ß, SB203580 for p38, SP600125 for JNK/SAPK, and roscovitine for cdk5) for the last hour of treatment with either taxol or nocodazole (Fig. 2)
. Only the JNK inhibitor SP600125 almost abolished the taxol- or nocodazole-derived major exogenous 72 kDa and endogenous 60 kDa PHF-1 bands. Taken together, these cell culture studies indicate that endogenous JNKs, when activated in the cytoplasm during mitosis, most likely hyperphosphorylate both R406W and WT tau at Ser396 and at the PHF-1 site.
CONCLUSIONS AND SIGNIFICANCE
In the present study, we investigated the mechanisms by which tau phosphorylation at the PHF-1 site is regulated physiologically and how the R406W mutation, a causative mutation of FTDP-17 in humans, disrupts such PHF-1 phosphorylation. We also demonstrated that cytoplasmic active JNKs can lead to hyperphosphorylation of R406W tau at this particular site, suggesting that conditions inducing such cytoplasmic JNK activation may be involved in the pathophysiology of tauopathies.
Physiological tau phosphorylation at the PHF-1 site is regulated in the cell cycle-dependent manner (Fig. 3
). In nonmitotic cells and postmitotic neurons, tau phosphorylation at the PHF-1 site is mediated mostly by GSK-3ß and Ser404 kinase(s) and apparently disrupted by the R406W mutation (Fig. 1)
. In mitotic cells, however, regulation of PHF-1 phosphorylation changes dramatically. This type of tau phosphorylation, referred to as "mitotic tau phosphorylation," has been studied extensively. Our cell culture studies demonstrate that cytoplasmic active JNK is involved in the mitotic hyperphosphorylation of both WT and R406W tau at the PHF-1 site (Ser396).
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The abnormal re-entry of neurons into the cell cycle has long been implicated as a possible mechanism of AD pathogenesis. This is partly supported by the fact that the mitotic tau hyperphosphorylation induces unique tau mobility changes similar to those observed in the abnormal tau hyperphosphorylation in AD. In this context, tau hyperphosphorylation at the PHF-1 site by JNK may be simply a consequence of the abnormal re-entry of neurons into the cell cycle. Relatively late appearance of the PHF-1 epitope in the affected neurons in AD may represent this idea.
Another important implication for the pathogenesis of R406W FTDP-17 and AD is that the cytoplasmic JNK activation may trigger the critical final step of the tangle formation (the PHF-1 accumulation) and cell death. For example, the neuronal PHF-1 accumulation in AD has been reported to correlate closely with the induction of fodrin caspase-cleavage product, a marker for the activation of apoptotic pathways. Since the cytoplasmic JNK activation may be associated with oxidative/nitration-related damage, disruption of calcium homeostasis, endoplasmic reticulum stress, CNS inflammation, and suppressed phosphatase activities, some of these conditions may be involved in the pathogenesis of tauopathies.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4362fje;
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