Okadaic acid mediates tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel

Molecular Brain Research 117 (2003) 145–151 www.elsevier.com / locate / molbrainres

Research report

Okadaic acid mediates tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel Fatma J. Ekinci 1 , Daniela Ortiz, Thomas B. Shea* Center for Cellular Neurobiology and Neurodegeneration Research, Department of Biological Sciences, University of Massachusetts Lowell, 1 University Avenue, Lowell, MA 01854, USA Accepted 13 June 2003

Abstract Accumulation of phosphorylated isoforms of the microtubule-associated protein tau is one hallmark of affected neurons in Alzheimer’s disease (AD). This increase has been attributed to increased kinase or decreased phosphatase activity. Prior studies indicate that one of the kinases that phosphorylates tau (mitogen-activated protein kinase, or MAP kinase) does so at least in part indirectly within intact neuronal cells by phosphorylating and activating the L-voltage-sensitive calcium channel. Resultant calcium influx then fosters tau phosphorylation via one or more calcium-activated kinases. We demonstrate herein that treatment of differentiated SH-SY-5Y human neuroblastoma with the phosphatase inhibitor okadaic acid (OA) similarly may increase tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel. OA increased phospho-tau as indicated by increased immunoreactivity towards an antibody (PHF-1) directed against paired helical filaments from AD brain. This increase was blocked by co-treatment with the channel antagonist nimodipine. OA treatment increased channel phosphorylation. The increases in calcium influx, PHF-1 immunoreactivity and channel phosphorylation were all attenuated by co-treatment with PD98059, which inhibits MAP kinase activity, suggesting that OA mediates these effects at least in part via sustained activation of MAP kinase. These findings underscore that divergent and convergent kinase and phosphatase activities regulate tau phosphorylation.  2003 Elsevier B.V. All rights reserved. Keywords: Okadaic acid; phosphatase; MAP kinase; L-voltage calcium channel; Alzheimer’s disease; Calcium; Neurodegeneration

1. Introduction One hallmark of affected neurons in Alzheimer’s disease (AD) is the accumulation of increased levels of phosphorylated forms of the microtubule-associated protein tau [13,17]. Several kinases have been implicated in regulation of tau phosphorylation [3,7,9,10,12,22,24,26,28,32,37,38] (Hanger et al., 1992). In addition, however, a number of studies indicate that phosphatase activities contribute to increased tau phosphorylation [8,19,25,27,5,31]. Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, increases levels of phospho-tau in culture and in situ [10,2,15,16,40,47,20,21,46,42,41]. It has been considered

*Corresponding author. Tel.: 11-978-934-2881; fax: 11-978-9343044. E-mail address: thomas [email protected] (T.B. Shea). ] 1 Present address: Department of Pharmacology, College of Medicine, University of Tennessee Center for Health Sciences, Memphis, TN 38163, USA. 0169-328X / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0169-328X(03)00294-8

that alterations in phosphatase activity may exert a more profound impact than that of kinase activity [44]. At the very least, it should be kept in mind that the combined impact of kinase and phosphatase activities are likely to be responsible for the net level of tau phosphorylation. Prior studies from our laboratory point towards a further complexity, in that one of the kinases reported to phosphorylate tau, mitogen-activated protein (MAP) kinase, may increase phospho-tau levels within cells at least in part by an indirect manner as follows. Following treatment of cultured neurons with Abeta, MAP kinase phosphorylates the L-voltage-sensitive calcium channel, inducing calcium influx [11]. Co-treatment with an antagonist of this channel or chelation of cytosolic calcium following channel activation each prevented Abeta-induced tau phosphorylation [11]. Thus, while MAP kinase can phosphorylate tau directly, these data suggested that the major influence of MAP kinase on tau phosphorylation was instead derived by MAP kinase-mediated activation of the L-voltage-sensitive calcium channel. Phosphorylation of

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tau itself may have then been carried out by activation of one or more calcium-dependent kinases following resultant calcium influx [11]. Inhibition of phosphatase activity, including that achieved by treatment with OA, potentiates MAP kinase activity by maintaining its phosphorylation [1,23]. We therefore investigated herein whether or not phosphatase inhibition might contribute to increased phospho-tau levels in an indirect manner by maintaining MAP kinase activity and / or activation of the L-voltage-sensitive calcium channel.

2. Experimental procedures

control cells were fixed for 15 min with 4% paraformaldehyde in 0.1 M phosphate buffer and immunostained by sequential reaction with a mouse monoclonal antibody (PHF-1; generous gift of Dr. Peter Davies) raised against tau from paired helical filaments from AD brains, followed by rhodamine-conjugated goat anti-mouse IgG and visualization by standard methods [11]. Identical results were obtained following substitution of methanol for paraformaldehyde. Additional controls, which yielded only background fluorescence, included substitution of non-immune murine IgG for PHF-1, or omission of primary antibody. Additional cultures were homogenized and subjected to immunoblot analyses with the phospho-dependent antibodies PHF-1, Alz-50 and the phospho-independent antitau antibody 5E2 (REF).

2.1. Cell culture and treatment SH-SY-5Y human neuroblastoma cells were cultured in DMEM (Cellgro) containing 10% fetal bovine serum in 5% CO 2 . Cultures were differentiated for 7 days with 10 mM retinoic acid, during which time they elaborate extensive neurites that exhibit characteristics of axons [43]. Cells were deprived of serum and treated for 2 h with one or more of the following: the phosphatase inhibitor OA (1 mM), the MAP kinase inhibitor PD98059 (10 mM; RBI, Natick, MA, USA [35]) or nimodipine (1 mM [45]), Prior studies have demonstrated efficacy of these concentrations for this cell system [41,11]. Cultures of SH-SY-5Y cells were radiolabeled by inclusion of 100 mCi 32 P-orthophosphate in culture medium during these 2 h incubations as described previously [11]. All supplies were from Sigma (St. Louis, MO, USA) unless otherwise specified.

2.4. Analysis of phosphorylation of the L-voltagesensitive calcium channel Cells were harvested by scraping with a rubber policeman and membrane preparations were generated by centrifugation of the homogenate for 15 min at 13,000 g [11]. The L-voltage calcium channel was immunoprecipitated from membrane preparations using a mouse anti-dihydropyridine antibody specific for this channel (UBI, Lake Placid, NY, USA) followed by protein GSepharose via standard procedures [11]. Immunoprecipitated material was subjected to sodium dodecyl sulfate (SDS)–gel electrophoresis. Dried gels were placed against X-Omat film (Kodak) to generate autoradiographs. Aliquots of fractions were also subjected to SDS–gel electrophoresis to visualize total protein.

2.2. Monitoring of intracellular calcium concentrations 2.5. Densitometric analyses Intracellular calcium concentration was monitored as described previously [11] by incubation with Fluo-3 (acetoxymethyl ester; Molecular Probes) for 30 min. Images were captured using a DAGE CCL-72 cooled CCD camera via a Scion LG-3 frame grabber operated by NIH Image analysis software and stored in a Macintosh Power PC 7100AV. Intracellular calcium was also monitored by flow cytometry as described [39]. Fluo-3 was added to two 25 cm 2 plates at a final concentration of 5 mM and cultures were incubated for 1 h at 37 8C. Medium was removed and cells were incubated in serum-free medium for 45 min at 37 8C. This medium was removed and cells received 200 ml trypsin. Following detachment from the culture dish, trypsin was quenched by the addition of 800 ml medium containing 10% fetal bovine serum. Detached cells were vortexed and analyzed in a Epics II Coulter cytometer. Analysis was carried out with NIH Image software.

2.3. Immunofluoresence and immunoblot analyses OA-, PD98059- and nimodipine-treated and untreated

Fifty to 100 cells in at least five randomly-selected microscopic fields processed as above for cytosolic calcium or tau immunoreactivity were scored for fluorescent intensity using NIH Image analysis software [7]. Representative background areas devoid of cells were similarly analyzed and subtracted from cell values to yield net densitometric values. All fields for each individual assay were illuminated, captured, and processed at the identical intensity. Autoradiographs were digitized with a UMAX flat-bed scanner and subjected to whole-band densitometric analyses using NIH Image as described; all radiolabeled bands were quantified and reported values represent the sum of all radiolabeled, immunoprecipitated species. The densitometric ‘plot profile’ of lanes of autoradiographs was carried out via NIH Image as described (e.g., Ref. [6]). Values were exported to Excel for statistical analyses via Student’s t-test. Radiolabeling and immunoprecipitation of the channel was carried out three times; all other experiments were carried out twice. Comparable results were obtained for all

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repeats; presented results are representative of all experiments.

3. Results

3.1. The phosphatase inhibitor OA enhances phosphorylation of the L-voltage-sensitive calcium channel Metabolic radiolabeling indicated that OA induced an approximate 100% increase in phosphorylation of the Lvoltage-sensitive calcium channel (Fig. 1; Table 1). While this effect could be due to prevention of dephosphorylation of the channel, OA also sustains activation of MAP kinase, a kinase known to phosphorylate this channel [11], by inhibition of dephosphorylation of the kinase itself. To examine whether or not sustained activation of MAP kinase could be responsible for the increase in channel phosphorylation following OA treatment, we co-treated additional cultures with 10 mM PD98059. PD98059 at this concentration has previously been shown to reduce MAP kinase activity by approximately 43%, and, in doing so, to inhibit MAP kinase-mediated phosphorylation of the Lvoltage-sensitive calcium channel [11]. When this was carried out, PD98059 attenuated the OA-mediated increase in channel phosphorylation by approximately half, confirming that sustained activation of MAP kinase played a role in OA-induced channel phosphorylation.

3.2. OA induces calcium influx via the L-voltagesensitive calcium channel Although phosphorylation is known to regulate activity of the L-voltage-sensitive calcium channel [14,18,33,34,49], we next examined more directly the influence of OA on channel function by quantifying whether or not OA treatment influenced cytosolic calcium levels and whether or not antagonists of the L-voltagesensitive calcium channel prevented any such increase. OA induced a marked increase in cytosolic calcium (Fig. 2). The addition of nimodipine, a specific inhibitor of the L-voltage-sensitive calcium channel, markedly prevented the OA-mediated increase in cytosolic calcium, confirming that OA mediated calcium influx at via this channel. In addition, PD98059 virtually completely blocked the OAmediated increase in cytosolic calcium. Since MAP kinase is known to regulate channel activity [11], this latter finding confirmed that OA-induced increase in cytosolic calcium was achieved by sustained activation of MAP kinase.

3.3. OA induces tau phosphorylation via the L-voltagesensitive calcium channel OA has been shown to increase levels of phospho-tau in

Fig. 1. OA induces phosphorylation of the L-voltage calcium channel. Left panel (‘Total’): Representative autoradiograph of equivalent (200 mg) aliquots of cultures with and without 2-h treatment with OA or OA1PD98059 as indicated. No apparent change in overall protein profile is apparent. Right panel (‘Precip.’): Representative autoradiograph of material immunoprecipitated by an antibody directed against the Lvoltage-sensitive calcium channel from SH-SY-5Y cells incubated with 32 P-orthophosphate for 2 h in the presence and absence of OA, PD98059, or both. The densitometric plot profile for immunoprecipitated material following treatment with OA versus OA1PD98059 is presented; note that co-treatment with PD98059 decreases phosphorylation of all major immunoprecipitated species. The accompanying graph presents densitometric analysis of three such samples (mean6standard error of the mean). Multiple species corresponding in migratory position to channel subunits (210, 170, 150, 125 and 84 kDa and 52 kDa) were immunoprecipitated (see also Ref. [11]); an additional 30 kDa radiolabeled species was detected at the front of the gel, which may represent a breakdown product. All immunoprecipitated bands were quantified; the value represents the sum of all densitometric values. Note that OA includes a near 100% increase in phosphorylation of immunoprecipitated proteins (P, 0.05 vs. control), and that co-treatment with PD98059 attenuated this increase (P,0.05 vs. OA alone), but that OA did not induce an overall increase in labeling of total proteins during this 2 h radiolabeling.

culture and in situ (e.g., see Refs. in the Introduction). We next examined whether or not activation of the L-voltagesensitive calcium channel played a role in OA-mediated tau phosphorylation by treatment with OA in the presence and absence of nimodipine and PD98059. Treatment with OA increased phospho-tau levels by approximately 50% as assayed by PHF-1 immunoreactivity (Fig. 3). This increase

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Table 1 OA induces phosphorylation of the L-voltage calcium channel Mw

210 175 150 125 84 52 30

Fogld increase vs. control

% Attenuation

OA

OA1PD98059

by PD98059

0.4 1.3 2.1 7.3 1.1 5.1 6.3

0.1 0.2 0.0 0.9 0.1 1.1 1.6

268 287 298 288 288 278 274

Values represent the fold increase in phosphorylation of immunoprecipitated proteins of the indicated apparent molecular weight following treatment with OA and OA1PD98059 calculated from the autoradiograph presented in Fig. 1. Also presented is the % attenuation of the OAmediated increase in the presence of PD98059. OA increased the association of radiolabeled phosphate with individual species to varying degrees; PD98059 attenuated phosphorylation of all species.

was markedly prevented by nimodipine, indicating that activation of the L-voltage-sensitive calcium channel and resultant calcium influx were essential for the influence of OA on phospho-tau levels. The OA-mediated increase in PHF-1 immunoreactivity was attenuated by 54.6626.8% in cells co-treated with PD98059 (Fig. 3), which is consistent with the extent of inhibition of OA-mediated channel phosphorylation by PD98059 (43612%; Fig. 1), as well as inhibition of MAP kinase activity by PD98059 under these conditions (43620% [11]). These findings were corroborated by immunoblot analyses: PHF-1 and Alz-50 immunoreactivity was increased by 50–60% following treatment with OA, while this increase was prevented by co-treatment with PD98059; moreover, no changes were observed in total tau levels (Fig. 3), indicating that the changes in phospho-tau immunoreactivity resulted specifically from changes in phosphorylation rather than overall tau levels.

4. Discussion The findings of the present study indicate that phosphatase inhibition sustains phosphorylation and activity of the L-voltage-sensitive calcium channel. These findings are consistent with prior studies indicating that multiple phosphatases regulate activity of this channel [14,18,33,34,49]. The twofold increase in 32 P labeling of channel subunits following OA addition suggests that this channel normally undergoes rapid cycles of phosphorylation and dephosphorylation. It is therefore not unexpected that inhibition of channel dephosphorylation would promote increased calcium influx and, in turn, down-stream pathological consequences of excessive calcium influx, including tau phosphorylation. OA could therefore mediate its effect on tau phosphorylation by (1) preventing dephosphorylation of tau, (2) preventing dephosphorylation of the channel, and / or (3)

Fig. 2. Phosphatase inhibition increases cytosolic calcium. Panel A presents representative phase-contrast and corresponding UV images of cultures in which cytosolic calcium was visualized by incubation with Flou-3 following 2 h with or without OA along with PD98059 or nimodipine as indicated. The accompanying graph presents densitometric data derived from multiple cells from duplicate cultures from duplicate experiments; values are presented as the % increase (mean6standard error of the mean) in cytosolic calcium in treated cultures over levels in untreated control cultures. Panel B presents the relative increase in cytosolic calcium as determined by flow cytometry of cultures treated as indicated. Note the increase in cytosolic calcium following OA treatment as ascertained by both methodologies, and the prevention of this increase by co-treatment with PD98059 or nimodipine.

preventing dephosphorylation of MAP kinase. In the latter case, there is yet a further possible bifurcation in mechanisms, since MAP kinase can phosphorylate both the channel [11] as well as tau [9,10,12,38,43,29] (Ledesma et al., 1992). Elucidation of responsible mechanism(s) was attempted by selective inhibition of channel function and

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Fig. 3. Phosphatase inhibition increases PHF-1 immunoreactivity. Panel A presents representative phase-contrast and corresponding UV images of cultures processed for PHF-1 immunoreactivity 2 h with or without OA along with PD98059 or nimodipine as indicated. The accompanying graph presents densitometric data derived from multiple cells from duplicate cultures from duplicate experiments; values are presented as the % increase in cytosolic calcium levels over levels in untreated control cultures (mean6standard error of the mean). Note the increase in PHF-1 following OA treatment, and the prevention of this increase by cotreatment with PD98059 or nimodipine. Panel B presents representative immunoblot analyses with PHF-1, Alz-50 and 5E2 of homogenates of cultures treated as indicated. The accompanying graph presents the densitometric values for cultures treated with OA or OA1PD98059 versus values obtained for untreated controls (mean6standard error of the mean). Note that OA increased phospho-tau but not total tau, and that co-treatment with PD98059 prevented the increase in phospho-tau.

MAP kinase activity. Co-treatment with nimodiprine, an antagonist of the L-voltage-sensitive calcium channel, markedly inhibited the OA-mediated increase in tau phosphorylation, indicating that (1) OA mediates increased tau phosphorylation via sustained activation of the channel and that (2) increased calcium influx is a critical component for OA-mediated tau phosphorylation. These findings further suggest that activation of one or more calcium-dependent kinases is required to increase tau phosphorylation following OA treatment of these cells as assayed by PHF-1 immunoreactivity. Calcium influx via sustained channel activation could also contribute to MAP kinase-mediated tau phosphorylation since prior phosphorylation of tau via the calcium-dependent kinases, including CaM kinase and protein kinase C, facilitates subsequent phosphorylation by MAP kinase ([10] and Refs. therein). The nature and extent of involvement of MAP kinase in these phenomena were further addressed by co-treatment of cells with the inhibitor of MAP kinase kinase activity, PD98059. The extent of reduction by PD98059 in OA-mediated tau phosphorylation (54.7626.8%) did not differ statistically

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from the reduction in OA-mediated channel phosphorylation (43.5612.2%; P.0.55, Student’s t-test). This suggests that the major impact of OA on increased tau phosphorylation is mediated by sustained phosphorylation (and therefore activation) of the channel via MAP kinase rather than by direct inhibition of tau dephosphorylation by OA. This conclusion is strengthened by the demonstration that blocking the L-voltage-sensitive calcium channel prevented the OA-induced increase tau phosphorylation. However, we cannot rule out the possibility that OA induced some increased phospho-tau levels via direct action of MAP kinase on tau due to the slightly greater, though not significantly increased, impact of PD98059 on OA-mediated phospho-tau levels versus that of PD98059 on OA-mediated channel phosphorylation (54.7 versus 43.5% inhibition). This concentration of PD98059 inhibited overall MAP kinase activity by 43620% [11]; to clarify these issues further, of interest would be to inhibit MAP kinase activity to a greater degree. Unfortunately, however, increasing the concentration of PD98059 and / or antisense oligonucleotides directed against MAP kinase was lethal [11], perhaps reflecting essential role(s) of MAP kinase for survival. It is also possible that the effect of sustained channel activation on tau phosphorylation could be augmented by direct prevention of tau dephosphorylation by OA, including that by calcium-dependent phosphatases (Shea and Ekinci, 1999). Interpretation of the net effect on tau phosphorylation is further complicated by the potential action of phosphatase PP2B, which also dephosphorylates tau [48]. The MAP kinase inhibitor PD98059 reduced OA-mediated phosphorylation of the L-voltage-sensitive calcium channel by approximately 43%, yet far more extensively blocked the OA-induced calcium influx. These data leave open the possibility that MAP kinase also regulates calcium influx into the cytosol by additional routes, including perhaps other plasma membrane channels, as well as efflux from intracellular stores such as the endoplasmic reticulum. However, nimodipine, which specifically inhibits the L-voltage-sensitive calcium channel, also markedly prevented the OA-mediated increase in cytosolic calcium, confirming that the major source of OA-mediated calcium influx is via the L-voltage-sensitive calcium channel. One interpretation of these data is that the impact of MAP kinase-mediated channel phosphorylation and calcium influx is not necessarily linear, and that a more profound impact is observed on cytosolic calcium levels than on channel phosphorylation. Additional time- and dose-dependent studies may clarify this possibility. Several additional candidate intracellular tau kinases have been identified, including glycogen synthase kinase 3b (GSK-3b; Hanger et al., 1992; [22,26,28]) cyclindependent kinase 5 (cdk5 [24,32]), calcium-calmodulin kinase (CaM kinase [3]) and protein kinase C (PKC [7,37,43]). The role of MAP kinase in intracellular tau phosphorylation remains controversial. While MAP kinase

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clearly phosphorylates tau in cell-free analyses and, in doing so, increases phospho-tau immunoreactivity [9,43] (Ledesma et al., 1992). It has been reported to phosphorylate tau within cells in some [10,12,38,29], but not all studies [26,28]. The findings of the present study, along with those of our prior study demonstrating increased phosphorylation of the L-voltage-sensitive calcium channel by MAP kinase following Abeta treatment [11], leave open the possibility that MAP kinase may indirectly promote tau phosphorylation by activating the L-voltage-sensitive calcium channel. Resultant calcium influx could foster tau phosphorylation by other calcium-dependent kinases. In addition, calcium influx could activate the calcium-dependent protease calpain, which is known to activate cdk5 by conversion of the cdk5 activator protein p35 to its constitutively-active form, p25 [36]. The findings of the present study also suggest that phosphatase activity can lead to increased tau phosphorylation by the same indirect mechanism. Given the pivotal role of tau in multiple neurodegenerative conditions including AD, progressive supranuclear palsy, Pick’s disease and corticobasal degeneration (for a review, see Ref. [13]), these findings underscore that multiple convergent and divergent signal transduction pathways impact neurodegeneration. The potential contribution of the L-voltage-sensitive calcium channel in neurodegeneration is highlighted by the recent demonstration that the product of lipid peroxidation, 4-hydroxynonenol, can lead to sustained activation of this channel [30].

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