TY - JOUR
AU - Yuan,, Tianli
AB - Abstract Microcystin-leucine-arginine (MC-LR) has been implicated as a potential environmental factor in Alzheimer’s disease because of its potent inhibition of protein phosphatase 2A (PP2A) activity, but experimental evidence to support its detailed neurotoxic effects and their underlying mechanisms has been lacking. The present study investigated the role of PP2A catalytic subunit (PP2Ac) demethylation and its link with glycogen synthase kinase-3β (GSK)-3β in tau hyperphosphorylation induced by MC-LR. The results showed that MC-LR treatment significantly increased demethylation of PP2Ac, with a concomitant increase in GSK-3β phosphorylation at Ser9 resulting in elevated tau hyperphosphorylation at PP2A-favorable sites in SH-SY5Y cells and rat hippocampus. Coimmunoprecipitation experiments showed that MC-LR treatment dissociated PP2Ac from Bα, making it incompetent in binding tau, thus causing tau hyperphosphorylation. Moreover, we found that inhibition of PP2A resulted in an increase in phosphorylation of GSK-3β at Ser9 and a decrease in GSK-3β activity, which further promoted demethylation of PP2Ac induced by MC-LR. These findings suggest a scenario in which MC-LR-mediated demethylation of PP2Ac is associated with GSK-3β phosphorylation at Ser9 and contributes to dissociation of Bα from PP2Ac, which would result in Bα degradation and disruption of PP2A/Bα-tau interactions, thus promoting tau hyperphosphorylation and paired helical filaments-tau accumulation and, consequently, axonal degeneration and cell death. microcystin-LR, protein phosphatase 2A, glycogen synthase kinase-3β, tau phosphorylation, demethylation, Alzheimer’s disease Microcystins (MCs), a group of monocyclic heptapeptide molecules, are produced by several freshwater cyanobacteria species. With the frequent outbreaks of cyanobacterial blooms, an increasing number of lakes and rivers are facing the threat of MC pollution. MCs pose a substantial health hazard to humans through contaminated drinking water and aquatic creatures. Among 80 MCs, MC-leucine-arginine (MC-LR) is the most common and most toxic variant (Hoeger et al., 2005; Meriluoto and Spoof, 2008). Microcystin-leucine-arginine preferentially accumulates in the liver because to abundant expression of organic anion-transporting polypeptides (rodent Oatp/human OATP) that are responsible for uptake of MC-LR into cell membrane. One of the known MC-LR transporters, OATP1A2, has been expressed in endothelial cells of the human blood-brain barrier (BBB) (Fischer et al., 2005), suggesting that MC-LR could cross the human BBB. The latter hypothesis is supported by data from MC-LR presence in hippocampi of rats and sequent impairment of memory and cognitive function associated with Alzheimer’s disease (AD) in rats via intraperitoneal injection with MC-LR (Li et al., 2012) as well as the observed inflammation in memory-related brain regions after oral administration of MC-LR (Li et al., 2014). Even though the potential neurotoxicity of MC-LR has been proposed, the molecular basis of MC-LR neurotoxicity remains elusive. It is well accepted that selective and potent inhibition of the serine/threonine (Ser/Thr) phosphatases 1 and 2A appears to be one of the main events in MC-LR-induced toxicity, and inhibition of protein phosphatase 2A (PP2A) is more efficient than inhibition of PP-1 by MC-LR. Tau hyperphosphorylation is an early event of neurofibrillary degeneration in AD, which is positively correlated with dementia in the patients. Tau phosphorylation is mainly regulated by the balance of kinases and phosphatases. Protein phosphatase 2A is the most important and major phosphatase implicated in tau dephosphorylation in the brain. Protein phosphatase 2A alone accounts for 70% of tau phosphatase activity in the human brain (Liu et al., 2005). Indeed, alterations in total PP2A activity (Liu et al., 2005), PP2A regulators (Tanimukai et al., 2005; Watkins et al., 2012), subunit expression (Vogelsberg-Ragaglia et al., 2001; Sontag et al., 2004a), methylation at Leu309 (Sontag et al., 2004b; Yoon et al., 2007; Zhou et al., 2008), and phosphorylation at Tyr307 (Liu et al., 2008) have been observed in AD brains repeatedly and contribute to tau hyperphosphorylation. Microcystin-leucine-arginine can effectively inhibit PP2A, which has been demonstrated to play a central role in tau (de)phosphorylation linked to Alzheimer’s disease; therefore, understanding how PP2A works will enable the elucidation of the neurotoxicity mechanisms induced by MC-LR. Protein phosphatase 2A is composed of a catalytic C subunit, a structural A subunit, and a variety of B subunits that regulate subcellular localization and enzyme specificity. Protein phosphatase 2A regulation is highly complex, involving not only holoenzyme composition and PP2A inhibitors but also methylation of the enzyme at Leu309 and phosphorylation at Tyr307 at its C-terminus of the catalytic subunit. There is overwhelming experimental evidence establishing the multifaceted deregulation of PP2A in AD and its link with the development of P-tau pathology (Martin et al., 2013; Sontag and Sontag, 2014; Voronkov et al., 2011). Among all events that underlie PP2A dysfunction, the interruption of PP2A methylation in AD is of particular interest, because it can lead to a loss of PP2A/Bα, which is the primary PP2A isoform that mediates tau dephosphorylation in brain. In vitro assays have established that the Bα subunit attaches the PP2A heterocomplex to microtubules, ideally positioning PP2A to dephosphorylate tau (Xu et al., 2008; Sontag et al., 2012). The importance of PP2A catalytic subunit (PP2Ac) methylation is further underlined by the observation that deregulation of PP2A methylation and concomitant loss of PP2A holoenzymes containing the regulatory Bα subunit disrupted normal PP2A-tau interaction, thereby preventing PP2A-mediated tau dephosphorylation while contributing to deregulation of normal tau distribution (Sontag et al., 2013) and neuritogenesis (Sontag et al., 2010) in AD pathogenesis. Another key enzyme involved in the regulation of tau phosphorylation is glycogen synthase kinase-3β (GSK-3β). GSK-3β is a proline-directed serine/threonine kinase and is the major tau kinase in the brain. The activity of GSK-3β is regulated by phosphorylation at Ser9 and Tyr216. Ser9 phosphorylation inhibits the kinase activity of the enzyme, whereas Tyr216 phosphorylation is required for its full activity. GSK-3β is involved in tau hyperphosphorylation, neurofibrillary tangle (NFT) formation, neuronal death, synaptic loss, and memory deficits in the AD brain (Kimura et al., 2008). Specific PP2A inhibition has been proved to result in deregulation of GSK-3β (Louis et al., 2011; Wang et al., 2010). Conversely, GSK-3β can in turn modulate PP2A via several mechanisms including demethylation of PP2A at Leu309 (Yao et al., 2012). Dysregulation of both PP2A and GSK-3β, respectively, the major tau phosphatase and kinase, were suggested to be involved in tau hyperphosphorylation and aggregation during the development of AD; therefore, identifying the relationship between PP2A and GSK-3β and the impact of their dysregulation on tau phosphorylation is critical to understand the molecular basis of MC-LR neurotoxicity. In the present study, we emphasized the role of PP2Ac demethylation and its link with GSK-3β in tau hyperphosphorylation induced by MC-LR. We demonstrate that MC-LR-mediated demethylation of PP2Ac contributes to PP2A loss of function leading to tau hyperphosphorylation and subsequently cell death. Furthermore, we investigate the relationship of PP2A demethylation and GSK-3β and show that GSK-3β inactivation can further promote MC-LR-induced demethylation of PP2A. MATERIALS AND METHODS Reagents and animals Purified MC-LR (purity >95%) was purchased from Alexis Biochemicals (Lausen, Switzerland). AR-A014418 was bought from MedChemExpress (TargetMol). Endo-Porter (EP) was obtained from Gene Tools. Antimicrocystin (clone MC10E7) monoclonal antibody was from Axxora (San Diego, California). Antibodies against tau phosphorylated at Ser202, Ser356, Ser 199, Ser 396, and Ser262 and against total tau were all purchased from Abcam (Cambridge, Massachusetts). An antibody against phospho-paired helical filaments (PHF)-tau (pSer202 + Thr205) (AT8) was purchased from ThermoFisher (Waltham, Massachusetts). Antibody (tau46) against total tau was purchased from Cell Signaling Technology (Beverly, Massachusetts). Antibodies against PP2A-A subunit, PP2A-B subunit, PP2A-C subunit, phosphorylated SAPK/JNK (Thr183/Tyr185), SAPK/JNK, phosphorylated p38 (Thr180/Tyr182), total p38 MAPK, phosphorylated GSK-3β (Ser9), and GSK-3β were all obtained from Cell Signaling Technology. An antibody against PP2A c phosphorylated at Tyr307 was obtained from ImmunoWay (Plano, Texas). Antibodies against demethylated PP2Ac and PP2A Bα, short interfering RNAs (siRNAs) against PP2A c and PP2A Bα, and all of the secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California). Alexa Fluor 488 goat antimouse IgG conjugate and Alexa Fluor 594 goat antirabbit IgG conjugate were from Molecular Probes/Invitrogen. A Serine/Threonine Phosphatase Assay kit was purchased from Promega (Madison, Wisconsin). Male Sprague Dawley rats (180–220 g, 7–8 weeks) were purchased from the Laboratory Animal Center, Medical School, Nantong University. They were kept in an air-conditioned environment at 24 °C ± 1 °C with a 12 h/12 h light/dark cycle and had free access to food and water. The animals were allowed to acclimatize to their surroundings for 3 days before experiments started. Principles of laboratory animal care were followed and all procedures were conducted according to the guidelines established by the National Institutes of Health, and every effort was made to minimize suffering. This study was approved by the Animal Experiment Committee of Nantong University (2012-0031). Cell culture and treatment Human SH-SY5Y neuroblastoma cells were cultured in DMEM/F-12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), l-glutamine, and penicillin/streptomycin (Invitrogen) at 37 °C in a humidified atmosphere with 5% CO2. Cells in the logarithmic growth phase were exposed to DMSO (vehicle) at a concentration not exceeding 0.1% or to MC-LR at final concentrations of 5 and 10 μM, respectively. Facilitated transport of MC-LR was performed with 6 μl/ml EP. Immunofluorescence on coverslips SH-SY5Y cells were plated onto cover slips in 12-well plates and treated with 0 or 10 μM MC-LR or 10 μM MC-LR with EP for 24 h. After treatment, the cells were washed twice with 1 ml PBS and fixed with 4% paraformaldehyde for 10 min. After 3 washes with PBS, the cells on the coverslips were blocked with PBS containing 10% FBS for 20 min at 37 °C. Following the blocking step, the cells were incubated overnight at 4 °C with primary antibodies at an appropriate dilution in PBS containing 0.1% saponin. After incubation, the cells were washed with PBS and incubated for 1 h with secondary Alexa Fluor 488- and Alexa Fluor 549-labeled antibodies (dilution 1:100) for 1 h at room temperature in the dark; the nuclei were stained by incubation with DAPI (1 μg/ml in PBS) for 10 min at room temperature. The coverslips were then washed 3 times with PBS and mounted with Flu-G (Southern Biotech) on slides. Immunofluorescence signals were visualized under a laser scanning microscope (63×) (Zeiss LSM 510 META). Immunoprecipitation After 24 h of MC-LR incubation, SH-SY5Y cells were washed twice with PBS, scraped and treated with lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 15 mM EDTA, 1 mM PMSF, 1 mM NaF, and 1 mM Na3VO4, and l complete protease inhibitor tablet (Roche, Indianapolis, Indiana). The cells were then lysed for 30 min on ice. The lysate solution was then centrifuged at 14 000 × g for 30 min at 4 °C. The supernatants were used for immunoprecipitation with anti-PP2A c rabbit monoclonal antibody at 4 °C and rotated overnight. A volume of 100 μl Protein A/G agarose beads (Santa Cruz) was added and incubated at 4 °C for 3 h. The beads were washed with lysis buffer twice and with PBS twice, then resuspended in SDS loading buffer and boiled prior to Western blotting. PP2A phosphatase activity assay Protein phosphatase 2A activity was assayed using the phosphatase kit V2460 (Promega). Briefly, total cellular proteins were extracted in phosphatase storage buffer (50 mM Tris-HCl pH 7.5, 0.05% Triton X-100, 0.1 mM EDTA, 0.5 mM PMSF, 0.05% β-mercaptoethanol, 10% glycerol) with protease inhibitor complex (Roche) for 40 min and purified by removing endogenous phosphate with the provided spin columns. Protein concentrations were determined using the BCA protein assay. The cell protein was then applied to a premixed reaction containing the specific peptide substrate RRA(pT)VA, PP2A-specific 5×  reaction buffer (250 mM imidazole pH 7.2, 1 mM EGTA, 0.1% β-mercaptoethanol, and 0.5 mg/ml BSA) and phosphatase storage buffer on a 96-well plate, and the reactions were incubated for 15 min at 37 °C. The reactions were terminated by adding a molybdate dye/additive mixture. Free phosphate which was generated from a synthetic phosphothreonine peptide RRA(pT)VA, an optimal substrate for PP2A, PP2B, and PP2C, but not for PP1 in a buffer optimized for PP2A activity while cation-dependent PP2B and PP2C were inhibited, was quantified by measuring molybdate/malachite green/phosphate complex at 630 nm. All determinations were performed in duplicate, and the absorbance of the reactions was corrected by determining the absorbance from duplicate reactions not treated with the phosphopeptide substrate. Protein phosphatase 2A activity was expressed as picomoles of free PO4 generated per min and per microgram of protein. Lactate dehydrogenase release assay Lactate dehydrogenase (LDH) leakage was measured using an LDH Detection kit (Beyotime, Haimen, Jiangsu, China) according to the manufacturer’s protocol. SH-SY5Y cells were plated in 96-well plates and treated with 0 and 10 μM MC-LR with EP for 24 h. After treatment, culture medium (120 μl) was transferred to a new 96-well plate. Substrate mixture (60 μl) was added to each well and allowed to react for 30 min in the dark at room temperature. The absorbance was read at 440 nm using a BioELISA Reader (BioTek Instruments, Winooski, Vermont). The absorbance was normalized based on the absorbance of negative controls, which consisted of cells not exposed to MC-LR. Experimental protocols in vivo A total of 30 (n = 30) rats were divided into 2 groups: Control (CO, n = 15) and MC-LR (MC, n = 15). All rats (CO: n= 15, MC: n = 15) were trained to navigate by using Morris water maze (MWM) task (MWM). After 5 continuous days of spatial acquisition training, the rats were tested on day 6. On day 7, rats were given a microinjection of MC-LR (MC-LR groups) or saline solution (control groups). After MWM testing on day 8, some rats (n = 5 per group) were perfused with 4% paraformaldehyde for immunofluorescence, and others (n = 5 per group) were sacrificed and had hippocampal samples collected for Western blotting. Injection of MC-LR The animals were anaesthetized with 500 mg/kg chloral hydrate, with bilateral injections performed stereotaxically into the upper leaf of the dentate gyrus in the dorsal hippocampus (–3.5 mm anteroposterior, ±2.0 mm mediolateral, and –2.7 mm dorsoventral from the dura, according to the bregma). The animals were injected with 3 μl of either saline solution or MC-LR (0.1 μg/μl in saline solution). MWM task To evaluate spatial memory, we conducted the MWM task using a procedure similar to that described previously (Muller et al., 2010). The rats (CO: n = 15, MC: n = 15) were first trained to find the hidden platform that was placed in 1 of the 4 quadrants throughout 5 consecutive days of the MWM testing. A trial was initiated by placing the rat in 1 of the other 3 quadrants (which did not contain the platform), with its head toward the wall of the tank. The rat was then allowed to search for the hidden platform. If the rat failed to find the platform in 90s, it was removed from the water, gently placed on the platform and allowed to rest for 20s. They were tested in 4 trials per day from 3 different locations, with an intertrial interval of 3–5 min. As the rats swam around the pool, various parameters, such as the time taken to reach the platform and the swimming paths, were recorded by a video imaging analysis system. Escape latency was defined as the time to find the platform during each trial and was used to assess acquisition of the water maze task. A probe test without the platform was performed, and the time spent and swimming distance in the target quadrant were used as indicators of memory retention. Immunofluorescence The saline solution- or MC-LR-injected rats were anaesthetized and perfused with 4% paraformaldehyde in PBS. The brains were removed and postfixed overnight at 4 °C. After successive anhydration in 20% and 30% sucrose until they sank, the brains were cut in 20-μm-thick coronal sections using a freezing microtome. The slices were incubated with primary antibodies and then with the secondary Alexa Fluor 488-conjugated antimouse antibody. Light and fluorescence images were captured under a laser scanning microscope (Zeiss LSM 510 META). Western blot analyses SH-SY5Y cells were lysed on ice for 30 min, and the hippocampal tissue samples were homogenized in lysis buffer (P0013, Beyotime, Haimen, Jiangsu, China). The lysate solution was then centrifuged at 14 000 × g for 20 min at 4 °C. The protein concentrations in the supernatants were determined by using a BCA protein assay kit. All samples were aliquoted and stored at −80 °C prior to electrophoresis. Aliquots from the supernatant containing 50 μg of protein each were mixed with equal volumes of 2× loading buffer. Protein samples were run on a 10% gradient SDS–PAGE gel, and the proteins were transferred to nitrocellulose membranes. The membranes were blocked at room temperature for 3 h in TBST (50 mM Tris-Cl, 150 mM NaCl, pH 7.6, 0.1% TWEEN 20) containing 5% nonfat milk and were then incubated overnight at 4 °C with the primary antibodies listed above. The membranes were rinsed with TBST 3 times for 10 min per rinse and then incubated for 2 h with HRP-conjugated secondary antibodies. Subsequently, the membranes were rinsed 3 times in TBST and then evenly covered with ECL chemiluminescence reagents for 1 min. The blots were exposed to X-ray film for radiographic detection of the bands. The autoradiograms were scanned, and the bands were quantified by densitometry using Quantity One from Bio-Rad (Hercules, California). Statistical analysis Data were expressed as the mean ± standard deviation (SD) and analyzed by the Student’s t test for 2 groups’ comparison and by 1-way ANOVA for multiple-groups analysis followed by Duncan’s post hoc test. p < .05 were considered statistically significant. RESULT MC-LR is Efficiently Transported Into SH-SY5Y Cells by EP, Increases Hyperphosphorylation of PP2A-Dependent Tau Sites, and Causes Cell Death Many cell types that lack OATPs are not sensitive to MC-LR. Therefore, a method to effectively transport MC-LR into these cell lines is crucial for evaluating MC-LR cytotoxicity. Endo-Porter, a peptide reagent that binds to the cell membrane, has been shown to facilitate delivery of MC-LR in the HepG2 and Jurkat cell lines (Jasionek et al., 2010). Therefore, in the present study, EP is used to transport MC-LR into SH-SY5Y cells. To examine the effective of EP at delivery, we analyzed the intracellular accumulation of MC-LR with an antibody against MC-LR after SH-SY5Y cells were incubated with MC-LR alone or with both MC-LR and EP. Because catalytic C subunit of PP2A and PP1 are the primary targets of MC-LR, adducts composed of MC-LR (1 kDa) bound to PP2Ac (36 kDa) and PP1 (37.5 kDa) are detected at 39 kDa on Western blots. We found that addition of EP significantly increased the quantity of accumulated MC-LR in cells, which suggested that EP is an effective reagent for the delivery of MC-LR into SH-SY5Y cells (Figs. 1A and 1B). Figure 1. Open in new tabDownload slide Accumulation of MC-LR in SH-SY5Y cells and its effects on tau phosphorylation and neuronal toxicity. MC-LR is transported into SH-SY5Y cells by EP. Cells were treated with one of 2 doses of MC-LR (5 or 10 μM) with or without EP for 24 h. Accumulation of MC-LR was detected by Western blotting using an antibody against MC-LR (A), and the quantity was normalized to GAPDH for analysis (B). MC-LR caused hyperphosphorylation of tau. Phosphorylation of tau at Ser202, Ser356, Ser262, Ser199, and Ser396 as well as total tau after MC-LR treatment was detected by Western blotting (C), and the quantity was normalized to total tau for analysis (D, E). MC-LR causes neuronal toxicity. Immunofluorescence staining using an antitau antibody (F, red), and an anti-PHF-tau antibody (F, green) in SH-SY5Y cells treated with MC-LR (10 μM) in the presence of EP (scale bar = 10 μm). Lactate dehydrogenase release was measured 24 h after MC-LR treatment (10 μM) in the presence of EP (G). Data are expressed as the mean ± SD for 3 separate experiments. *p < .05, **p < .01 versus the control. Figure 1. Open in new tabDownload slide Accumulation of MC-LR in SH-SY5Y cells and its effects on tau phosphorylation and neuronal toxicity. MC-LR is transported into SH-SY5Y cells by EP. Cells were treated with one of 2 doses of MC-LR (5 or 10 μM) with or without EP for 24 h. Accumulation of MC-LR was detected by Western blotting using an antibody against MC-LR (A), and the quantity was normalized to GAPDH for analysis (B). MC-LR caused hyperphosphorylation of tau. Phosphorylation of tau at Ser202, Ser356, Ser262, Ser199, and Ser396 as well as total tau after MC-LR treatment was detected by Western blotting (C), and the quantity was normalized to total tau for analysis (D, E). MC-LR causes neuronal toxicity. Immunofluorescence staining using an antitau antibody (F, red), and an anti-PHF-tau antibody (F, green) in SH-SY5Y cells treated with MC-LR (10 μM) in the presence of EP (scale bar = 10 μm). Lactate dehydrogenase release was measured 24 h after MC-LR treatment (10 μM) in the presence of EP (G). Data are expressed as the mean ± SD for 3 separate experiments. *p < .05, **p < .01 versus the control. Next, phosphorylation of serine (Ser) 202, Ser356, and Ser262 was analyzed relative to total tau using site-specific phospho-tau antibodies and the antitau antibody detecting total tau. We found that phosphorylation of Ser202, Ser356, and Ser262 increased with the concentration of accumulated MC-LR (Figs. 1C and 1D), confirming that the phosphorylation of these epitopes is regulated by PP2A. Phosphorylation of Ser199 and Ser396, however, did not increase in cells treated with MC-LR (Figs. 1C and 1E), which confirms previous observations that Ser396 and Ser199 are not efficient PP2A targets (Liu et al., 2005, 2007). It is known that abnormal hyperphosphorylation of tau is responsible for neuronal toxicity and loss of biological activity, which causes dissociation of tau from microtubules and further promotes its aggregation into PHFs in AD. We therefore examined whether MC-LR-induced hyperphosphorylation of tau would produce neuronal toxicity. Immunohistofluorescent analysis revealed that the addition of 10 μM MC-LR with EP caused translocation of tau protein from the neurites to the cell body as well as increased PHF-tau staining; correspondingly, neurites degeneration occurred in SH-SY5Y cells (Figure 1F). Using a LDH release assay, we also observed that 10 μM MC-LR with EP caused cell death (Figure 1G). Taken together, this evidence indicates that MC-LR-induced phosphorylation of tau promotes dissociation of tau from microtubules and aggregation of PHF-tau, which may consequently lead to neurites degeneration and cell death. MC-LR-Induced PP2A Inhibition Is Mainly Attributable to Demethylation of PP2Ac Considering that PP2A inhibition is 1 of the main events in MC-LR toxicity and is also the key player of tau phosphorylation, we then investigated whether accumulated MC-LR can inhibit PP2A activity. As shown in Figure 2A, the activity of PP2A was inhibited gradually from 52.7 ± 8.5 at 5 µM MC-LR to 39.8 ± 8.8 at 10 µM MC-LR and from 23.6 ± 6.7 at 5 µM MC-LR with EP to 11.6 ± 2.8 at 10 µM MC-LR with EP (Figure 2A). These results demonstrated that MC-LR induced a concentration-dependent inhibition of PP2A activity, which coincided with progressive uptake of MC-LR. Figure 2. Open in new tabDownload slide Effect of MC-LR on activity-related modifications of PP2A. A, Inhibition of PP2A activity. Cells were incubated with the indicated concentrations of MC-LR for 24 h. After incubation, intracellular PP2A activity was assessed by measuring phosphate release in picomoles as described in “Materials and methods”. The values are expressed as a percentage of the control (untreated cells; 100% PP2A activity). Alterations of various PP2A subunit levels. SH-SY5Y cells were treated with one of 2 doses of MC-LR (5 or 10 μM) with or without EP for 24 h. The levels of the PP2A A, B, Bα, and C subunits and of demethylated (DM-PP2Ac) and phosphorylated PP2Ac (p-PP2Ac) were detected by Western blot using corresponding antibodies (B), and the quantities were normalized to GAPDH (C) or PP2Ac for analysis (D). Alteration in binding of PP2Ac to Bα. An immunoprecipitation assay was performed in SH-SY5Y cell lysate using an anti-PP2Ac antibody, and the precipitated material was detected by Western blotting with PP2A-Bα (E). The amount of Bα associated with PP2Ac was normalized to immunoprecipitated PP2Ac (F). Data are expressed as the mean ± SD for 3 separate experiments. *p < .05, **p < .01 versus the control. Figure 2. Open in new tabDownload slide Effect of MC-LR on activity-related modifications of PP2A. A, Inhibition of PP2A activity. Cells were incubated with the indicated concentrations of MC-LR for 24 h. After incubation, intracellular PP2A activity was assessed by measuring phosphate release in picomoles as described in “Materials and methods”. The values are expressed as a percentage of the control (untreated cells; 100% PP2A activity). Alterations of various PP2A subunit levels. SH-SY5Y cells were treated with one of 2 doses of MC-LR (5 or 10 μM) with or without EP for 24 h. The levels of the PP2A A, B, Bα, and C subunits and of demethylated (DM-PP2Ac) and phosphorylated PP2Ac (p-PP2Ac) were detected by Western blot using corresponding antibodies (B), and the quantities were normalized to GAPDH (C) or PP2Ac for analysis (D). Alteration in binding of PP2Ac to Bα. An immunoprecipitation assay was performed in SH-SY5Y cell lysate using an anti-PP2Ac antibody, and the precipitated material was detected by Western blotting with PP2A-Bα (E). The amount of Bα associated with PP2Ac was normalized to immunoprecipitated PP2Ac (F). Data are expressed as the mean ± SD for 3 separate experiments. *p < .05, **p < .01 versus the control. Protein phosphatase 2A shows efficient enzymatic activity when PP2Ac is methylated at Leu309 and unphosphorylated at Tyr307. To understand the mechanism underlying MC-LR-induced inhibition of PP2A, we measured phosphorylation of PP2Ac at Tyr307, demethylation of PP2Ac at Leu309, and the levels of the PP2A A, B and C subunits. We found a dramatic increase in demethylation at Leu309 (inactivated form) and phosphorylation of PP2Ac at Tyr307 (inactivated form) (Figs. 2B and 2D), but we did not detect any significant changes in the level of the PP2A A, B, or C subunit in SH-SY5Y cells treated with MC-LR (Figs. 2B and 2C). Demethylation of PP2Ac has been proved to increase tyrosine phosphorylation of PP2Ac (Dudiki et al., 2015). We observed that the degree of PP2Ac demethylation was greater than phosphorylation compared with the control in the same MC-LR treatment group (Figure 2D), indicating that demethylation may promote phosphorylation of PP2Ac. Because the demethylation of PP2Ac at Leu309 interrupts the binding of PP2Ac to its regulatory subunit Bα and further results in a loss of Bα, we performed coimmunoprecipitation experiments using an antitotal PP2Ac antibody and analyzed the amount of Bα that is associated with PP2Ac in the absence or presence of both 10 μM MC-LR and EP. Addition of 10 μM MC-LR and EP led to a clear decrease in binding between PP2Ac and Bα (Figs. 2E and 2F). Concomitantly, we observed a remarkable decrease in PP2A-Bα (Figs. 2B and 2C). Taken together, these results indicate that demethylation of PP2Ac induced by MC-LR renders it incompetent in binding PP2A-Bα, resulting in the degradation of PP2A-Bα, and that demethylation may promote phosphorylation of PP2Ac and thus inhibits PP2A activity. MC-LR Causes Alterations of Tau Protein Kinases Regulated by PP2A Protein phosphatase 2A can regulate phosphorylation of tau directly and indirectly via the activation of several tau protein kinases that are regulated by it. We therefore measured levels of total and activated/inactivated forms of several PP2A-regulated protein kinases, such as GSK-3β and members of the mitogen-activated protein kinase family (JNK and p38). As shown in Figures 3A and 3B, exposure to MC-LR resulted in an increase in JNK phosphorylation at Thr183/Tyr185 (activatory phosphorylation) and P38 phosphorylation at Thr180/Tyr182 (activatory phosphorylation). To our surprise, MC-LR treatment increased the level of GSK-3β phosphorylated at Ser9, which is known to be an inactive form of the enzyme (Figs. 3A and 3B). Figure 3. Open in new tabDownload slide Effect of MC-LR on tau protein kinase. A, Phosphorylation of JNK at Thr183/Tyr185 and total JNK, phosphorylation of P38 at Thr180/Tyr182 and total P38, phosphorylation of GSK-3β at Ser9 and total GSK-3β, or phosphorylation of β-catenin at Ser33/Ser37/Thr41 and total catenin after MC-LR treatment were detected by Western blot. Corresponding densitometric analyses of the levels of phosphorylated JNK, P38, GSK-3β (B) and β-catenin (C) were normalized to total protein. Data are expressed as the mean ± SD for 3 separate experiments. *p < .05, **p < .01 versus the control. Figure 3. Open in new tabDownload slide Effect of MC-LR on tau protein kinase. A, Phosphorylation of JNK at Thr183/Tyr185 and total JNK, phosphorylation of P38 at Thr180/Tyr182 and total P38, phosphorylation of GSK-3β at Ser9 and total GSK-3β, or phosphorylation of β-catenin at Ser33/Ser37/Thr41 and total catenin after MC-LR treatment were detected by Western blot. Corresponding densitometric analyses of the levels of phosphorylated JNK, P38, GSK-3β (B) and β-catenin (C) were normalized to total protein. Data are expressed as the mean ± SD for 3 separate experiments. *p < .05, **p < .01 versus the control. To further assess MC-LR-induced changes in GSK-3β activity, we analyzed MC-LR-induced phosphorylation of β-catenin, a well-known GSK-3β substrate. GSK-3β is well known to phosphorylate β-catenin at Ser33, Ser37, and Thr41 and thereby facilitates its degradation by the proteasome (D'Mello et al., 2017; Valério et al., 2016). We found no significant change in the level of phosphorylated β-catenin between control and MC-LR-treated cells (Figs. 3A and 3C). Furthermore, we observed that MC-LR treatment did not affect the phosphorylation of tau at Ser396 or Ser199 (Figs. 1C and 1E), which are target sites for GSK-3β (Liu et al., 2007). These data indicated that MC-LR globally induces GSK-3β inactivation. Demethylation of PP2Ac Caused by MC-LR Is Related Closely to GSK-3β Inactivation To determine the functional interaction of PP2Ac and GSK-3β, we knocked down PP2Ac using a siRNA and then measured the phosphorylation of GSK-3β. We found that knockdown of PP2Ac increased phosphorylation of GSK-3β at Ser9 significantly (Figs. 4A and 4B), confirming the regulation of GSK-3β phosphorylation at Ser9 by PP2Ac. Next, we preincubated cells with the GSK-3β inhibitor AR-A014418 before treatment with MC-LR, then measured the demethylation of PP2Ac and the total level of PP2Ac. The Western blot results showed that the level of DM-PP2Ac was significantly increased in the cells treated with AR-A014418 (Figs. 4C and 4D). More importantly, we found that the level of DM-PP2Ac was significantly greater in the cells receiving MC-LR combined with AR than in the MC-LR group, without any change in the total PP2Ac (Figs. 4C and 4D), which indicated that inhibition of GSK-3β can promote MC-LR-induced demethylation of PP2Ac. Figure 4. Open in new tabDownload slide GSK-3β interacts with PP2Ac. The inhibition of PP2Ac promoted phosphorylation of GSK-3β at Ser9. SH-SY5Y cells were transfected with an siRNA against PP2Ac for 48 h. Levels of PP2Ac and pSer9-GSK-3β were determined by Western blots (A) and normalized to total GAPDH and GSK-3β, respectively (B). Inhibition of GSK-3β enhanced demethylation of PP2Ac induced by MC-LR. SH-SY5Y cells were treated with 20 μM AR-A014418 for 4 h and then treated with 10 μM MC-LR for 24 h. Levels of DM-PP2Ac and PP2Ac were measured by Western blots (C). The level of DM-PP2Ac was normalized to total PP2Ac (D). Data are expressed as the mean ± SD for 3 separate experiments. **p < .01 versus the control. ##p < .01 versus MC-LR group. E, DM-PP2Ac colocalized with p-GSK-3β. The immunoreactivity levels of demethylated PP2Ac (green) and GSK-3β phosphorylated at Ser9 (red) were examined by double immunofluorescent staining. The nuclei were stained with DAPI. Scale bar = 25 μm. Figure 4. Open in new tabDownload slide GSK-3β interacts with PP2Ac. The inhibition of PP2Ac promoted phosphorylation of GSK-3β at Ser9. SH-SY5Y cells were transfected with an siRNA against PP2Ac for 48 h. Levels of PP2Ac and pSer9-GSK-3β were determined by Western blots (A) and normalized to total GAPDH and GSK-3β, respectively (B). Inhibition of GSK-3β enhanced demethylation of PP2Ac induced by MC-LR. SH-SY5Y cells were treated with 20 μM AR-A014418 for 4 h and then treated with 10 μM MC-LR for 24 h. Levels of DM-PP2Ac and PP2Ac were measured by Western blots (C). The level of DM-PP2Ac was normalized to total PP2Ac (D). Data are expressed as the mean ± SD for 3 separate experiments. **p < .01 versus the control. ##p < .01 versus MC-LR group. E, DM-PP2Ac colocalized with p-GSK-3β. The immunoreactivity levels of demethylated PP2Ac (green) and GSK-3β phosphorylated at Ser9 (red) were examined by double immunofluorescent staining. The nuclei were stained with DAPI. Scale bar = 25 μm. To further demonstrate the relevance of PP2Ac demethylation and GSK-3β phosphorylation, we conducted colabeling studies using a confocal microscope. Compared with the control, the immunoreactivity levels of demethylated PP2Ac (green, Figure 4E) and Ser9-phosphorylated GSK-3β (red, Figure 4E) were increased in SH-SY5Y cells treated with both 10 µM MC-LR and 10 µM MC-LR with EP; concomitantly, the demethylated PP2Ac was colocalized with the phosphorylated GSK-3β (yellow, Figure 4E). These data together strongly suggested the existence of an interaction between phosphorylation of GSK-3β at Ser9 and demethylation of PP2Ac induced by MC-LR in SH-SY5Y cells. MC-LR-Induced PP2Ac Demethylation Causes Dissociation of B55α From Tau and Consequent Hyperphosphorylation of Tau To validate the role of B55α in MC-LR-induced tau hyperphosphorylation, tau phosphorylation state was analyzed in SH-SY5Y cells with reduced B55α protein levels. Transfection of SH-SY5Y cells with B55α siRNA led to a reduction of 50% in B55α protein in immunoblots, when compared with scrambled siRNA, and B55α siRNA elevated the phosphorylation levels of tau at Ser202 in the absence of MC-LR treatment (Figs. 5A and 5B). Furthermore, the Western blot results showed that phosphorylation of tau at Ser202 was significantly elevated in cells treated with B55α siRNA and MC-LR compared with those receiving only MC-LR, whereas the total tau level remained constant (Figs. 5A and 5B). This result demonstrated a crucial role of B55α in tau phosphorylation induced by MC-LR. Figure 5. Open in new tabDownload slide Impact of PP2A/B55α on MC-LR-induced tau phosphorylation at Ser202. Knockdown of B55α promotes MC-LR-induced tau phosphorylation. SH-SY5Y cells were transfected with B55α siRNA or scrambled siRNA for 48 h and then incubated in the presence (+) or absence (−) of 10 µM MC-LR for 24 h. The levels of B55α and phosphorylated tau in the cell lysates were measured by Western blots (A). The levels of B55α and phosphorylated tau were normalized to GAPDH and total tau, respectively. Data are expressed as the mean ± SD for 3 separate experiments. **p < .01 versus the control. ##p < .01 versus MC-LR group (B). (C) Tau colocalized with B55α. Tau, red channel; B55α, green channel; merged images show the colocalization of tau and B55α. The nuclei were stained with DAPI. Scale bar = 10 μm. Figure 5. Open in new tabDownload slide Impact of PP2A/B55α on MC-LR-induced tau phosphorylation at Ser202. Knockdown of B55α promotes MC-LR-induced tau phosphorylation. SH-SY5Y cells were transfected with B55α siRNA or scrambled siRNA for 48 h and then incubated in the presence (+) or absence (−) of 10 µM MC-LR for 24 h. The levels of B55α and phosphorylated tau in the cell lysates were measured by Western blots (A). The levels of B55α and phosphorylated tau were normalized to GAPDH and total tau, respectively. Data are expressed as the mean ± SD for 3 separate experiments. **p < .01 versus the control. ##p < .01 versus MC-LR group (B). (C) Tau colocalized with B55α. Tau, red channel; B55α, green channel; merged images show the colocalization of tau and B55α. The nuclei were stained with DAPI. Scale bar = 10 μm. Given that PP2A/B55α can directly bind to tau via a domain responsible for the microtubule-binding properties of tau, resulting in tau dephosphorylation, we examined whether reducing PP2A methylation by MC-LR, and consequently decreasing the binding of PP2Ac with B55α, would disrupt normal PP2A/B55α-tau interactions. In untreated cells, double immunofluorescence showed that a large proportion of PP2A/B55α (green) was well colocalized with tau (red), implying the importance of PP2A/B55α in the regulation of tau phosphorylation (Figure 5C). However, compared with the controls, the immunoreactivity of B55α was dramatically reduced, and PP2A/B55α and tau showed visibly abated colocalization in SH-SY5Y cells treated with 10 µM MC-LR with EP, indicating the disruption of PP2A/B55α-tau interactions by MC-LR (Figure 5C). MC-LR Injection Induces an Increase in Demethylation of PP2Ac and Phosphorylation of GSK-3β and Tau in the Rat Hippocampus To validate our observations obtained from cultured cells, we administered MC-LR bilaterally into rat hippocampi and first ascertained the accumulation of MC-LR in hippocampus. Western blotting analysis, using an antibody against MC-LR, indicated that MC-LR injection at 0.1 μg/μl for 24 h in rats resulted in accumulation in the hippocampus (Figure 6B). Next, we examined the effect of MC-LR injection on demethylation of PP2Ac as well as phosphorylation of GSK-3β at Ser9 and tau at Ser202. As shown in Figures 6C and 6D, demethylation of PP2Ac and phosphorylation of GSK-3β at Ser9 were increased markedly in MC-LR-injected rats compared with vehicle-injected rats, whereas their total protein levels remained unchanged (Figs. 6C and 6D). Correspondingly, phosphorylation of tau at Ser202 was found to be dramatically elevated after MC-LR injection as detected by Western blotting assays (Figs. 6C and 6D). Figure 6. Open in new tabDownload slide Effect of MC-LR on PP2A signaling in the rat hippocampus. Bilateral injections of MC-LR and accumulation in hippocampus. Microinjections were given in the bilateral dorsal hippocampus (A). Rats were injected with either 3 μl of MC-LR (0.1 μg/μl) or saline only. Accumulation of MC-LR in the hippocampus was detected by a Western blot using antibodies against MC-LR (B). Demethylation of PP2Ac and phosphorylation of GSK-3β and tau in rat hippocampi injected with MC-LR. Demethylation of PP2Ac (DM-PP2Ac) and phosphorylation of GSK-3β at Ser9 (p-GSK-3β) and tau at Ser202 (P-tau) after MC-LR injection were detected by Western blotting (C), and the quantities were normalized to total PP2Ac, GSK-3β and tau, respectively, for analysis (D). *p < .05, **ρ < 0.01 versus the control. E, Immunofluorescence staining using an anti-PHF-tau antibody (green) in the hippocampal CA3 (scale bar = 100 μm). Figure 6. Open in new tabDownload slide Effect of MC-LR on PP2A signaling in the rat hippocampus. Bilateral injections of MC-LR and accumulation in hippocampus. Microinjections were given in the bilateral dorsal hippocampus (A). Rats were injected with either 3 μl of MC-LR (0.1 μg/μl) or saline only. Accumulation of MC-LR in the hippocampus was detected by a Western blot using antibodies against MC-LR (B). Demethylation of PP2Ac and phosphorylation of GSK-3β and tau in rat hippocampi injected with MC-LR. Demethylation of PP2Ac (DM-PP2Ac) and phosphorylation of GSK-3β at Ser9 (p-GSK-3β) and tau at Ser202 (P-tau) after MC-LR injection were detected by Western blotting (C), and the quantities were normalized to total PP2Ac, GSK-3β and tau, respectively, for analysis (D). *p < .05, **ρ < 0.01 versus the control. E, Immunofluorescence staining using an anti-PHF-tau antibody (green) in the hippocampal CA3 (scale bar = 100 μm). Immunofluorescence confocal microscopy was used to further explore the in situ distribution of tau phosphorylation. As displayed in Figure 6E, the immunoreactivity of PHF-tau (Ser202/Thr205) was significantly increased in the hippocampal neurons of the MC-LR-injected rats compared with the control subjects. Together, these results revealed that the effect of MC-LR on PP2A signaling in vivo in the rat brain was similar to the effect we observed in SH-SY5Y cells. In Vivo Bilateral Injection of MC-LR Into Rat Hippocampi Leads to a Spatial Memory Deficit To analyze the effects of MC-LR on spatial memory, we subjected the rats to the MWM task. During the acquisition trials (5 days), each group showed improvements in their performances across days and took progressively less time to find the hidden platform (Figure 7A). In the retention session, the time spent in the target quadrant and the ratio of swimming distance in the target quadrant to the total distance were used to estimate performance. The MWM test showed that there was no difference in time or percentage of swimming distance spent in the target quadrant between the control group on day 6 (before saline injection) and the control group on day 8 (after saline injection). However, compared with day 6 in the MC-LR group, the time and percentage of swimming distance spent in the target quadrant were significantly reduced after MC-LR injection on day 8 (Figs. 7B and 7C). Similarly, typical swimming paths on day 8 indicate that rats in the MC-LR group displayed a weak ability to search for the removed platform after MC-LR microinjection, but the motion trails of the control rats did not change (Figure 7D). Overall, the behavioral data obtained in the MWM test demonstrated that intrahippocampal injection of MC-LR is able to induce memory impairment in rats. Figure 7. Open in new tabDownload slide Effect of MC-LR on spatial memory of rats. Morris water maze task on day 6 (before injection) and day 8 (after injection) for rats in the control group and the MC-LR group. The latency to find the platform was used to assess learning ability (A). Time spent in the target quadrant (B). Ratio of the swimming distance in target quadrant to the total distance for each group (C). Representative spatial search path (D). Data are expressed as the mean ± SD. **p < .01 versus MC-LR group on day 6. Figure 7. Open in new tabDownload slide Effect of MC-LR on spatial memory of rats. Morris water maze task on day 6 (before injection) and day 8 (after injection) for rats in the control group and the MC-LR group. The latency to find the platform was used to assess learning ability (A). Time spent in the target quadrant (B). Ratio of the swimming distance in target quadrant to the total distance for each group (C). Representative spatial search path (D). Data are expressed as the mean ± SD. **p < .01 versus MC-LR group on day 6. DISCUSSION The present study demonstrated the neurotoxic potential of MC-LR in vitro and in vivo, characterized by neurites disintegration and cell death in the SH-SY5Y cell line as well as spatial learning and memory impairment in rats. Using SH-SY5Y cells and hippocampal tissues, we showed that MC-LR induced PP2A demethylation, GSK-3β inactivation and tau hyperphosphorylation at PP2A-dependent epitopes in vitro and in vivo. Specifically, we revealed that the demethylation of PP2Ac was associated with GSK-3β and contributed to the dissociation of B55α from PP2Ac, which resulted in B55α degradation and consequent hyperphosphorylation of tau in MC-LR-induced neurotoxicity. Based on the research of Jasionek et al. (2010) in which EP has proved to be the most suitable transporter for MC-LR, we confirmed that EP facilitated the delivery of MC-LR into SH-SY5Y cells by comparing the quantities of accumulated MC-LR and its cytotoxic actions such as PP2A inhibition and tau hyperphosphorylation in SH-SY5Y cells incubated with MC-LR in the presence of EP and with MC-LR alone. It is well accepted that the toxicity of MC-LR leads to a specific and highly effective inhibition of PP2A activity, which has been suggested to responsible for the abnormal tau phosphorylation and aggregation linked to AD (D'Mello et al., 2017; Valério et al., 2016). The present results also show a marked increase in the abnormal hyperphosphorylation of tau at Ser 202, Ser 359, and Ser262 with a concomitant inhibition of PP2A activity in MC-LR-induced SH-SY5Y cells. All these phosphorylation sites are known to be involved in PP2A-dependent epitopes (Liu et al., 2005, 2007). Furthermore, PP2A inhibition induced by MC-LR did not affect phosphorylation of tau at Ser199 and Ser396, which are not favorable sites for dephosphorylation by PP2A (Liu et al., 2005, 2007). The correlative changes both PP2A and tau implied that the inhibition of PP2A induced by MC-LR is a critical factor in tau hyperphosphorylation. However, we cannot exclude the possibility that the effect of MC-LR on tau phosphorylation results from its inhibition of other protein phosphatases, including PP1, PP4, and PP5, which are potent targets of MC-LR action (Swingle et al., 2007). Nonetheless, PP2A has been shown to be the major tau phosphatase in the brain and accounts for approximately 70% of the total tau phosphatase activity, whereas PP1, PP2B, and PP5 each account for only approximately 10% of the total tau phosphatase activity (Liu et al., 2005). Additionally, Ser262 was hyperphosphorylated in our study, but is not a preferred site for PP1 and PP5, and Ser356 is not a site for dephosphorylation by PP1 and PP5 (Liu et al., 2006). Therefore, based on the results from the present study, MC-LR-induced tau phosphorylation might mainly result from the inhibition of PP2A activity. Ser202, Ser262, and Ser356 are among the critical phosphorylation sites that convert tau to a toxic molecule that sequesters normal microtubule-associated proteins from microtubules (Liu et al., 2006). The breakdown of the microtubule network in the affected neurons compromises axonal transport and leads to retrograde degeneration, which, in turn, results in neuronal death. Indeed, we also observed that MC-LR produced neurotoxicity, characterized by translocation of tau protein from the axons to the cell body, disintegration of the axon, and eventual cell death. Thus, taken together, these results suggest that MC-LR is potent a neurotoxin that can be delivered into cells by EP, where it targets PP2A, causing hyperphosphorylation of PP2A-dependent tau sites and consequent axon degradation as well as cell death. On the basis of the key role that PP2A inhibition plays in tau hyperphosphorylation, elucidating the primary events that underlie PP2A inhibition is important for determining the possible causes of the tau hyperphosphorylation induced by MC-LR. Methylation of the PP2A C-terminal L309 indirectly activates PP2A activity by controlling the binding of regulatory B subunits to AC dimmers, and the demethylation of the PP2A C-terminal L309 has an opposite effect (Tolstykh et al., 2000). Protein phosphatase 2A methylation is decreased and PP2A demethylation is increased in the brains of patients with AD. Moreover, the low activity of PP2A observed in the brains of patients with AD may be also partially related to the decrease in PP2A methylation (Janssens et al., 2008; Zhou et al., 2008). In the present study, we found that the inhibition of PP2A activity could mainly be attributed to the demethylation of PP2Ac in SH-SY5Y cells treated with MC-LR. Consistent with our work in vitro, the study in vivo also showed that demethylated PP2Ac was increased, with concomitant tau hyperphosphorylation and memory deficits in rats injected with MC-LR. Considering the results of our present study and others reports concerning the important roles of PP2A methylation in regulating PP2A activity towards tau in subjects with AD, we chose to focus on assessing the potential role of PP2Ac demethylation in MC-LR-mediated tau phosphorylation and toxicity. Protein phosphatase 2A methylation is thought to play a critical role in modulating the biogenesis and stabilization of PP2A/Bα, a heterotrimer containing the catalytic C, scaffolding A, and regulatory Bα subunits. Many groups have reported that the methylation of PP2Ac promotes the binding of Bα to the PP2A core enzyme and thus increases the activity of PP2A, whereas mutant methylation defects of PP2Ac and down-regulation of methylated PP2Ac expression levels interfere with the ability of PP2Ac to associate with Bα, leading to a loss of Bα, which is unstable as a monomer and becomes targeted for degradation (Janssens et al., 2008; Longin et al., 2007; Nunbhakdi-Craig et al., 2007; Sontag et al., 2008). In line with these other reports, this study also found that demethylation of PP2Ac induced by MC-LR dissociated Bα from PP2Ac, thus resulting in Bα degradation. PP2A/Bα is the primary PP2A isoform that mediates tau dephosphorylation (Sontag et al., 1996, 1999; Xu et al., 2008). Specific inhibition of PP2A/Bα is connected with tau hyperphosphorylation at many AD-like phosphoepitopes and the consequent inability of tau to bind to and stabilize microtubules (Sontag et al., 1996). In the present study, we first demonstrated the importance of Bα in MC-LR-induced phosphorylation of tau, and we subsequently used an immunofluorescence approach to confirm the disruption of PP2A/Bα-tau interactions by MC-LR. Taken together, the results of the present study suggest that demethylation of PP2Ac facilitates a series of biochemical events including dissociation of Bα from PP2Ac, the disruption of PP2A/Bα-Tau interactions and tau hyperphosphorylation. Several studies have indicated that increased GSK-3β activity is critical in the pathogenesis of AD, mediating hyperphosphorylation of tau and aggregation of the protein into NFT, impairment of neurogenesis, alterations in synaptic plasticity and memory deficits, all of which are observed in the AD brain (Medina and Avila, 2013, 2014). However, we found that MC-LR treatment increased the level of GSK-3β phosphorylated at Ser9 (inactivated form) and caused a dramatic increase in tau phosphorylation at PP2A-sensitive and GSK-3β-insensitive sites, such as Ser 202, 262, and 356, but no increase at GSK-3β-sensitive and PP2A-insensitive sites including Ser199 and Ser396. In line with in vitro observations, elevated phosphorylation of GSK-3β at Ser9 and tau at Ser202 was observed in the rat hippocampus after injection with MC-LR. The conflict between the several suggested roles played by GSK-3β in AD pathogenesis and the status of GSK-3β inactivation in the present study encouraged us to investigate whether GSK-3β is involved in MC-LR-induced phosphorylation of PP2A-dependent tau sites. Using pharmacological and molecular biological approaches, we found that inhibition of PP2A may lead to an increase in GSK-3β phosphorylation at Ser9 and a decrease in GSK-3β activity, which may further promote PP2A demethylation induced by MC-LR. Furthermore, we used an immunofluorescence approach to confirm that the PP2Ac demethylation was well colocalized with GSK-3β phosphorylation at Ser9. Therefore, the present study demonstrated that dysregulation of GSK-3β is involved in MC-LR neurotoxicity by its interaction with PP2A demethylation. The finding is supported by other studies that found increased levels of GSK-3β phosphorylated at Ser9 in postmortem samples (Ferrer et al., 2002; Swatton et al., 2004). Based on the results of the current study, tau hyperphosphorylation corresponds to an increase in PP2A demethylation and deregulated GSK-3β phosphorylation in MC-LR-induced neurotoxicity in SH-SY5Y cells and the rat brain. These findings are consistent with earlier study reporting that OA, a PP2A inhibitor similar to MC-LR and an experiment tool to study the mechanism of AD pathology (Kamat et al., 2013, 2014), induced a significant increase in PP2A demethylation, GSK-3β phosphorylation at Ser9 and tau phosphorylation at PHF-1 sites in wild-type and APPswe N2a neuroblastoma cells (Zhou et al., 2008), as well as metabolically active rat brain slices (Wang et al., 2015). The similarity of MC-LR and OA actions suggests a common mechanism in which PP2A demethylation may be one of the key initial factors that reduces PP2A activity, subsequently comprising its capacity to dephosphorylate tau. Moreover, deregulated PP2A activity results in increased GSK-3β phosphorylation at Ser9, which may further promote PP2A demethylation and decrease PP2A activity. In conclusion, cyanobacterial toxin MC-LR exerts its potential neurotoxicity through the PP2A demethylation and associated GSK-3β phosphorylation that cause the dissociation of Bα from PP2Ac, resulting in Bα degradation to promote Tau phosphorylation at PP2A-dependent sites and PHF-Tau accumulation and, consequently, neurites degeneration and cell death (Figure 8). Our novel findings shed light on the underlying mechanism of MC-LR-induced neurotoxicity. Future experiments are needed to identify the critical role of demethylated PP2Ac in an animal model of tauopathies induced by MC-LR. Figure 8. Open in new tabDownload slide A proposed schematic representation of a possible mechanism leading to the neurotoxicity of MC-LR. MC-LR-mediated demethylation of PP2Ac was associated with GSK-3β phosphorylation at Ser9 and contributed to the dissociation of Bα from PP2Ac. This dissociation resulted in Bα degradation and disruption of PP2A/Bα-tau interactions, thus promoting tau hyperphosphorylation at PP2A-dependent sites and PHF-tau accumulation and, consequently, neurites degeneration and cell death. Phosphorylation of GSK-3β at ser9 is not directly but indirectly involved in MC-LR-induced tau phosphorylation at PP2A-dependent sites by its interaction with PP2A demethylation. Figure 8. Open in new tabDownload slide A proposed schematic representation of a possible mechanism leading to the neurotoxicity of MC-LR. MC-LR-mediated demethylation of PP2Ac was associated with GSK-3β phosphorylation at Ser9 and contributed to the dissociation of Bα from PP2Ac. 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TI - Microcystin-Leucine-Arginine Induces Tau Pathology Through Bα Degradation via Protein Phosphatase 2A Demethylation and Associated Glycogen Synthase Kinase-3β Phosphorylation
JO - Toxicological Sciences
DO - 10.1093/toxsci/kfx271
DA - 2018-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/microcystin-leucine-arginine-induces-tau-pathology-through-b-62mkADtxU6
SP - 475
VL - 162
IS - 2
DP - DeepDyve
ER -