Phosphatase 2A Inhibition Affects Endoplasmic ...

Mol Neurobiol DOI 10.1007/s12035-015-9640-1

Phosphatase 2A Inhibition Affects Endoplasmic Reticulum and Mitochondria Homeostasis Via Cytoskeletal Alterations in Brain Endothelial Cells Ana I. Plácido 1,2 & Cláudia M. F. Pereira 1,2 & Sónia C. Correira 1,3 & Cristina Carvalho 1,3 & Catarina R. Oliveira 1,2 & Paula I. Moreira 1,2

Received: 30 July 2015 / Accepted: 15 December 2015 # Springer Science+Business Media New York 2016

Abstract The loss of endothelial cells (ECs) homeostasis is a trigger for cerebrovascular dysfunction that is a common event in several neurodegenerative disorders such as Alzheimer’s disease (AD). The present work addressed the role of phosphatase 2A (PP2A) in cytoskeleton rearrangement, endoplasmic reticulum (ER) homeostasis, ER–mitochondria communication and mitochondrial dynamics in brain ECs. For this purpose, rat brain endothelial (RBE4) cells were exposed to okadaic acid, a well-known inhibitor of PP2A activity. An increase in the levels of tau phosphorylated on Ser396 and Thr181 residues was observed upon PP2A inhibition, concomitantly with the rearrangement of microtubules and actin cytoskeleton. Under these conditions, an increase in the levels of ER stress markers, namely GRP78, XBP1, peIF2αSer51, and ERO1α, was observed. Moreover, PP2A inhibition upregulated the Sigma-1 receptor, an ER chaperone located at the ER–mitochondria interface, and enhanced interorganelle Ca2+ transfer, culminating in mitochondrial Ca2+ overload and activation of mitochondria-dependent apoptosis. The inhibition of PP2A activity also promoted an alteration of the structural and spatial mitochondria network due to upregulation of mitochondrial fission (Drp1 and Fis1) and fusion

* Cláudia M. F. Pereira [email protected] * Paula I. Moreira [email protected]; [email protected]

1

CNC—Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3004-504, Portugal

2

Faculty of Medicine, University of Coimbra, Coimbra 3000-548, Portugal

3

Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal

(Mfn1, Mfn2 and OPA1) proteins, suggesting detrimental changes in mitochondrial dynamics. In accordance with our in vitro observations, brain vessels from 3xTg-AD mice showed a significant decrease in PP2A protein levels accompanied by an increase in tau phosphorylated on Ser396 and GRP78 protein levels. Collectively, these results suggest that the loss of cerebrovascular homeostasis that occurs in AD might be a downstream event of the compromised activity and/or expression of PP2A, which is observed in the brain of individuals affected with this devastating neurodegenerative disorder. Keywords Protein phosphatases 2A . Endoplasmic reticulum stress . Mitochondria dynamics . Cytoskeleton

Introduction Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine threonine (Ser/Thr) phosphatase that regulates the function of microtubule (MTs)-associated proteins, such as tau, supporting that PP2A plays a pivotal role in the stability of the cytoskeleton [1, 2]. In endothelial cells (ECs), this phosphatase is present in MTs-enriched fractions [3] and its inhibition was shown to potentiate the effect of nocodazole on transendothelial electrical resistance and to change the organization of the cytoskeleton, leading to MTs dissolution and actin rearrangements suggesting that PP2A have a pivotal role in regulation of ECs function and barrier properties [3–5]. Cytoskeletal perturbations can affect organelle homeostasis, namely of the endoplasmic reticulum (ER) [6] and mitochondria [7], as well as inter-organelle crosstalk through mitochondria-associated ER membranes (MAMs), which allows their mutual regulation [8, 9]. Early ER stress was demonstrated to increase the mitochondrial and reticular network

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in a MTs-dependent manner, thereby promoting mitochondrial respiration and bioenergetics [10, 11]. However, mitochondrial metabolism is compromised under prolonged ER stress [10, 11]. Due to their pivotal function as powerhouses of the cells, mitochondria are highly dynamic organelles that continuously repeat cycles of fission and fusion that determine mitochondrial morphology and size [12, 13]. Despite the role of ER in mitochondria fission lacks clarity, recent data suggest that mitochondrial fission occurs preferentially at ER contact sites enriched in the inverted forming 2 (INF2), a protein that accelerates both actin polymerization and depolymerisation [7, 14]. The role of PP2A in mitochondria fission/fusion events is presently controversial. According to Merrill and colleagues, PP2A inhibition is a trigger for mitochondria fusion [15]. However, Cho and collaborators demonstrated that the inhibition of PP2A by okadaic acid (OA) enhances mitochondrial fission [16]. Taking into account that brain ECs are highly enriched in mitochondria and the fact that cytoskeleton perturbations impair mitochondria function and their communication with the ER, it is not surprising that these events lead to ECs dysfunction and microvascular abnormalities such as those observed in Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS) [17–19]. These microvascular alterations decrease cerebral blood flow, lead to an inefficient supply of oxygen, energetic substrates and nutrients to neuronal and glial cells, and impair clearance of neurotoxins that accumulate in the brain’s interstitial fluid [20]. Based on the above findings and considering that the integrity of ECs is crucial to the cerebrovascular homeostasis, we hypothesized that destabilization of the cytoskeleton through the inhibition of PP2A, perturbs the ER–mitochondria communication, which in turn causes ER stress and altered mitochondrial dynamics, culminating in ECs dysfunction and death. In this study, we demonstrated that PP2A inhibition promotes the rearrangement of actin filaments as well as the destabilization of MTs, and causes ER and oxidative stress, deregulates ER and mitochondria Ca2+ homeostasis and affects the levels of proteins that modulate mitochondrial fission and fusion, culminating in mitochondrial fragmentation and activation of mitochondria-mediated apoptotic cell death in brain ECs. Our in vitro observations are supported by ex vivo studies demonstrating that brain vessels from triple transgenic mice for AD (3xTg-AD) are characterized by ER stress, a phenomenon associated with lower protein levels of PP2A and increased phosphorylation of tau.

Material and Methods Fetal bovine serum (FBS), geneticin (G480), HAM’s F-10, and MEM-alpha medium with Glutamax-1 (α-MEM) were purchased from Gibco-Invitrogen (Grand Island, NY, USA).

Fura2-acetoxymethyl ester (Fura2-AM), rhodamine2acetoxymethyl ester (Rhod2-AM), alexa fluor-488 phalloidin, and dihydroethidium (DHE) were purchased from Molecular Probes-Invitrogen (Grand Island, NY, USA). Basic fibroblast growth factor (bFGF), anti-α-tubulin, anti-β-actin and antiacetylated-α-tubulin antibodies, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) and Pluronic® F-127, cytochalasin B, paclitaxel (taxol), and nocodazole were obtained from Sigma (St. Louis, MO, USA). Rat-tail collagen was purchased from Roche Diagnostics (Mannheim, Germany). Anti-TOM 20, anti-phospho-Tau (Ser396), antiphospho-Tau (Thr181), anti-total Tau, anti-Mfn1, anti-Mfn2, anti-phospho-eIF2α, anti-total eIF2α, anti-ERO1α, and antiSigma-1R antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-GRP78 and anti-cytochrome c antibodies, N-acetyl-Asp-Glu-ValAsp-p-nitroanilide (Ac-DEVD- pNA) Ac-Leu-His-Asp-pnitroanilide (Ac-LEHD-pNA) colorimetric caspase substrates were obtained from Calbiochem, Merck KGaA (Darmstadt, Germany). Anti-phospho-DRP1 (Ser 616) antibody was obtained from Cell Signaling Technology (Danvers, MA, USA), anti-total DRP1, and anti-OPA1 antibodies were obtained from BD Biosciences (San Jose, USA), anti-Fis1 antibody was obtained from Imgenex (Colorado, USA) and antiXBP1 was obtained from Abcam (Cambridge, USA). All the other chemicals were of the highest grade of purity that is commercially available. Cell Culture and Treatments The rat brain endothelial cell line (RBE4) was kindly provided by Dr. Jon Holy (University of Minnesota, Duluth, MN, USA) and cultured as previously described [21]. For plating, the number of viable cells in suspension was quantified by counting trypan blue-excluding cells in a hemocytometer chamber. Cells were used for experiments 1 day after plating (80 % confluency) and were treated for 6 and 24 h with OA (5–15 nM), which was added from a 5-mM stock prepared in ethanol. To modulate cytoskeleton dynamics, RBE4 cells were treated for 24 h with 10 nM taxol, 100 nM nocodazole, and 10 μg/mL cytochalasin B. For all experimental procedures, controls were performed in the absence of those agents. Assessment of Cell Viability Cell viability was analyzed using the MTT assay [22]. Control and treated cells were washed with Krebs buffer [(in millimolar): 132 NaCl, 4 KCl, 1.2 NaH2PO4, 1.4 MgCl2, 6 glucose, 10 HEPES, and 1 CaCl2 (pH 7.4)] and incubated with MTT (0.5 mg/ml) for 3 h at 37 °C. The blue formazan crystals formed were dissolved in an equal volume of 0.04 M HCl in isopropanol and quantified spectrophotometrically by measuring the absorbance at 570 nm using a microplater reader

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(SpectraMax Plus 384, Molecular Devices, San Francisco, CA, USA). Results were expressed as the percentage of the absorbance determined in control cells. Measurement of Calcium Levels Reticular Ca2+ levels were measured using a spectrofluorometric assay, as previously described [22]. Briefly, control and treated RBE4 cells were washed twice in Krebs buffer and loaded with 10 μM Fura2-AM supplemented with 0.02 % (v/v) pluronic acid (Pluronic® F-127) and 1 % (w/v) BSA in Krebs buffer for 45 min at 37 °C. Thereafter, cells were washed in Ca2+-free Krebs buffer and incubated for 30 min in medium free of Ca2+ and of dye to allow the hydrolysis of the acetoxymethylester. Ca2+-induced Fura2 fluorescence was recorded upon excitation at both 340 and 380 nm using a temperature-controlled SPEX 1681 Fluorolog spectrofluorometer (HORIBA, Jobin Yvon Inc., Edison, NJ, USA). Cells were stimulated with thapsigargin (5 μM) in the absence of extracellular Ca2+, to empty the ER store. The peak amplitude of Fura2 fluorescence (340/380 ratio) upon stimulation was used to evaluate ER Ca2+ levels. To monitor mitochondrial Ca2+ content, cells were incubated at 37 °C with 10 μM of the fluorescent membrane permeable probe Rhod2-AM and Ca2+-induced Rhod2 fluorescence was measured (581-nm excitation and 581-nm emission). Mitochondrial maximal Ca2+ uptake was assessed by challenging cells with the Ca2+ ionophore A23187 (5 μM), as described previously [23]. The peak amplitude of Rhod2 fluorescence upon stimulation was used to evaluate mitochondrial Ca2+ levels. After ER and mitochondrial Ca2+ measurement, Krebs medium was removed and cells were scraped in buffer containing 25 mM HEPES, 1 mM EDTA, 1 mM EGTA, 2 mM MgCl2, and protease inhibitors [0.1 M PMSF, 0.2 M DTT, 1 % (v/v) Triton X-100, and 1:1,000 protease-inhibitor cocktail containing chymostatin, pepstatin A, leupeptin, and antipain at 1 μg/ml] for protein quantification using the Pierce’s BCA protein assay kit. Results were normalized to protein levels and were expressed relatively to control values. Measurement of Caspase-3- and Caspase-9-Like Activities Activation of caspase-3 and caspase-9 was monitored using a colorimetric method as previously described [21]. Control and treated cells were washed twice with phosphate buffered saline (PBS) and were then lysed in cold lysis buffer, containing 25 mM HEPES-Na, 10 % (w/v) sucrose, 10 mM DTT, and 0.1 % (w/v) CHAPS, pH 7.4. Cells were harvested through scraping and frozen/defrozen three times. The lysates were centrifuged for 10 min at 20,800×g (2–16 K Sigma-Aldrich Co., St. Louis, MO, USA) at 4 °C. The supernatant was collected and protein concentration was measured using the BioRad protein dye assay reagent. Cell extracts (50 μg protein)

were incubated at 37 °C for 2 h in 25 mM HEPES, pH 7.5, containing 0.1 % (w/v) CHAPS, 10 % (w/v) sucrose, 2 mM DTT, and 40 μM DEVD-pNA (caspase-3 substrate) or 40 μM Ac-LEHD-pNA (caspase-9 substrate). Caspase-like activities were determined by measuring substrate cleavage at 405 nm in a microplate reader (SpectraMax Plus 384, Molecular Devices). Results were expressed relatively to the control values. Measurement of Intracellular Reactive Oxygen Species The accumulation of reactive oxygen species (ROS) was evaluated using dihydroethidium (DHE), a dye that exhibits blue fluorescence in the cytosol and intercalates within the cell’s DNA after oxidation by superoxide anion radicals, staining its nucleus with a bright red fluorescence [24]. Control and treated RBE4 cells were loaded with 10 μM DHE in Krebs buffer for 30 min at 37 °C in the dark. Thereafter, cells were washed with Krebs buffer and the ROS-induced fluorescence was monitored for 1 h at 518-nm excitation and 605-nm emission, using a temperature-controlled SPEX 1681 Fluorolog spectrofluorometer (HORIBA, Jobin Yvon Inc., Edison, NJ, USA). Then, the Krebs medium was removed and the cells were scraped in buffer containing 25 mM HEPES, 1 mM EDTA, 1 mM EGTA, 2 mM MgCl2, and protease inhibitors [0.1 M PMSF, 0.2 M DTT, 1 % (v/v) Triton X-100, and 1:1,000 protease-inhibitor cocktail] for protein quantification using the Pierce’s BCA protein assay kit. Results were expressed relatively to baseline, then normalized to protein levels and, finally, normalized to control values. Western Blot Analysis In order to prepare whole cell extracts, RBE4 cells were washed twice with PBS, scraped and resuspended in icecold lysis buffer (25 mM HEPES-Na, 2 mM MgCl2, 1 mM EDTA, and 1 mM EGTA) supplemented with 0.1 M PMSF, 0.2 M DTT, and 1:1,000 of protease inhibitor cocktail (containing chymostatin, pepstatin A, leupeptin, and antipain, 1 μg/ml), and were then frozen and thawed three times. The protein content in each sample was quantified using the BioRad protein dye assay reagent. To isolate cytosolic and mitochondrial fractions, cells were washed with PBS, scraped in a buffer containing 250 mM sucrose, 20 mM Hepes, 1 mM EDTA, 1 mM EGTA, and protease inhibitors (0.1 M PMSF, 0.2 M DTT, and 1:1,000 dilution of protease inhibitor cocktail) and were then homogenized. Cells were frozen/defrozen three times on liquid nitrogen and the lysate was centrifuged at 500×g for 12 min at 4 °C. The supernatant was centrifuged at 10,600×g for 10 min at 4 °C to separate a crude mitochondrial fraction in the pellet and the cytosolic fraction in the supernantant. Protein content was measured using the Pierce’s BCA protein assay kit. Samples were resolved by

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electrophoresis in 8–12 % (v/v) SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding was blocked by gentle agitation in 5 % (w/v) BSA and 0.1 % (v/v) Tween in Tris-buffered saline (TBS) for 1 h at RT. Membranes were subsequently incubated overnight at 4 °C under gentle agitation with a mouse monoclonal antibody (mAB) anti-GRP78 (1:750), mouse mAB anti-cytochrome c (1:500), rabbit polyclonal antibody (pAB) anti-XBP1 (1:500), rabbit pAB anti-Fis1 (1:1,000), rabbit pAB anti-phospho-DRP1 (1:1,000), mouse pAB anti-DRP1 (1:1,000), mouse mAB anti-OPA1 (1:1,000), rabbit pAB antieIF2α (1:1,000), mouse pAB anti-phospho-eIF2α (1:1000), mouse mAB anti-Mfn2 (1:1,000), rabbit pAB anti-Mfn1 (1:1, 000), rabbit pAB anti-phospho-TauSer396 (1:1,000), rabbit pAB anti-phospho-TauThr181 (1:1,000), mouse pAB anti-total Tau (1:1,000), mouse mAB anti-actin (1:2,000), rabbit pAB anti-tubulin (1:1,000), mouse pAB anti acetylated-tubulin (1:1,000), rabbit pAB anti-TOM20 (1:1,000), goat pAB antiSigma 1R (1:1,000). Isolation of Soluble and Polymeric Tubulin In order to prepare soluble and polymeric tubulin fractions, RBE4 cells were gently washed twice with MTs-stabilizing buffer (0.1 M N-morpholino ethanesulfonic acid, pH 6.75, 1 mM MgSO4, 2 mM EGTA, 0.1 mM EDTA, and 4 M glycerol). After incubation during 4–6 min at 37 °C in MTstabilizing buffer supplemented with 0.1 % (v/v) Triton X-100, soluble proteins were collected. Polymeric proteins were then obtained after scraping in 25 mM Tris (pH 6.8) with 0.5 % (v/v) SDS and frozen/defrozen three times in liquid nitrogen [25]. Protein content was measured using the Pierce’s BCA Protein Assay kit. Isolation of F- and G-Actin For preparation of F and G actin fractions, cells were gently washed with PBS before lysis with actin-stabilization buffer [0.1 M PIPES, pH 6.9, 30 % (v/v) glycerol, 5 % (v/v) DMSO, 1 mM MgSO4, 1 mM EGTA, 1 % (v/v Triton X-100, 1 mM ATP and protease inhibitor cocktail] on ice for 10 min. Cells were dislodged by scraping and the extract was centrifuged at 4 °C for 75 min at 16,000×g. The supernatant containing Gactin was recovered and the F-actin pellet was solubilized with actin-depolymerization buffer (0.1 M PIPES, pH 6.9, 1 mM MgSO4, 10 mM CaCl2, and 5 μM cytochalasin D) [26]. Protein content was measured using the Pierce’s BCA protein assay kit. Immunocytochemistry Cells were washed twice with PBS and fixed in 4 % (w/v) paraformaldehyde for 30 min at RT. Then, cells were

permeabilized for 2 min at RT with 0.2 % Triton X-100 (v/v) in PBS and blocked for 30 min in PBS containing 3 % (w/v) BSA. Cells were incubated for 1 h with the following primary antibodies prepared in PBS with 3 % (w/v) BSA: rabbit polyclonal anti-TOM 20 antibody (1:100 dilution), mouse polyclonal anti-tubulin, Alexa Fluor 488-phalloidin. Then, cells were washed with PBS and incubated with Alexa Fluor 488 goat anti-rabbit IgG antibody conjugate (1:200 dilution in 3 % BSA-containing PBS) and Alexa Fluor 594 goat anti-mouse IgG antibody conjugate (1:200 dilution in 3 % BSAcontaining PBS) for 1 h at RT. Finally, cells were washed twice with PBS and treated with Dako Cytomation Fluorescent mounting solution on a microscope slide. Images were acquired on a Zeiss LSM510 META confocal microscope (×63 1.4NA plan-apochromat oil immersion lens) by using Zeiss LSM510 v3.2 software. Alexa Fluor 488phalloidin fluorescence was quantified using imageJ software (Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MA, USA). Results were expressed as corrected total cell fluorescence (CTCF) according with the calculation: CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings).

Isolation of Mice Brain Vessels Brain vessels were isolated from 11-month-old male wild-type (WT) and 3xTg-AD mice by following a previously described method [27]. Briefly, mice were decapitated, and the whole brain minus the cerebellum was rapidly removed, washed, minced, and homogenized at 4 °C in phosphate buffer [PBS; 0.01 M (8.5 g/L NaCl and 1.42 g/L Na2HPO4; pH = 7.4)] and then centrifuged three times at 720×g for 5 min (Sigma 3-16 K refrigerated centrifuge with a Swing-out rotor, Sigma 11133). The pellet was then resuspended in PBS and layered over dextran 16 % (w/v) and then centrifuged at 4,500×g for 20 min. The supernatant and the middle layer were collected and the above steps repeated. Finally, the two resulting pellets were resuspended in PBS and stored at −80 °C. Protein concentration was determined by the biuret method calibrated with bovine serum albumin.

Data Analysis Data represent mean ± SEM of at least three independent experiments performed in duplicate (in vitro studies) or of five mice/group (ex vivo studies). Statistical significance was obtained in the GraphPad prism software (San Diego, CA, USA) using the unpaired one-tailed t test or the oneway analysis of variance (ANOVA) test, followed by Dunnett’s post hoc test.

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Results OA Compromises Brain Endothelial Cells Survival The shellfish toxin OA is a highly selective inhibitor of the serine/threonine protein phosphatase activity, and specifically inhibits PP2A activity at low concentrations (