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, p-eIF2αSer51, and ERO1α, was observed. Moreover, PP2A inhibition upregulated the Sigma-1 receptor, an ER chaperone located at the ER–mitochondria interface, and enhanced inter-organelle 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 (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.
Similar content being viewed by others
References
Tang HL, Le AH, Lung HL (2006) The increase in mitochondrial association with actin precedes Bax translocation in apoptosis. Biochem J 396(1):1–5
Tar K et al (2006) Role of protein phosphatase 2A in the regulation of endothelial cell cytoskeleton structure. J Cell Biochem 98(4):931–53
Tar K et al (2004) Phosphatase 2A is involved in endothelial cell microtubule remodeling and barrier regulation. J Cell Biochem 92(3):534–46
Kasa A et al (2013) Protein phosphatase 2A activity is required for functional adherent junctions in endothelial cells. Microvasc Res 89:86–94
Menzel D et al (1995) Protein phosphatase 2A, a potential regulator of actin dynamics and actin-based organelle motility in the green alga Acetabularia. Eur J Cell Biol 67(2):179–87
Terasaki M, Chen LB, Fujiwara K (1986) Microtubules and the endoplasmic reticulum are highly interdependent structures. J Cell Biol 103(4):1557–68
Korobova F, Ramabhadran V, Higgs HN (2013) An actin-dependent step in mitochondrial fission mediated by the ER-associated formin INF2. Science 339(6118):464–7
Ebneth A, Godemann R, Stamer K, Illenberger S, Trinczek B, Mandelkow E (1998) Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer’s disease. J Cell Biol 143(3):777–94
Schon EA, Area-Gomez E (2013) Mitochondria-associated ER membranes in Alzheimer disease. Mol Cell Neurosci 55:26–36
Bravo R et al (2011) Increased ER-mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress. J Cell Sci 124(Pt 13):2143–52
Wang X et al (2011) ER stress modulates cellular metabolism. Biochem J 435(1):285–96
Moreira PI et al (2007) Brain mitochondrial dysfunction as a link between Alzheimer’s disease and diabetes. J Neurol Sci 257(1–2):206–14
Murphy E (2015) Solving mitochondrial mysteries. J Mol Cell Cardiol 78:1–2
Friedman JR et al (2010) ER sliding dynamics and ER-mitochondrial contacts occur on acetylated microtubules. J Cell Biol 190(3):363–75
Merrill RA, Slupe AM, Strack S (2013) N-terminal phosphorylation of protein phosphatase 2A/Bbeta2 regulates translocation to mitochondria, dynamin-related protein 1 dephosphorylation, and neuronal survival. FEBS J 280(2):662–73
Cho MH et al (2012) Increased phosphorylation of dynamin-related protein 1 and mitochondrial fission in okadaic acid-treated neurons. Brain Res 1454:100–10
Guan J et al (2013) Vascular degeneration in Parkinson’s disease. Brain Pathol 23(2):154–64
Zhong Z et al (2008) ALS-causing SOD1 mutants generate vascular changes prior to motor neuron degeneration. Nat Neurosci 11(4):420–2
Zipser BD et al (2007) Microvascular injury and blood–brain barrier leakage in Alzheimer’s disease. Neurobiol Aging 28(7):977–86
Zlokovic BV (2011) Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders. Nat Rev Neurosci 12(12):723–38
Placido AI et al (2015) Enhanced amyloidogenic processing of amyloid precursor protein and cell death under prolonged endoplasmic reticulum stress in brain endothelial cells. Mol Neurobiol 51(2):571–90
Ma J et al (2014) Synthesis and high sensing properties of a single Pd-doped SnO2 nanoribbon. Nanoscale Res Lett 9(1):503
Deniaud A et al (2008) Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene 27(3):285–99
Whiteman M et al (2008) Detection and measurement of reactive oxygen intermediates in mitochondria and cells. Methods Mol Biol 476:29–50
Joshi HC, Cleveland DW (1989) Differential utilization of beta-tubulin isotypes in differentiating neurites. J Cell Biol 109(2):663–73
Rasmussen I et al (2010) Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia. BMC Immunol 11:44
Carvalho C et al (2013) Type 2 diabetic and Alzheimer’s disease mice present similar behavioral, cognitive, and vascular anomalies. J Alzheimers Dis 35(3):623–35
Cohen P, Klumpp S, Schelling DL (1989) An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett 250(2):596–600
Butler D et al (2007) Microtubule-stabilizing agent prevents protein accumulation-induced loss of synaptic markers. Eur J Pharmacol 562(1–2):20–7
Mitsuda T et al (2011) Sigma-1Rs are upregulated via PERK/eIF2alpha/ATF4 pathway and execute protective function in ER stress. Biochem Biophys Res Commun 415(3):519–25
Hayashi T, Su TP (2007) Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival. Cell 131(3):596–610
Ishikawa R et al (1992) Characterization of smooth muscle caldesmon as a microtubule-associated protein. Cell Motil Cytoskeleton 23(4):244–51
Moraga DM et al (1993) A tau fragment containing a repetitive sequence induces bundling of actin filaments. J Neurochem 61(3):979–86
Sattilaro RF, Dentler WL, LeCluyse EL (1981) Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro. J Cell Biol 90(2):467–73
Guay J et al (1997) Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27. J Cell Sci 110(Pt 3):357–68
Gusev NB, Bogatcheva NV, Marston SB (2002) Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins. Biochemistry (Mosc) 67(5):511–9
Lavoie JN et al (1995) Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27. Mol Cell Biol 15(1):505–16
Lee TY, Gotlieb AI (2003) Microfilaments and microtubules maintain endothelial integrity. Microsc Res Tech 60(1):115–27
Liu F et al (2005) Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation. Eur J Neurosci 22(8):1942–50
Martin L, Latypova X, Terro F (2011) Post-translational modifications of tau protein: implications for Alzheimer’s disease. Neurochem Int 58(4):458–71
Gong CX et al (1993) Phosphoprotein phosphatase activities in Alzheimer disease brain. J Neurochem 61(3):921–7
Wang JZ, Grundke-Iqbal I, Iqbal K (1996) Restoration of biological activity of Alzheimer abnormally phosphorylated tau by dephosphorylation with protein phosphatase-2A, −2B and −1. Brain Res Mol Brain Res 38(2):200–8
Wang JZ, Grundke-Iqbal I, Iqbal K (2007) Kinases and phosphatases and tau sites involved in Alzheimer neurofibrillary degeneration. Eur J Neurosci 25(1):59–68
Wang JZ, Liu F (2008) Microtubule-associated protein tau in development, degeneration and protection of neurons. Prog Neurobiol 85(2):148–75
Wang Y et al (2015) Cross talk between PI3K-AKT-GSK-3beta and PP2A pathways determines tau hyperphosphorylation. Neurobiol Aging 36(1):188–200
Nakagawa S et al (2007) Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain endothelial cells. Cell Mol Neurobiol 27(6):687–94
Cambray-Deakin MA, Burgoyne RD (1987) Acetylated and detyrosinated alpha-tubulins are co-localized in stable microtubules in rat meningeal fibroblasts. Cell Motil Cytoskeleton 8(3):284–91
Elie A et al (2015) Tau co-organizes dynamic microtubule and actin networks. Sci Rep 5:9964
Fulga TA et al (2007) Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal degeneration in vivo. Nat Cell Biol 9(2):139–48
He HJ et al (2009) The proline-rich domain of tau plays a role in interactions with actin. BMC Cell Biol 10:81
Frandemiche ML et al (2014) Activity-dependent tau protein translocation to excitatory synapse is disrupted by exposure to amyloid-beta oligomers. J Neurosci 34(17):6084–97
Yu JZ, Rasenick MM (2006) Tau associates with actin in differentiating PC12 cells. FASEB J 20(9):1452–61
Dreier L, Rapoport TA (2000) In vitro formation of the endoplasmic reticulum occurs independently of microtubules by a controlled fusion reaction. J Cell Biol 148(5):883–98
Joensuu M et al (2014) ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays. Mol Biol Cell 25(7):1111–26
Chambers JE, et al (2015) Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2alpha dephosphorylation. Elife 4.
Ho YS et al (2012) Endoplasmic reticulum stress induces tau pathology and forms a vicious cycle: implication in Alzheimer’s disease pathogenesis. J Alzheimers Dis 28(4):839–54
Perreault S et al (2009) Increased association between rough endoplasmic reticulum membranes and mitochondria in transgenic mice that express P301L tau. J Neuropathol Exp Neurol 68(5):503–14
Anelli T et al (2012) Ero1alpha regulates Ca(2+) fluxes at the endoplasmic reticulum-mitochondria interface (MAM). Antioxid Redox Signal 16(10):1077–87
Bhandary B et al (2012) An involvement of oxidative stress in endoplasmic reticulum stress and its associated diseases. Int J Mol Sci 14(1):434–56
Higa A, Chevet E (2012) Redox signaling loops in the unfolded protein response. Cell Signal 24(8):1548–55
Tu BP, Weissman JS (2002) The FAD- and O(2)-dependent reaction cycle of Ero1-mediated oxidative protein folding in the endoplasmic reticulum. Mol Cell 10(5):983–94
Jacobson J, Duchen MR (2002) Mitochondrial oxidative stress and cell death in astrocytes—requirement for stored Ca2+ and sustained opening of the permeability transition pore. J Cell Sci 115(Pt 6):1175–88
Lock JT, Sinkins WG, Schilling WP (2012) Protein S-glutathionylation enhances Ca2+-induced Ca2+ release via the IP3 receptor in cultured aortic endothelial cells. J Physiol 590(Pt 15):3431–47
Brookes PS et al (2004) Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol 287(4):C817–33
Dagda RK et al (2008) The spinocerebellar ataxia 12 gene product and protein phosphatase 2A regulatory subunit Bbeta2 antagonizes neuronal survival by promoting mitochondrial fission. J Biol Chem 283(52):36241–8
Dickey AS, Strack S (2011) PKA/AKAP1 and PP2A/Bbeta2 regulate neuronal morphogenesis via Drp1 phosphorylation and mitochondrial bioenergetics. J Neurosci 31(44):15716–26
Patergnani S, Pinton P (2015) Mitophagy and mitochondrial balance. Methods Mol Biol 1241:181–94
DuBoff B, Gotz J, Feany MB (2012) Tau promotes neurodegeneration via DRP1 mislocalization in vivo. Neuron 75(4):618–32
Moreira PI et al (2010) Mitochondria: a therapeutic target in neurodegeneration. Biochim Biophys Acta 1802(1):212–20
Acknowledgments
Ana I. Plácido has a PhD fellowship from the Fundação para a Ciência e Tecnologia (SFRH/BD/73388/2010). The work was supported by PEst-C/SAU/LA0001/2013—Strategic Project—LA 1-2013-2014; Gabinete de Apoio à Investigação (GAI)—Faculty of Medicine, University of Coimbra & Banco Santader Totta (project MOREIRA05.01.13).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare no conflicts of interest.
Rights and permissions
About this article
Cite this article
Plácido, A.I., Pereira, C.M.F., Correira, S.C. et al. Phosphatase 2A Inhibition Affects Endoplasmic Reticulum and Mitochondria Homeostasis Via Cytoskeletal Alterations in Brain Endothelial Cells. Mol Neurobiol 54, 154–168 (2017). https://doi.org/10.1007/s12035-015-9640-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12035-015-9640-1