Oxidative Stress Modifies the Levels and Phosphorylation State of Tau Protein in Human Fibroblasts

Alejandro Ibáñez-Salazar, Bernardo Bañuelos-Hernández, Ildefonso Rodríguez-Leyva, Erika Chi-Ahumada, Elizabeth Monreal-Escalante, María E Jiménez-Capdeville, Sergio Rosales-Mendoza, Alejandro Ibáñez-Salazar, Bernardo Bañuelos-Hernández, Ildefonso Rodríguez-Leyva, Erika Chi-Ahumada, Elizabeth Monreal-Escalante, María E Jiménez-Capdeville, Sergio Rosales-Mendoza

Abstract

Since the tau protein is closely involved in the physiopathology of Alzheimer's disease (AD), studying its behavior in cellular models might lead to new insights on understanding this devastating disease at molecular levels. In the present study, primary cultures of human fibroblasts were established and used to determine the expression and localization of the tau protein in distinct phosphorylation states in both untransfected and tau gene-transfected cells subjected to oxidative stress. Higher immunopositivity to phospho-tau was observed in cell nuclei in response to oxidative stress, while the levels of total tau in the cytosol remained unchanged. These findings were observed in both untransfected cells and those transfected with the tau gene. The present work represents a useful model for studying the physiopathology of AD at the cellular level in terms of tau protein implications.

Keywords: Alzheimer's disease; fibroblasts; oxidative stress; phosphorylation state; tau protein.

Figures

Figure 1
Figure 1
Confocal microscopy analysis of untransfected human fibroblasts. Cells isolated from a skin biopsy showed a positive reactivity for Tau5 (cytoplasm), PHF (nucleus), and AT8 anti-serum (cytoplasm and nucleus). Nuclei were stained with Sytox (in red), while Tau was detected through FITC conjugated to the secondary antibody (in green). Sytox/FITC signal overlap is observed in yellow.
Figure 2
Figure 2
Map and restriction profile of the expression vector to overexpress the tau gene. (A) Physical map of the pVAX-Tau vector, which is based in the pVAX backbone and drives the expression of tau gene by the CMV promoter. (B) Restriction profiles of the constructed pVAX-Tau vector; the release of the 1.4 kb fragment upon EcoRI/ApaI restriction indicated the presence of the tau gene, which was confirmed by conventional sequencing.
Figure 3
Figure 3
Cell viability assay of human fibroblasts exposed to oxidative stress. Graph of resazurin experiment. Eight concentrations of hydrogen peroxide at four-time points were assessed. Cell viability is reported as percentage of control (considered as 100%).
Figure 4
Figure 4
Behavior of tau in fibroblasts upon oxidative stress and overexpression. Fibroblasts cultures were subjected to transfection with the tau gene and exposed to H2O2 to compare the behavior of Tau to that of untrasfected fibroblasts (UnFs). (A) Tau5 labeling revealed increased cytoplasmic reactivity in TFs exposed to 20 and 60 μM H2O2. PHF reactivity in UnFs was located into the nucleus and is increased in TFs. (B) AT8 labeling of UnFs revealed high cytoplasmic reactivity, which increased in TFs and appears into the nucleus. Anti-AT8 serum showed a similar pattern of that of the AT8 antibody but with a higher signal. Scale bar 2,000 pixels, equivalent to 100 μm. Nuclei were stained with DAPI (in blue), while Tau was detected through FITC conjugated to the secondary antibody (in green). DAPI/FITC signal overlap is observed in green/white.
Figure 5
Figure 5
Different levels of distinct forms of Tau in fibroblasts. Quantification of mean fluorescence intensity in microphotographs corresponding to labeling with distinct anti-Tau antibodies was performed with the Zen Lite 2012 software. Bars represent mean ± SD of a total of 135 cells counted in 9 microphotographies from each slide. (A) Nuclear levels of Tau detected with PHF antibody. (B) Cytosolic levels of Tau detected with Tau5 antibody. (C) Cytosolic levels of Tau detected with AT8 antibody. (D) Cytosolic levels of Tau detected with anti-AT8 serum. Asterisks right above columns indicate statistical significant differences between UnFs and TFs. Bars denote statistical significant differences between cells subjected to distinct H2O2 treatments. *p = 0.01; **p = 0.001; ***p = 0.0001.
Figure 6
Figure 6
Assessment of Tau overexpression by Western blot. Total protein extracts from fibroblasts under distinct experimental conditions were subjected to immunodetection using Tau5 antibody. β-actin immunodetection was performed as loading control. UnFs, untransfected fibroblasts; TFs, fibroblasts transfected with pVAX-Tau.

References

    1. Aguzzi A., Baumann F., Bremer J. (2008). The prion's elusive reason for being. Annu. Rev. Neurosci. 31, 439–477. 10.1146/annurev.neuro.31.060407.125620
    1. Auburger G., Klinkenberg M., Drost J., Marcus K., Morales-Gordo B., Kunz W. S., et al. . (2012). Primary skin fibroblasts as a model of Parkinson's Disease. Mol. Neurobiol. 46, 20–27. 10.1007/s12035-012-8245-1
    1. Audrey S., Fabrice N., Marie V., Séverine B., Anne L., Smail T., et al. . (2011). Nuclear Tau, a key player in Neuronal DNA protection. J. Biol. Chem. 286, 4566–4575. 10.1074/jbc.M110.199976
    1. Bayreuther K., Francz P. I., Gogol J., Hapke C., Maier M., Meinrath H. G. (1991). Differentiation of primary and secondary fibroblasts in cell culture systems. Mutat. Res. 256, 233–242. 10.1016/0921-8734(91)90014-3
    1. Braak H., Alafuzoff I., Arzberger T., Kretzschmar H., Del Tredici K. (2006). Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol. 112, 389–404. 10.1007/s00401-006-0127-z
    1. Calissano P., Matrone C., Amadoro G. (2009). Apoptosis and in vitro Alzheimer disease neuronal models. Com. Int. Biol. 2, 163–169. 10.4161/cib.7704
    1. Choi S. H., Kim Y. H., Quinti L., Rudolph E. T., Kim D. Y. (2016). 3D culture models of Alzheimer's disease: a road map to a “cure-in-a-dish.” Mol. Neurodegen. 75, 1–11. 10.1186/s13024-016-0139-7
    1. Corlier F., Rivals I., Lagarde J., Hamelin L., Corne H., Dauphinot L., et al. (2015). Modifications of the endosomal compartment in peripheral blood mononuclear cells and fibroblasts from Alzheimer's disease patients. Transl. Psychiatry 5e, 595 10.1038/tp.2015.87
    1. Cross D. C., Munoz J. P., Hernandez P., Maccioni R. B. (2000). Nuclear and cytoplasmic tau proteins from human nonneuronal cells share common structural and functional features with brain tau. J. Cell. Biochem. 78, 305–317. 10.1002/(SICI)1097-4644(20000801)78:2<305::AID-JCB12>;2-W
    1. Fraser G., Stalder A. K., Beibel M., Staufenbiel M. (2009). Transmission and spreading of tauopathy in transgenic mouse brain. Nat. Cell. Biol. 11, 909–913. 10.1038/ncb1901
    1. Freshney R. I. (2010). Culture of Animal Cells: a Manual of Basic Technique and Specialized Applications. 6th Edn. Hoboken, NJ: Wiley-BlackWell.
    1. Frost B., Hemberg M., Lewis J., Feany M. B. (2014). Tau promotes neurodegeneration through global chromatin relaxation. Nat. Neurosci. 17, 357–366. 10.1038/nn.3639
    1. Hardy J. (2006). A hundred years of Alzheimer's disease research. Neuron 52, 3–13. 10.1016/j.neuron.2006.09.016
    1. Hua Q., He R. Q. (2003). Effect of phosphorylation and aggregation on tau binding to DNA. Biochim. Biophys. 1645, 205–211. 10.1016/S1570-9639(02)00538-1
    1. Karpenko A. A., Rozanova I. A., Poveshchenko O. V., Lykov A. P., Bondarenko N. A., Kim I. I., et al. . (2015). Effect of extracellular matrix components on adhesion of bone marrow multipotent mesenchymal stromal cells to polytetrafluoroethylene. J. Angiol. Sosud. Khir. 21, 178–184.
    1. Klenyaeva A. N., Chuprov-Netochin R. N., Marusich E. I., Tatarnikova O. G., Orlov M. A., Bobkova N. V. (2014). Development of mouse fibroblast cell line expressing human Tau protein and evaluation of Tau-dependent cytotoxity. Biochem. (Mosc). Suppl. Ser A. Membr. Cell. Biol. 8, 232–239. 10.1134/S1990747814020111
    1. Kruman I. I., Wersto R. P., Cardozo-Pelaez F., Smilenov L., Chan S. L., Chrest F. J., et al. . (2004). Cell cycle activation linked to neuronal cell death initiated by DNA damage. Neuron 41, 549–561. 10.1016/S0896-6273(04)00017-0
    1. LaFerla F. M., Green K. N. (2012). Animal models of Alzheimer disease. Cold Spring Harb. Perspect. Med. 2, 1–13. 10.1101/cshperspect.a006320
    1. Loo A. E., Halliwell B. (2012). Effects of hydrogen peroxide in a keratinocyte-fibroblast co-culture model of wound healing. Biochem. Biophys. Res. Commun. 423, 253–258. 10.1016/j.bbrc.2012.05.100
    1. Macedo N. D., Buzin A. R., Abreu de Araujo I. B., Nogueira B. V., de Andrade T. U., Endringer D. C., et al. . (2017). Objective detection of apoptosis in rat renal tissue sections using light microscopy and free image analysis software with subsequent machine learning Detection of apoptosis in renal tissue. Tissue Cell 49, 22–27. 10.1016/j.tice.2016.12.006
    1. Mastroeni D., Grover A., Delvaux E., Whiteside C., Coleman P. D., Rogers J. (2011). Epigenetics mechanisms in Alzheimer's disease. Neurobiol. Aging 32, 1161–1180. 10.1016/j.neurobiolaging.2010.08.017
    1. Miyoshi N., Oubrahim H., Chock P. B., Stadtman E. R. (2006). Age-dependent cell death and the role of ATP in hydrogen peroxide-induced apoptosis and necrosis. Proc. Natl. Acad. Sci. U.S.A. 103, 1727–1731. 10.1073/pnas.0510346103
    1. Muramatsu K., Hashimoto Y., Uemura T. (2008). Neuron-specific crecombination by Cre recombinase inserted into the murine tau locus. Biochem. Biophys. Res. Commun. 370, 419–423. 10.1016/j.bbrc.2008.03.103
    1. NOM-062-ZOO (1999). Especificaciones Técnicas Para la Producción, Cuidado y Uso de los Animales de Laboratorio. NOM-062-ZOO.
    1. O'Brien J., Wilson I., Orton T., Pognan F. (2000). Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426. 10.1046/j.1432-1327.2000.01606.x
    1. Qiu C., Kivipelto M., von Strauss E. (2009). Epidemiology of Alzheimer's disease: occurrence, determinants, and strategies toward intervention. Dialogues Clin. Neurosci. 11, 111–128.
    1. Rodríguez-Leyva I., Chi-Ahumada E., Calderón–Garcidue-as A. L., Medina-Mier V., Santoyo Martha E., Martel-Gallegos G., et al. (2015). Presence of phosphorylated tau protein in the skin of Alzheimer's disease patients. J. Mol. Biomark. Diagn. S6:005 10.4172/2155-9929.S6-005
    1. Rossi G., Dalpra,‘ L., Crosti F., Lissoni S., Sciacca F. L., Catania M., et al. . (2008). A new function of microtubule-associated protein tau: involvement in chromosome stability. Cell Cycle 7, 1788–1794. 10.4161/cc.7.12.6012
    1. Sanchez-Mut J. V., Gräff J. (2015). Epigenetic Alterations in Alzheimer's Disease. Front. Behav. Neurosci. 9:347. 10.3389/fnbeh.2015.00347
    1. Selkoe D. J. (2004). Cell biology of protein misfolding: the examples of Alzheimer's and Parkinson's diseases. Nat. Cell Biol. 6, 1054–1061. 10.1038/ncb1104-1054
    1. Seung-Jae L., Hee-Sun L., Eliezer M., He-Jin L. (2011). Protein aggregate spreading in neurodegenerative diseases: problems and perspectives. Neurosci. Res. 70, 339–348. 10.1016/j.neures.2011.05.008
    1. Sjoberg M. K., Shestakova E., Mansuroglu Z., Maccioni R. B., Bonnefoy E. (2006). Tau protein binds to pericentromeric DNA: a putative role for nuclear tau in nucleolar organization. J. Cell Sci. 119, 2025–2034. 10.1242/jcs.02907
    1. Spillantini M. G., Goedert M. (2013). Tau pathology and neurodegeneration. Lancet Neurol. 12, 609–622. 10.1016/S1474-4422(13)70090-5
    1. Wang Y., Loomis P. A., Zinkowski R. P., Binder L. I. (1997). A novel tau transcript in cultured human neuroblastoma cells expressing nuclear tau. J. Cell Biol. 121, 257–267. 10.1083/jcb.121.2.257
    1. Woerman A. L., Aoyagi A., Patel S., Kazmi S. A., Lobach I., Grinberg L. T., et al. . (2016). Tau prions from Alzheimer's disease and chronic traumatic encephalopathy patients propagate in cultured cells. Proc. Natl. Acad. Sci. U.S.A. 113, E8187–E8196. 10.1073/pnas.1616344113
    1. Xiao-Hong L., Bing-Ling L., Jia-Zhao X., Jing L., Xin-Wen Z., Jian-Zhi W. (2012). AGEs induce Alzheimer-like tau pathology and memory deficit via RAGE-mediated GSK-3 activation. Neurobiol. Aging 33, 1400–1410. 10.1016/j.neurobiolaging.2011.02.003
    1. Yoshida H., Ihara Y. (1993). Tau in paired helical fi laments is functionally distinct from fetal tau: assembly incompetence of paired helical filament-tau. J. Neurochem. 61, 1183–1186. 10.1111/j.1471-4159.1993.tb03642.x

Source: PubMed

Sponsor e collaboratori

Condizioni mediche

Interventi farmacologici

3
Sottoscrivi