Modification of lipid rafts by extracellular vesicles carrying HIV-1 protein Nef induces redistribution of amyloid precursor protein and Tau, causing neuronal dysfunction
2020; Elsevier BV; Volume: 295; Issue: 38 Linguagem: Inglês
10.1074/jbc.ra120.014642
ISSN1083-351X
AutoresMichael Ditiatkovski, Nigora Mukhamedova, Dragana Dragoljevic, Anh Hoang, Hann Low, Tatiana Pushkarsky, Ying Fu, Irena Carmichael, Andrew F. Hill, Andrew Murphy, Michael Bukrinsky, Dmitri Sviridov,
Tópico(s)Reproductive System and Pregnancy
ResumoHIV-associated neurocognitive disorders (HANDs) are a frequent outcome of HIV infection. Effective treatment of HIV infection has reduced the rate of progression and severity but not the overall prevalence of HANDs, suggesting ongoing pathological process even when viral replication is suppressed. In this study, we investigated how HIV-1 protein Nef secreted in extracellular vesicles (exNef) impairs neuronal functionality. ExNef were rapidly taken up by neural cells in vitro, reducing the abundance of ABC transporter A1 (ABCA1) and thus cholesterol efflux and increasing the abundance and modifying lipid rafts in neuronal plasma membranes. ExNef caused a redistribution of amyloid precursor protein (APP) and Tau to lipid rafts and increased the abundance of these proteins, as well as of Aβ42. ExNef further potentiated phosphorylation of Tau and activation of inflammatory pathways. These changes were accompanied by neuronal functional impairment. Disruption of lipid rafts with cyclodextrin reversed the phenotype. Short-term treatment of C57BL/6 mice with either purified recombinant Nef or exNef similarly resulted in reduced abundance of ABCA1 and elevated abundance of APP in brain tissue. The abundance of ABCA1 in brain tissue of HIV-infected human subjects diagnosed with HAND was lower, and the abundance of lipid rafts was higher compared with HIV-negative individuals. Levels of APP and Tau in brain tissue correlated with the abundance of Nef. Thus, modification of neuronal cholesterol trafficking and of lipid rafts by Nef may contribute to early stages of neurodegeneration and pathogenesis in HAND. HIV-associated neurocognitive disorders (HANDs) are a frequent outcome of HIV infection. Effective treatment of HIV infection has reduced the rate of progression and severity but not the overall prevalence of HANDs, suggesting ongoing pathological process even when viral replication is suppressed. In this study, we investigated how HIV-1 protein Nef secreted in extracellular vesicles (exNef) impairs neuronal functionality. ExNef were rapidly taken up by neural cells in vitro, reducing the abundance of ABC transporter A1 (ABCA1) and thus cholesterol efflux and increasing the abundance and modifying lipid rafts in neuronal plasma membranes. ExNef caused a redistribution of amyloid precursor protein (APP) and Tau to lipid rafts and increased the abundance of these proteins, as well as of Aβ42. ExNef further potentiated phosphorylation of Tau and activation of inflammatory pathways. These changes were accompanied by neuronal functional impairment. Disruption of lipid rafts with cyclodextrin reversed the phenotype. Short-term treatment of C57BL/6 mice with either purified recombinant Nef or exNef similarly resulted in reduced abundance of ABCA1 and elevated abundance of APP in brain tissue. The abundance of ABCA1 in brain tissue of HIV-infected human subjects diagnosed with HAND was lower, and the abundance of lipid rafts was higher compared with HIV-negative individuals. Levels of APP and Tau in brain tissue correlated with the abundance of Nef. Thus, modification of neuronal cholesterol trafficking and of lipid rafts by Nef may contribute to early stages of neurodegeneration and pathogenesis in HAND. HIV-associated neurocognitive disorders (HANDs) include three levels of neurocognitive dysfunction in HIV infection: asymptomatic neurocognitive impairment, mild neurocognitive disorder, and HIV-associated dementia (1Saylor D. Dickens A.M. Sacktor N. Haughey N. Slusher B. Pletnikov M. Mankowski J.L. Brown A. Volsky D.J. McArthur J.C. HIV-associated neurocognitive disorder: pathogenesis and prospects for treatment.Nat. Rev. Neurol. 2016; 12 (26965674): 234-24810.1038/nrneurol.2016.27Crossref PubMed Scopus (340) Google Scholar, 2Antinori A. Arendt G. Becker J.T. Brew B.J. 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Cholesterol metabolism and dysregulation of lipid rafts were shown to play a key role in pathogenesis of neural damage in many neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease, and prion diseases (5Schengrund C.L. Lipid rafts: keys to neurodegeneration.Brain. Res. Bull. 2010; 82 (20206240): 7-1710.1016/j.brainresbull.2010.02.013Crossref PubMed Scopus (98) Google Scholar, 6Björkhem I. Leoni V. Meaney S. Genetic connections between neurological disorders and cholesterol metabolism.J. Lipid Res. 2010; 51 (20466796): 2489-250310.1194/jlr.R006338Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 7Grassi S. Giussani P. Mauri L. Prioni S. Sonnino S. Prinetti A. Lipid rafts and neurodegeneration: structural and functional roles in physiologic aging and neurodegenerative diseases.J. Lipid Res. 2020; 61 (31871065): 636-65410.1194/jlr.TR119000427Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). 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Human immunodeficiency virus impairs reverse cholesterol transport from macrophages.PLos Biol. 2006; 4 (17076584): e36510.1371/journal.pbio.0040365Crossref PubMed Scopus (234) Google Scholar). We recently demonstrated that changes in ABCA1 and lipid rafts inflicted by HIV Nef in macrophages were almost identical to those found in neurons infected by prions (11Cui H.L. Guo B. Scicluna B. Coleman B.M. Lawson V.A. Ellett L. Meikle P.J. Bukrinsky M. Mukhamedova N. Sviridov D. Hill A.F. Prion infection impairs cholesterol metabolism in neuronal cells.J. Biol. Chem. 2014; 289 (24280226): 789-80210.1074/jbc.M113.535807Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Cells do not need to be infected with HIV to be affected by Nef, because Nef is secreted from HIV-infected cells in extracellular vesicles (EVs). These have the same effect on cholesterol metabolism in "bystander" cells in uninfected tissues as Nef in HIV-infected cells (12Cui H.L. Ditiatkovski M. Kesani R. Bobryshev Y.V. Liu Y. Geyer M. Mukhamedova N. Bukrinsky M. Sviridov D. HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis.FASEB J. 2014; 28 (24642731): 2828-283910.1096/fj.13-246876Crossref PubMed Scopus (32) Google Scholar, 13Mukhamedova N. Hoang A. Dragoljevic D. Dubrovsky L. Pushkarsky T. Low H. Ditiatkovski M. Fu Y. Ohkawa R. Meikle P.J. Horvath A. Brichacek B. Miller Y.I. Murphy A. Bukrinsky M. et al.Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.PLoS Pathog. 2019; 15 (31344124): e100790710.1371/journal.ppat.1007907Crossref PubMed Scopus (35) Google Scholar). Nef-containing EVs ("extracellular" Nef, exNef) were found in relatively high levels in blood and, importantly, in the CSF of HIV-infected subjects treated with anti-retroviral therapy (ART) (14Lee J.H. Schierer S. Blume K. Dindorf J. Wittki S. Xiang W. Ostalecki C. Koliha N. Wild S. Schuler G. Fackler O.T. Saksela K. Harrer T. Baur A.S. HIV-Nef and ADAM17-containing plasma extracellular vesicles induce and correlate with immune pathogenesis in chronic HIV infection.EBioMedicine. 2016; 6 (27211553): 103-11310.1016/j.ebiom.2016.03.004Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 15Ferdin J. Goricar K. Dolzan V. Plemenitas A. Martin J.N. Peterlin B.M. Deeks S.G. Lenassi M. Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA.PLoS One. 2018; 13 (29364927): e019161310.1371/journal.pone.0191613Crossref PubMed Scopus (30) Google Scholar, 16Raymond A.D. Campbell-Sims T.C. Khan M. Lang M. Huang M.B. Bond V.C. Powell M.D. HIV Type 1 Nef is released from infected cells in CD45+ microvesicles and is present in the plasma of HIV-infected individuals.AIDS Res. Hum. Retroviruses. 2011; 27 (20964480): 167-17810.1089/aid.2009.0170Crossref PubMed Scopus (103) Google Scholar). Several studies have suggested a role for systemically available Nef in pathogenesis of HAND, but the proposed mechanisms assumed direct neurotoxicity (16Raymond A.D. Campbell-Sims T.C. Khan M. Lang M. Huang M.B. Bond V.C. Powell M.D. HIV Type 1 Nef is released from infected cells in CD45+ microvesicles and is present in the plasma of HIV-infected individuals.AIDS Res. Hum. Retroviruses. 2011; 27 (20964480): 167-17810.1089/aid.2009.0170Crossref PubMed Scopus (103) Google Scholar, 17Saribas A.S. Cicalese S. Ahooyi T.M. Khalili K. Amini S. Sariyer I.K. HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity.Cell Death Dis. 2017; 8 (28079886): e254210.1038/cddis.2016.467Crossref PubMed Scopus (66) Google Scholar, 18Raymond A.D. Diaz P. Chevelon S. Agudelo M. Yndart-Arias A. Ding H. Kaushik A. Jayant R.D. Nikkhah-Moshaie R. Roy U. Pilakka-Kanthikeel S. Nair M.P. Microglia-derived HIV Nef+ exosome impairment of the blood–brain barrier is treatable by nanomedicine-based delivery of Nef peptides.J. Neurovirol. 2016; 22 (26631079): 129-13910.1007/s13365-015-0397-0Crossref PubMed Scopus (65) Google Scholar). Although direct toxicity of Nef has been demonstrated in vitro (17Saribas A.S. Cicalese S. Ahooyi T.M. Khalili K. Amini S. Sariyer I.K. HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity.Cell Death Dis. 2017; 8 (28079886): e254210.1038/cddis.2016.467Crossref PubMed Scopus (66) Google Scholar, 19Fujii Y. Otake K. Tashiro M. Adachi A. Soluble Nef antigen of HIV-1 is cytotoxic for human CD4+ T cells.FEBS Lett. 1996; 393 (8804432): 93-9610.1016/0014-5793(96)00859-9Crossref PubMed Scopus (114) Google Scholar, 20Khan M.B. Lang M.J. Huang M.B. Raymond A. Bond V.C. Shiramizu B. Powell M.D. Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aβ1-42 secretion in SH-SY5Y neural cells.J. Neurovirol. 2016; 22 (26407718): 179-19010.1007/s13365-015-0383-6Crossref PubMed Scopus (49) Google Scholar), the level of toxicity of very low concentrations of Nef found in ART-treated subjects is unlikely to be a primary mechanism of neural damage. In this study, we tested a hypothesis that Nef-containing EVs contribute to the neuronal damage by reorganizing lipid rafts and causing accumulation of amyloidogenic proteins in these membrane domains. In this study, we used EVs produced by HEK293 cells transfected with either Nef or GFP (control). As established in our previous studies (13Mukhamedova N. Hoang A. Dragoljevic D. Dubrovsky L. Pushkarsky T. Low H. Ditiatkovski M. Fu Y. Ohkawa R. Meikle P.J. Horvath A. Brichacek B. Miller Y.I. Murphy A. Bukrinsky M. et al.Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.PLoS Pathog. 2019; 15 (31344124): e100790710.1371/journal.ppat.1007907Crossref PubMed Scopus (35) Google Scholar, 21Dubrovsky L. Ward A. Choi S.-H. Pushkarsky T. Brichacek B. Vanpouille C. Adzhubei A.A. Mukhamedova N. Sviridov D. Margolis L. Jones R.B. Miller Y.I. Bukrinsky M. Inhibition of HIV replication by apolipoprotein A-I binding protein targeting the lipid rafts.mBio. 2020; 11 (31964734): e02919-e02956Google Scholar), these EVs, designated as exNef and exGFP, respectively, have predominant sizes of 120–150 nm, were positive for ALIX, tetraspanin CD63 and Hsp70 (cytosolic marker), and negative for cytochrome c, thus satisfying International Society for Extracellular Vesicles criteria for extracellular vesicles (22Théry C. Witwer K.W. Aikawa E. Alcaraz M.J. Anderson J.D. Andriantsitohaina R. Antoniou A. Arab T. Archer F. Atkin-Smith G.K. Ayre D.C. Bach J.-M. Bachurski D. Baharvand H. Balaj L. et al.Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.J. Extracell. Vesicles. 2018; 7 (30637094): 153575010.1080/20013078.2018.1535750Crossref PubMed Scopus (2361) Google Scholar). They did not contain Nef-coding mRNA (13Mukhamedova N. Hoang A. Dragoljevic D. Dubrovsky L. Pushkarsky T. Low H. Ditiatkovski M. Fu Y. Ohkawa R. Meikle P.J. Horvath A. Brichacek B. Miller Y.I. Murphy A. Bukrinsky M. et al.Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.PLoS Pathog. 2019; 15 (31344124): e100790710.1371/journal.ppat.1007907Crossref PubMed Scopus (35) Google Scholar). ExNef contained Nef at an average concentration of 0.5 ng Nef/1 μg of total EV protein (13Mukhamedova N. Hoang A. Dragoljevic D. Dubrovsky L. Pushkarsky T. Low H. Ditiatkovski M. Fu Y. Ohkawa R. Meikle P.J. Horvath A. Brichacek B. Miller Y.I. Murphy A. Bukrinsky M. et al.Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.PLoS Pathog. 2019; 15 (31344124): e100790710.1371/journal.ppat.1007907Crossref PubMed Scopus (35) Google Scholar), Nef content of EVs was measured separately for each batch, and control exGFP was added to cells in total EV protein concentration matching that of exNef. Unless indicated otherwise, the concentration of exNef used in this study was 0.4 ng/ml of Nef, which is lower than the concentration of Nef detected in the blood of ART-treated individuals with undetectable viral load (15Ferdin J. Goricar K. Dolzan V. Plemenitas A. Martin J.N. Peterlin B.M. Deeks S.G. Lenassi M. Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA.PLoS One. 2018; 13 (29364927): e019161310.1371/journal.pone.0191613Crossref PubMed Scopus (30) Google Scholar, 23Raymond A.D. Lang M.J. Chu J. Campbell-Sims T. Khan M. Bond V.C. Pollard R.B. Asmuth D.M. Powell M.D. Plasma-derived HIV Nef+ exosomes persist in ACTG384 study participants despite successful virological suppression.bioRxiv. 2019; 10.1101/708719Google Scholar). It was demonstrated in our previous study that the effects of EVs produced by Nef-expressing cells on cholesterol metabolism and lipid rafts are similar to the effects of EVs produced by HIV-infected myeloid cells, whereas EVs from cells infected with ΔNefHIV did not have such effects (13Mukhamedova N. Hoang A. Dragoljevic D. Dubrovsky L. Pushkarsky T. Low H. Ditiatkovski M. Fu Y. Ohkawa R. Meikle P.J. Horvath A. Brichacek B. Miller Y.I. Murphy A. Bukrinsky M. et al.Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.PLoS Pathog. 2019; 15 (31344124): e100790710.1371/journal.ppat.1007907Crossref PubMed Scopus (35) Google Scholar). To determine whether differentiated SH-SY5Y human neuroblastoma cells could take up the EVs, we labeled EVs with fluorescent dye PKH67. EV uptake was slow over the first 4 h but increased in a linear fashion over the following 4 h (Fig. 1, A and B). No fluorescence was found inside the cells when BSA instead of EVs was added to the stain solution and incubated with cells for 8 h (Fig. 1A, last panel), suggesting that EVs were taken up by SH-SY5Y cells. To determine whether EVs could deliver Nef into cells, we incubated EVs containing Nef tagged with GFP (exGFP-Nef) with the cells for 24 h and used anti-GFP antibody to enhance the detection signal. GFP-Nef was detected inside the cells after a 24-h incubation and intracellular abundance of Nef continued to rise for at least another 24 h (Fig. 1, C and D). Thus, exNef was actively taken up by SH-SY5Y cells delivering Nef to the cell interior. We have previously demonstrated that Nef expressed in macrophages infected with HIV-1 or transfected with heterologous Nef, as well as delivered to cells by incubation with the recombinant Nef or Nef-containing EVs, reduces the abundance of ABCA1 and the rate of cholesterol efflux in macrophages (10Mujawar Z. Rose H. Morrow M.P. Pushkarsky T. Dubrovsky L. Mukhamedova N. Fu Y. Dart A. Orenstein J.M. Bobryshev Y.V. Bukrinsky M. Sviridov D. Human immunodeficiency virus impairs reverse cholesterol transport from macrophages.PLos Biol. 2006; 4 (17076584): e36510.1371/journal.pbio.0040365Crossref PubMed Scopus (234) Google Scholar, 13Mukhamedova N. Hoang A. Dragoljevic D. Dubrovsky L. Pushkarsky T. Low H. Ditiatkovski M. Fu Y. Ohkawa R. Meikle P.J. Horvath A. Brichacek B. Miller Y.I. Murphy A. Bukrinsky M. et al.Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.PLoS Pathog. 2019; 15 (31344124): e100790710.1371/journal.ppat.1007907Crossref PubMed Scopus (35) Google Scholar). Similar effects were observed when we tested the effects of 48 h of incubation with exNef (final concentration, 0.4 ng/ml) on SH-SY5Y neural cells. The abundance of total and cell surface ABCA1 and the rate of cholesterol efflux were reduced by 35% (n = 3) and 3-fold, respectively (Fig. 2, A and B). Another effect of Nef was elevation of the abundance of lipid rafts. Incubation of exNef with SH-SY5Y cells for 48 h also led to elevation of the abundance of lipid rafts as determined by confocal microscopy after staining with fluorescently tagged cholera toxin subunit B (CTB) (Fig. 2, C and D). To ensure that the effects of exNef can be attributed to Nef and not to a Nef-induced factor that may be carried by EVs produced by Nef-expressing cells, we incubated SH-SY5Y cells with myristoylated recombinant Nef (final concentration, 100 ng/ml). Recombinant Nef also caused elevation of the abundance of lipid rafts as determined by CTB binding and confocal microscopy (Fig. 2E) or flow cytometry (Fig. 2F), although the effects were less pronounced than with exNef and required higher concentration of Nef, similar to what was seen with macrophages (13Mukhamedova N. Hoang A. Dragoljevic D. Dubrovsky L. Pushkarsky T. Low H. Ditiatkovski M. Fu Y. Ohkawa R. Meikle P.J. Horvath A. Brichacek B. Miller Y.I. Murphy A. Bukrinsky M. et al.Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.PLoS Pathog. 2019; 15 (31344124): e100790710.1371/journal.ppat.1007907Crossref PubMed Scopus (35) Google Scholar). Next, we labeled cells with [3H]cholesterol and isolated lipid rafts from plasma membranes using density gradient centrifugation. Fractions containing the highest concentrations of [3H]cholesterol and lipid raft marker flotillin-1 were designated as lipid rafts; both markers co-distributed along the density gradient (Fig. 2, G and H). When we compared lipid rafts isolated from cells treated with exNef and exGFP, we found that lipid raft fractions from cells treated with exNef contained more cholesterol and more flotillin-1 than fractions from cells treated with exGFP. Furthermore, the density of the fractions containing maximum amounts of cholesterol and flotillin-1 was lower (lighter fractions) in cells treated with exNef (Fig. 2, G and H). These findings are consistent with lipid rafts in cells treated with exNef being more abundant and possibly bigger. Elevation of the abundance of lipid rafts may cause elevation of the abundance of lipid raft-associated amyloidogenic proteins. We tested the effect of exNef on the abundance of several amyloidogenic proteins in cell lysates and found elevated abundance of APP and Tau in cells treated with exNef (Fig. 3, A and B). This was confirmed with an independent method, confocal microscopy. Staining for APP and Tau on cell membranes was elevated in cells treated with exNef as compared with cells treated with exGFP (Fig. 3, C and D). We then used ELISA to evaluate the amount of amyloid Aβ42. The abundance of Aβ42 in cells treated with exNef tripled compared with cells treated with exGFP (Fig. 3E); the abundance of Aβ42 in the medium after 24 and 48 h of incubation was below the detection limit of the method. There was no difference in the abundance of α-synuclein and l-neurofilament in the cells (not shown). Several reports indicated that an essential step in formation of Tau microfilaments is hyperphosphorylation of Tau (24Stoothoff W.H. Johnson G.V. Tau phosphorylation: physiological and pathological consequences.Biochim. Biophys. Acta. 2005; 1739 (15615646): 280-29710.1016/j.bbadis.2004.06.017Crossref PubMed Scopus (325) Google Scholar, 25Alonso A.C. Zaidi T. Grundke-Iqbal I. Iqbal K. Role of abnormally phosphorylated Tau in the breakdown of microtubules in Alzheimer disease.Proc. Natl. Acad. Sci. U.S.A. 1994; 91 (8202528): 5562-556610.1073/pnas.91.12.5562Crossref PubMed Scopus (542) Google Scholar). Therefore, we assessed the abundance of phosphorylated Tau (pTau) in the plasma membranes of SH-SY5Y neural cells using Western blotting. Treatment of cells with exNef resulted in elevation in the abundance of pTau in parallel with increased total abundance of Tau (Fig. 3F). Interestingly, we also found that treatment with exNef increased the abundance of total and phosphorylated of ERK 1/2 (Fig. 3F), which may contribute, on the one hand, to hyperphosphorylation of Tau (26Qi H. Prabakaran S. Cantrelle F.-X. Chambraud B. Gunawardena J. Lippens G. Landrieu I. Characterization of neuronal Tau protein as a target of extracellular signal–regulated kinase.J. Biol. Chem. 2016; 291 (26858248): 7742-775310.1074/jbc.M115.700914Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and, on the other hand, to the elevated inflammatory status characteristic for brains of HIV-infected persons. The increased pro-inflammatory and pro-apoptotic signaling induced by exNef was further supported by elevation in phosphorylation of p38 MAPK in cells treated with exNef (Fig. 3G). Next, we assessed the cellular localization of APP and Tau in SH-SY5Y cells using confocal microscopy. Localization of APP and Tau in the SH-SY5Y cells is shown in Fig. 4 (A and B, respectively); most of APP and Tau localized at the plasma membranes and co-localized with lipid rafts. The co-localization of these proteins with lipid rafts was measured as Manders' co-localization coefficient (M2) (27Costes S.V. Daelemans D. Cho E.H. Dobbin Z. Pavlakis G. Lockett S. Automatic and quantitative measurement of protein–protein colocalization in live cells.Biophys. J. 2004; 86 (15189895): 3993-400310.1529/biophysj.103.038422Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). We found that treatment of cells with exNef increased co-localization of both APP and Tau with lipid rafts (Fig. 4, A–D). We then isolated lipid rafts by density gradient centrifugation and determined the abundance of flotillin-1 and APP in individual fractions. Higher abundance of APP was found in lipid raft fractions of cells treated with exNef compared with that of cells treated with exGFP (Fig. 4E). We next probed the abundance of flotillin-1, APP, and phosphorylated Tau by Western blotting in combined raft or nonraft fractions. There was considerably higher abundance of flotillin-1 in raft fractions; rafts from exNef-treated cells had more flotillin-1 than rafts from cells treated with exGFP (Fig. 4F). All detectable APP was found in raft fractions of exNef-treated cells; APP abundance in nonraft fractions of exNef-treated cells, as well as in both raft and nonraft fractions of exGFP-treated cells, was very low. Phosphorylated Tau was found in both raft and nonraft fractions with higher abundance in exNef-treated cells (Fig. 4F). Saribas et al. (17Saribas A.S. Cicalese S. Ahooyi T.M. Khalili K. Amini S. Sariyer I.K. HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity.Cell Death Dis. 2017; 8 (28079886): e254210.1038/cddis.2016.467Crossref PubMed Scopus (66) Google Scholar) found no effect of Nef expression in SH-SY5Y cells on the level of phosphorylated Tau. However, that study analyzed phosphorylated Tau in cell lysates as opposed to plasma membranes analyzed in our experiments. Previously, the neuropathic effects of Nef were attributed to its toxicity at high concentrations. However, when we tested for toxic effects of exNef at concentration and conditions of this study, exNef did not cause elevation of the rates of necrosis (Fig. 5A) or apoptosis (Fig. 5B) in SH-SY5Y cells. Two functional assays were employed to test whether exNef causes functional impairment in neural cells. First, we tested excitotoxicity by measuring susceptibility of SH-SY5Y cells to glutamate-induced apoptosis. This pathway of cell injury plays an integral role in pathogenesis of a number of neurodegenerative disorders, including AD and Parkinson's disease (28Choi D.W. Excitotoxic cell death.J. Neurobiol. 1992; 23 (1361523): 1261-127610.1002/neu.480230915Crossref PubMed Scopus (1997) Google Scholar, 29Tzschentke T.M. Glutamatergic mechanisms in different disease states: overview and therapeutical implications: an introduction.Amino Acids. 2002; 23 (12373529): 147-15210.1007/s00726-001-0120-8Crossref PubMed Scopus (47) Google Scholar). Consistent with lack of general toxicity, exNef did not cause an elevation of the proportion of dead cells in culture in the absence of glutamate (Fig. 5C). However, when treated with 50 μm glutamate, the apoptosis of neural cells pretreated with exNef was significantly increased (Fig. 5C). Second, we tested the activity of acetylcholine esterase (AChE), a measure of cholinergic hypofunction, which was linked to cognitive decline (30Madhusudan A. Sidler C. Knuesel I. Accumulation of reelin-positive plaques is accompanied by a decline in basal forebrain projection neurons during normal aging.Eur. J. Neurosci. 2009; 30 (19735296): 1064-107610.1111/j.1460-9568.2009.06884.xCrossref PubMed Scopus (21) Google Scholar). The activity of AChE in cells pretreated with exNef was significantly higher than in cells pretreated with exGFP (Fig. 5D). To confirm that functional changes in neural cells caused by exNef were due to modification of lipid rafts, we tested whether reversal of the effects on lipid rafts would also reverse functional impairment. We used methyl-β-cyclodextrin (MβCD), a nonspecific cholesterol acceptor well-known for its capacity to disrupt lipid rafts. Treatment with 0.25 mm of MβCD for 60 min, a condition previously reported as sufficient to disrupt rafts without causing toxic effects on cells (31Zidovetzki R. Levitan I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies.Biochim. Biophys. Acta. 2007; 1768 (17493580): 1311-132410.1016/j.bbamem.2007.03.026Crossref PubMed Scopus (713) Google Scholar), effectively reversed the elevation of the abundance of lipid rafts
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