Modification of lipid rafts by extracellular vesicles carrying HIV-1 protein Nef induces redistribution of amyloid precursor protein and Tau, causing neuronal dysfunction

Abstract

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) 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. Byrd D.A. Cherner M. Clifford D.B. Cinque P. Epstein L.G. Goodkin K. Gisslen M. Grant I. Heaton R.K. Joseph J. Marder K. et al.Updated research nosology for HIV-associated neurocognitive disorders.Neurology. 2007; 69 (17914061): 1789-179910.1212/01.WNL.0000287431.88658.8bCrossref PubMed Scopus (1703) Google Scholar). Effective treatment of HIV infection has reduced the rate of progression and severity of HAND symptoms, but the overall prevalence of HAND (up to 50% of HIV-infected subjects) has remained unchanged (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, 3Cysique L.A. Brew B.J. Prevalence of non-confounded HIV-associated neurocognitive impairment in the context of plasma HIV RNA suppression.J. Neurovirol. 2011; 17 (21416169): 176-18310.1007/s13365-011-0021-xCrossref PubMed Scopus (115) Google Scholar, 4Brew B.J. Crowe S.M. Landay A. Cysique L.A. Guillemin G. Neurodegeneration and ageing in the HAART era.J. Neuroimmune Pharmacol. 2009; 4 (19067177): 163-17410.1007/s11481-008-9143-1Crossref PubMed Scopus (138) Google Scholar). With aging of HIV-infected population and dramatically extended life expectancy of people living with HIV (PLWH), this condition constitutes a significant health problem. HAND has clinical features characteristic for a neurodegenerative disorder: a spectrum of declining cognitive functions, behavioral changes, and motor impairment. Pathologically, there may or may not be focal damage to the gray matter, and there are abnormalities in the white matter (4Brew B.J. Crowe S.M. Landay A. Cysique L.A. Guillemin G. Neurodegeneration and ageing in the HAART era.J. Neuroimmune Pharmacol. 2009; 4 (19067177): 163-17410.1007/s11481-008-9143-1Crossref PubMed Scopus (138) Google Scholar). Overall, HAND is consistent with the definition of a neurodegenerative disease as a condition with progressive neuronal damage and chronic loss of neurons (4Brew B.J. Crowe S.M. Landay A. Cysique L.A. Guillemin G. Neurodegeneration and ageing in the HAART era.J. Neuroimmune Pharmacol. 2009; 4 (19067177): 163-17410.1007/s11481-008-9143-1Crossref PubMed Scopus (138) Google Scholar). In treated patients, however, pathological features may be modest, pointing to more subtle changes to the synaptodendritic architecture manifesting in functional impairment. However, the pathogenetic mechanisms of neural impairment in PLWH are not fully understood. 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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D. impairs cholesterol 4 PubMed Scopus Google Scholar). changes in ABCA1 and lipid rafts by HIV Nef in were to in neurons by B. B. L. M. N. D. infection impairs cholesterol metabolism in neuronal 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). not to be with HIV to be by Nef is secreted HIV-infected cells in extracellular vesicles These the on cholesterol metabolism in cells in as Nef in HIV-infected cells M. M. N. M. D. HIV protein Nef and of cells in of J. 2014; PubMed Scopus Google Scholar, N. A. D. L. Pushkarsky H. M. A. B. A. M. et HIV protein Nef lipid rafts inflammatory in PubMed Scopus Google Scholar). were in levels in in the of HIV-infected subjects treated with S. K. J. S. N. S. G. K. and plasma extracellular vesicles and with pathogenesis in chronic HIV 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, J. K. V. A. M. protein Nef is in plasma of of HIV-infected with plasma HIV PubMed Scopus Google Scholar, M. M. HIV Nef is cells in and is in the plasma of HIV-infected Res. 2011; PubMed Scopus Google Scholar). a role for Nef in pathogenesis of but the mechanisms M. M. HIV Nef is cells in and is in the plasma of HIV-infected Res. 2011; PubMed Scopus Google Scholar, S. K. S. HIV-1 Nef is in extracellular vesicles for PubMed Scopus Google Scholar, P. S. M. A. H. A. S. M.P. HIV impairment of the is by of Nef Neurovirol. 2016; PubMed Scopus Google Scholar). of Nef has in S. K. S. HIV-1 Nef is in extracellular vesicles for PubMed Scopus Google Scholar, K. M. A. Nef of HIV-1 is for human PubMed Scopus Google Scholar, A. B. Nef the plasma of with HIV-associated dementia in neural Neurovirol. 2016; PubMed Scopus Google the level of of of Nef in subjects is to be a of neural In this study, we a contribute to the neuronal damage by lipid rafts and accumulation of in these membrane In this study, we by cells with either Nef or in N. A. D. L. Pushkarsky H. M. A. B. A. M. et HIV protein Nef lipid rafts inflammatory in PubMed Scopus Google Scholar, L. A. Pushkarsky B. A.A. N. D. L. M. of HIV replication by protein the lipid 2020; these as exNef and of were for and and for thus for for extracellular vesicles A. D. H. L. et for of extracellular vesicles a of the for and of the PubMed Scopus Google Scholar). not N. A. D. L. Pushkarsky H. M. A. B. A. M. et HIV protein Nef lipid rafts inflammatory in PubMed Scopus Google Scholar). ExNef Nef of of protein N. A. D. L. Pushkarsky H. M. A. B. A. M. et HIV protein Nef lipid rafts inflammatory in PubMed Scopus Google Nef of was for and was to cells in protein of the of exNef in this was of is the of Nef in the of with viral J. K. V. A. M. protein Nef is in plasma of of HIV-infected with plasma HIV PubMed Scopus Google Scholar, J. M. HIV in Scholar). was in the of by cells on cholesterol metabolism and lipid rafts are to the of by HIV-infected cells with not such N. A. D. L. Pushkarsky H. M. A. B. A. M. et HIV protein Nef lipid rafts inflammatory in PubMed Scopus Google Scholar). human cells up the we with was the 4 but increased in a the 4 and was the cells when of was to the and with cells for suggesting were taken up by Nef we Nef with with the cells for and to the was the cells a and intracellular abundance of Nef to for and Thus, exNef was taken up by cells Nef to the Nef in with HIV-1 or with as well as to cells by with the recombinant Nef or reduces the abundance of ABCA1 and the rate of cholesterol efflux in (10Mujawar Z. Rose H. Morrow M.P. Pushkarsky L. N. A. M. D. impairs cholesterol 4 PubMed Scopus Google Scholar, N. A. D. L. Pushkarsky H. M. A. B. A. M. et HIV protein Nef lipid rafts inflammatory in PubMed Scopus Google Scholar). were when we the of of with exNef on neural The abundance of and ABCA1 and the rate of cholesterol efflux were reduced by and and of Nef was of the abundance of lipid of exNef with cells for to of the abundance of lipid rafts as by with and the of exNef be to Nef and not to a may be by by we cells with recombinant Nef Nef caused of the abundance of lipid rafts as by and or the were with exNef and higher of to was with N. A. D. L. Pushkarsky H. M. A. B. A. M. et HIV protein Nef lipid rafts inflammatory in PubMed Scopus Google Scholar). we cells with and lipid rafts plasma the of and lipid were as lipid the and we compared lipid rafts cells treated with exNef and we lipid cells treated with exNef more cholesterol and more cells treated with the of the of cholesterol and was in cells treated with exNef and These are consistent with lipid rafts in cells treated with exNef more and of the abundance of lipid rafts may of the abundance of lipid the of exNef on the abundance of in and elevated abundance of APP and Tau in cells treated with exNef and was with for APP and Tau on was elevated in cells treated with exNef as compared with cells treated with and to the of amyloid Aβ42. The abundance of in cells treated with exNef compared with cells treated with the abundance of in the and of was the of the was in the abundance of and in the cells in of Tau is of Tau Tau and pathological PubMed Scopus Google Scholar, I. K. of Tau in the of in PubMed Scopus Google Scholar). we the abundance of Tau in the plasma of neural cells of cells with exNef resulted in in the abundance of in with increased abundance of Tau we treatment with exNef increased the abundance of and of may on the to of Tau H. S. B. J. G. I. of neuronal Tau protein as a of extracellular 2016; Full Text Full Text PDF PubMed Scopus Google on the to the elevated inflammatory characteristic for of HIV-infected The increased and by exNef was further by in phosphorylation of in cells treated with exNef we the cellular of APP and Tau in cells of APP and Tau in the cells is shown in 4 and of APP and Tau the plasma and with lipid The of these with lipid rafts was as D. Z. G. S. and of in J. 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In the HIV in cells and as well as in but not in neurons (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). neuronal impairment and neuronal characteristic for neurodegenerative disorders are not caused by the but are through of HIV on HIV infection in the brain in treated a HIV A. The HIV in and PubMed Scopus Google and HIV-infected cells extracellular vesicles viral and S. K. S. HIV-1 Nef is in extracellular vesicles for PubMed Scopus Google Scholar, A. B. Nef the plasma of with HIV-associated dementia in neural Neurovirol. 2016; PubMed Scopus Google Scholar). In this study, we a of these proteins, to the neuronal impairment through on lipid metabolism and lipid treatment of neural cells with exNef resulted in the of exNef by the cells a in the abundance of ABCA1 and the rate of cholesterol in elevated the abundance and the of lipid play a key role in the pathogenesis of amyloid precursor protein and were increased in lipid rafts neural increased abundance of and phosphorylation of Tau in cells treated with was with functional of neural cells to was as was and these were reversed by of lipid treatment of cells with exNef to increased activation of and with through the and a between HIV replication and the Res. 2010; PubMed Scopus Google Scholar). In mice with the abundance of ABCA1 in brain was and the abundance of APP were shown to the S. S. G. The role of in PubMed Scopus Google Scholar). reduced levels of ABCA1 and increased levels of lipid rafts in brain tissue of HIV-infected human subjects diagnosed with HAND. Tau and APP were increased in HAND and to with Nef These are consistent with the key of the exNef impairs ABCA1 and of lipid rafts in brain to accumulation of in and neuronal to the HAND The HAND is a of chronic brain to chronic HIV infection. of in the and dysfunction and and be in HIV M. the neuronal and PubMed Scopus Google Scholar). However, to pathogenesis of role as a of HAND has not the role of cholesterol and of lipid in is and Res. 2016; PubMed Scopus Google Scholar). Thus, neurodegeneration and in HIV infection may a in lipid impairment. the of Nef on lipid rafts may neurodegeneration and of secreted by HIV-infected cells may contribute to of neurons is a key of stages of neurodegeneration and HIV proteins, such as and caused in neuronal cells in M. the neuronal and PubMed Scopus Google Scholar). In Nef was in and of PLWH and was in pathogenesis of HAND S. K. S. HIV-1 Nef is in extracellular vesicles for PubMed Scopus Google Scholar, A. B. Nef the plasma of with HIV-associated dementia in neural Neurovirol. 2016; PubMed Scopus Google Scholar, D.J. Rose HIV Nef in with neurological Neurovirol. PubMed Scopus Google Scholar). However, of HIV be in the brain treatment to through may be to the impairment of cholesterol metabolism and lipid rafts A. J. P. metabolism to through of membrane and 4 PubMed Scopus Google Scholar). HAND has many with Alzheimer's disease, such as A. Volsky D.J. in brain tissue with HIV-associated neurocognitive Alzheimer's disease, and Neuroimmune Pharmacol. PubMed Scopus Google and increased abundance and of intracellular A. A. G. HIV infection contribute to increased and to neurodegeneration and Alzheimer's Neurovirol. 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