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Antiretroviral drugs induce oxidative stress and neuronal damage in the central nervous system.

Akay C, Cooper M, Odeleye A, Jensen BK, White MG, Vassoler F, Gannon PJ, Mankowski J, Dorsey JL, Buch AM, Cross SA, Cook DR, Peña MM, Andersen ES, Christofidou-Solomidou M, Lindl KA, Zink MC, Clements J, Pierce RC, Kolson DL, Jordan-Sciutto KL - J. Neurovirol. (2014)

Bottom Line: HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery.Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death.These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Dental Medicine, University of Pennsylvania, 240 S. 40th St, Rm 312 Levy Bldg, Philadelphia, PA, 19104-6030, USA.

ABSTRACT
HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery. Of note, potential toxicity of antiretroviral drugs in the central nervous system (CNS) remains remarkably underexplored and may contribute to the persistence of HAND in the cART era. Previous studies have shown antiretrovirals (ARVs) to be neurotoxic in the peripheral nervous system in vivo and in peripheral neurons in vitro. Alterations in lipid and protein metabolism, mitochondrial damage, and oxidative stress all play a role in peripheral ARV neurotoxicity. We hypothesized that ARVs also induce cellular stresses in the CNS, ultimately leading to neuronal damage and contributing to the changing clinical and pathological picture seen in HIV-positive patients in the cART era. In this report, we show that ARVs are neurotoxic in the CNS in both pigtail macaques and rats in vivo. Furthermore, in vitro, ARVs lead to accumulation of reactive oxygen species (ROS), and ultimately induction of neuronal damage and death. Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death. These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.

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Antiretroviral drugs induce neuronal damage in vivo. a–d Formalin-fixed, paraffin-embedded tissue sections from hippocampus of pig-tailed macaques that were either uninfected (n = 6), SIV infected but not cART treated (n = 7), or SIV infected and treated with cART (tenofovir, atazanavir, saquinavir, and L-870812a; n = 4) were prepared for immunofluorescent analysis and were triple labeled for MAP2 (red), synaptophysin (green), and GFAP. Sections were visualized by laser confocal microscopy and images were quantified for MAP2, synaptophysin and GFAP expression. a Representative composite images of two cases per group which were stained with MAP2 and synaptophysin are shown. Scale bar = 30 μm. b Quantification shows the resolution of GFAP immunoreactivity in SIV(+)/cART group, compared with SIV(+)/placebo group (one-way ANOVA, *p < 0.05). No changes were observed in MAP2 expression between groups (c), but there were statistically significant decreases in synaptophysin immunoreactivity (d) in SIV(+)/cART group, as compared with SIV(+)/untreated and uninfected groups (one-way ANOVA, *p < 0.05, ns not significant). e, f Fresh-frozen tissue sections from the frontal cortex of pig-tailed macaques that were either uninfected (n = 3), SIV infected but not cART treated (n = 6), or SIV infected and cART treated (n = 6) were used for standard protein extraction and subsequent immunoblotting for the expression of CaMKII. Actin was used as a loading control. A representative immunoblot is shown. Quantification shows statistically significant decreases in CaMKII in the cART-treated group, as compared with the uninfected group or the SIV(+)/untreated group (one-way ANOVA, *p < 0.05). g Whole cell lysates prepared from hippocampus of rats treated for 7 days with AZT/Rit/Saq (n = 4) or vehicle (n = 2) were immunoblotted for synaptophysin and MAP2. A band from the coomassie blue staining is included to control for equal loading and protein degradation
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Fig1: Antiretroviral drugs induce neuronal damage in vivo. a–d Formalin-fixed, paraffin-embedded tissue sections from hippocampus of pig-tailed macaques that were either uninfected (n = 6), SIV infected but not cART treated (n = 7), or SIV infected and treated with cART (tenofovir, atazanavir, saquinavir, and L-870812a; n = 4) were prepared for immunofluorescent analysis and were triple labeled for MAP2 (red), synaptophysin (green), and GFAP. Sections were visualized by laser confocal microscopy and images were quantified for MAP2, synaptophysin and GFAP expression. a Representative composite images of two cases per group which were stained with MAP2 and synaptophysin are shown. Scale bar = 30 μm. b Quantification shows the resolution of GFAP immunoreactivity in SIV(+)/cART group, compared with SIV(+)/placebo group (one-way ANOVA, *p < 0.05). No changes were observed in MAP2 expression between groups (c), but there were statistically significant decreases in synaptophysin immunoreactivity (d) in SIV(+)/cART group, as compared with SIV(+)/untreated and uninfected groups (one-way ANOVA, *p < 0.05, ns not significant). e, f Fresh-frozen tissue sections from the frontal cortex of pig-tailed macaques that were either uninfected (n = 3), SIV infected but not cART treated (n = 6), or SIV infected and cART treated (n = 6) were used for standard protein extraction and subsequent immunoblotting for the expression of CaMKII. Actin was used as a loading control. A representative immunoblot is shown. Quantification shows statistically significant decreases in CaMKII in the cART-treated group, as compared with the uninfected group or the SIV(+)/untreated group (one-way ANOVA, *p < 0.05). g Whole cell lysates prepared from hippocampus of rats treated for 7 days with AZT/Rit/Saq (n = 4) or vehicle (n = 2) were immunoblotted for synaptophysin and MAP2. A band from the coomassie blue staining is included to control for equal loading and protein degradation

Mentions: cART-induced peripheral neuropathy is well documented (Power et al. 2009), and it is likely that cART triggers similar damage to neurons in the CNS. To determine the neurotoxic potential of cART in the CNS, we assessed the effects of an ARV regimen on the expression of synaptophysin and MAP2, indicators of synaptic damage and neuronal loss, respectively, utilizing post-mortem tissue from a well-characterized SIV/pigtail macaque model of HIV CNS disease, which was designed to address the efficacy of CNS penetrant antiretroviral therapy in reducing viral load in the CNS (Zink et al. 2010). In this study, animals infected with SIV either received no cART or received early cART treatment that included tenofovir (NRTI), atazanavir (PI), saquinavir (PI), and L-870812a (integrase inhibitor) 12 days after the virus inoculation. Without cART, 90 % of animals develop neurologic disease within 3 months postinoculation (p.i.). By contrast, animals receiving cART do not develop SIV encephalitis. Rather, they show a rapid reduction in their plasma and CSF viral load followed by continued suppression of SIV replication with maintenance of CD4+ T cell counts until elective euthanasia around day 160 p.i. Additionally, cART-treated animals do not exhibit any outward signs of neurological deficits. Further, our quantitative immunofluorescent analysis of hippocampal tissue sections revealed reduced astrogliosis in the hippocampus of SIV-infected, cART-treated animals (SIV(+)/cART), compared with SIV-infected macaques that did not receive cART ((SIV(+)/placebo)) (Fig. 1b). However, we observed statistically significant decreases in synaptophysin expression in the hippocampi of the SIV(+)/cART group, compared with that in either the uninfected or the SIV(+)/placebo group (Fig. 1a, d). In addition, examination of the expression of a second marker of synaptodendritic integrity, calmodulin kinase II (CaMKII), by immunoblotting showed that CaMKII levels were significantly lower in the frontal cortex in the SIV(+)/cART macaques than in their SIV(+)/placebo counterparts (Fig. 1e, f). CaMKII is highly expressed in neurons of macaque hippocampus and frontal cortex, whereas its expression in other cell types, including microglia, infiltrating macrophages, and multinucleated giant cells, is minimal; thus, the differences in CaMKII expression in frontal cortex of the animals from our experimental groups are neuron specific (Gupta et al. 2010). Our results demonstrate synaptic injury in the presence of cART despite effective control of SIV replication in the periphery and CNS. Interestingly, we did not observe changes in MAP2 fluorescence in the hippocampus of infected and/or cART-treated animals compared with untreated/uninfected animals (Fig. 1a, c).Fig. 1


Antiretroviral drugs induce oxidative stress and neuronal damage in the central nervous system.

Akay C, Cooper M, Odeleye A, Jensen BK, White MG, Vassoler F, Gannon PJ, Mankowski J, Dorsey JL, Buch AM, Cross SA, Cook DR, Peña MM, Andersen ES, Christofidou-Solomidou M, Lindl KA, Zink MC, Clements J, Pierce RC, Kolson DL, Jordan-Sciutto KL - J. Neurovirol. (2014)

Antiretroviral drugs induce neuronal damage in vivo. a–d Formalin-fixed, paraffin-embedded tissue sections from hippocampus of pig-tailed macaques that were either uninfected (n = 6), SIV infected but not cART treated (n = 7), or SIV infected and treated with cART (tenofovir, atazanavir, saquinavir, and L-870812a; n = 4) were prepared for immunofluorescent analysis and were triple labeled for MAP2 (red), synaptophysin (green), and GFAP. Sections were visualized by laser confocal microscopy and images were quantified for MAP2, synaptophysin and GFAP expression. a Representative composite images of two cases per group which were stained with MAP2 and synaptophysin are shown. Scale bar = 30 μm. b Quantification shows the resolution of GFAP immunoreactivity in SIV(+)/cART group, compared with SIV(+)/placebo group (one-way ANOVA, *p < 0.05). No changes were observed in MAP2 expression between groups (c), but there were statistically significant decreases in synaptophysin immunoreactivity (d) in SIV(+)/cART group, as compared with SIV(+)/untreated and uninfected groups (one-way ANOVA, *p < 0.05, ns not significant). e, f Fresh-frozen tissue sections from the frontal cortex of pig-tailed macaques that were either uninfected (n = 3), SIV infected but not cART treated (n = 6), or SIV infected and cART treated (n = 6) were used for standard protein extraction and subsequent immunoblotting for the expression of CaMKII. Actin was used as a loading control. A representative immunoblot is shown. Quantification shows statistically significant decreases in CaMKII in the cART-treated group, as compared with the uninfected group or the SIV(+)/untreated group (one-way ANOVA, *p < 0.05). g Whole cell lysates prepared from hippocampus of rats treated for 7 days with AZT/Rit/Saq (n = 4) or vehicle (n = 2) were immunoblotted for synaptophysin and MAP2. A band from the coomassie blue staining is included to control for equal loading and protein degradation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3928514&req=5

Fig1: Antiretroviral drugs induce neuronal damage in vivo. a–d Formalin-fixed, paraffin-embedded tissue sections from hippocampus of pig-tailed macaques that were either uninfected (n = 6), SIV infected but not cART treated (n = 7), or SIV infected and treated with cART (tenofovir, atazanavir, saquinavir, and L-870812a; n = 4) were prepared for immunofluorescent analysis and were triple labeled for MAP2 (red), synaptophysin (green), and GFAP. Sections were visualized by laser confocal microscopy and images were quantified for MAP2, synaptophysin and GFAP expression. a Representative composite images of two cases per group which were stained with MAP2 and synaptophysin are shown. Scale bar = 30 μm. b Quantification shows the resolution of GFAP immunoreactivity in SIV(+)/cART group, compared with SIV(+)/placebo group (one-way ANOVA, *p < 0.05). No changes were observed in MAP2 expression between groups (c), but there were statistically significant decreases in synaptophysin immunoreactivity (d) in SIV(+)/cART group, as compared with SIV(+)/untreated and uninfected groups (one-way ANOVA, *p < 0.05, ns not significant). e, f Fresh-frozen tissue sections from the frontal cortex of pig-tailed macaques that were either uninfected (n = 3), SIV infected but not cART treated (n = 6), or SIV infected and cART treated (n = 6) were used for standard protein extraction and subsequent immunoblotting for the expression of CaMKII. Actin was used as a loading control. A representative immunoblot is shown. Quantification shows statistically significant decreases in CaMKII in the cART-treated group, as compared with the uninfected group or the SIV(+)/untreated group (one-way ANOVA, *p < 0.05). g Whole cell lysates prepared from hippocampus of rats treated for 7 days with AZT/Rit/Saq (n = 4) or vehicle (n = 2) were immunoblotted for synaptophysin and MAP2. A band from the coomassie blue staining is included to control for equal loading and protein degradation
Mentions: cART-induced peripheral neuropathy is well documented (Power et al. 2009), and it is likely that cART triggers similar damage to neurons in the CNS. To determine the neurotoxic potential of cART in the CNS, we assessed the effects of an ARV regimen on the expression of synaptophysin and MAP2, indicators of synaptic damage and neuronal loss, respectively, utilizing post-mortem tissue from a well-characterized SIV/pigtail macaque model of HIV CNS disease, which was designed to address the efficacy of CNS penetrant antiretroviral therapy in reducing viral load in the CNS (Zink et al. 2010). In this study, animals infected with SIV either received no cART or received early cART treatment that included tenofovir (NRTI), atazanavir (PI), saquinavir (PI), and L-870812a (integrase inhibitor) 12 days after the virus inoculation. Without cART, 90 % of animals develop neurologic disease within 3 months postinoculation (p.i.). By contrast, animals receiving cART do not develop SIV encephalitis. Rather, they show a rapid reduction in their plasma and CSF viral load followed by continued suppression of SIV replication with maintenance of CD4+ T cell counts until elective euthanasia around day 160 p.i. Additionally, cART-treated animals do not exhibit any outward signs of neurological deficits. Further, our quantitative immunofluorescent analysis of hippocampal tissue sections revealed reduced astrogliosis in the hippocampus of SIV-infected, cART-treated animals (SIV(+)/cART), compared with SIV-infected macaques that did not receive cART ((SIV(+)/placebo)) (Fig. 1b). However, we observed statistically significant decreases in synaptophysin expression in the hippocampi of the SIV(+)/cART group, compared with that in either the uninfected or the SIV(+)/placebo group (Fig. 1a, d). In addition, examination of the expression of a second marker of synaptodendritic integrity, calmodulin kinase II (CaMKII), by immunoblotting showed that CaMKII levels were significantly lower in the frontal cortex in the SIV(+)/cART macaques than in their SIV(+)/placebo counterparts (Fig. 1e, f). CaMKII is highly expressed in neurons of macaque hippocampus and frontal cortex, whereas its expression in other cell types, including microglia, infiltrating macrophages, and multinucleated giant cells, is minimal; thus, the differences in CaMKII expression in frontal cortex of the animals from our experimental groups are neuron specific (Gupta et al. 2010). Our results demonstrate synaptic injury in the presence of cART despite effective control of SIV replication in the periphery and CNS. Interestingly, we did not observe changes in MAP2 fluorescence in the hippocampus of infected and/or cART-treated animals compared with untreated/uninfected animals (Fig. 1a, c).Fig. 1

Bottom Line: HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery.Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death.These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, School of Dental Medicine, University of Pennsylvania, 240 S. 40th St, Rm 312 Levy Bldg, Philadelphia, PA, 19104-6030, USA.

ABSTRACT
HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery. Of note, potential toxicity of antiretroviral drugs in the central nervous system (CNS) remains remarkably underexplored and may contribute to the persistence of HAND in the cART era. Previous studies have shown antiretrovirals (ARVs) to be neurotoxic in the peripheral nervous system in vivo and in peripheral neurons in vitro. Alterations in lipid and protein metabolism, mitochondrial damage, and oxidative stress all play a role in peripheral ARV neurotoxicity. We hypothesized that ARVs also induce cellular stresses in the CNS, ultimately leading to neuronal damage and contributing to the changing clinical and pathological picture seen in HIV-positive patients in the cART era. In this report, we show that ARVs are neurotoxic in the CNS in both pigtail macaques and rats in vivo. Furthermore, in vitro, ARVs lead to accumulation of reactive oxygen species (ROS), and ultimately induction of neuronal damage and death. Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death. These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.

Show MeSH
Related in: MedlinePlus