Dataset Information

HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons.

ABSTRACT:

Introduction

Mitochondrial dysregulation is a key event in HIV-1 infection. Recent studies have suggested that age-related neurodegenerative disorders are associated with increased mitochondrial DNA (mtDNA) damage. As accelerated ageing was found in HIV-1 patients, we hypothesized that HIV-1 infection or HIV-1 proteins can lead to mtDNA damage. Unrepaired mtDNA impairs mitochondrial function, which can lead to oxidative stress and cell death. Investigations of mechanisms of mtDNA damage are limited by the lack of available human models.

Methods

We compared mtDNA or nDNA (nuclear DNA) damage in human cortical neurons and PBMC cells. Primary neuronal cultures were incubated with conditioned media from HIV-1 infected PBMC, or HIV-1 viral proteins Tat or Vpr. Total genomic DNA (nuclear and mtDNA) was isolated using the QIAamp Kit. Nuclear and mtDNA were amplified using the long q-PCR/Gene Amp XL Kit. Real-Time RT-PCR using mitochondrial energy metabolism array was performed to assess mitochondrial energy metabolism markers. Superoxide dismutase (SOD) activity in neuronal cells was measured by the OxiSelect SOD Activity Assay. Reactive oxygen species (ROS) were determined by the confocal microscopy. ATP levels were analyzed using ATP determination biochemical assay. Mitochondrial, cytoplasmic and nuclear proteins were studied by quantitative western-blot assay.

Results

We show that both treatment of neuronal cells with HIV-1 conditioned media, or infection of PBMC with HIV-1 increase mtDNA damage in cells. mtDNA damage was also seen in neuronal cells, incubated with HIV-1 proteins, Tat and Vpr. Next, we confirmed that mtDNA damage was also increased in neuronal cells transfected by Tat expressing plasmids. We showed that mtDNA was not damaged in neuronal cells following treatment with heat inactivated HIV-1 or Tat protein. Further, we demonstrated that HIV-1 or Tat caused more mtDNA damage compared to nuclear DNA damage in neuronal cells. Finally, we showed that Tat dysregulates RNA expression of several genes regulating mitochondrial energy metabolism, suggesting involvement of Tat in mitochondrial bioenergetics in human neurons. Finally, our hypothesis was confirmed by qWestern analysis of mitochondrial and apoptotic proteins demonstrating the accumulation of apoptotic Bax and Bad proteins in mitochondrial fraction of Tat-treated neuronal cells, suggesting toxic effects of Tat on mitochondrial survival.

Conclusion

We showed an increase of mtDNA damage in primary neurons, treated with HIV-1 proteins and in PBMC, infected with HIV-1. Increased mtDNA damage can lead to neurodegeneration, and cause neuronal apoptosis. Our system presents a suitable model to study mtDNA changes during HIV-1 infection.

SUBMITTER: Darbinian N

PROVIDER: S-EPMC7837619 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Publications

HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons.

Darbinian Nune N Darbinyan Armine A Merabova Nana N Selzer Michael E ME Amini Shohreh S

Journal of HIV and AIDS 20200831 1

<h4>Introduction</h4>Mitochondrial dysregulation is a key event in HIV-1 infection. Recent studies have suggested that age-related neurodegenerative disorders are associated with increased mitochondrial DNA (mtDNA) damage. As accelerated ageing was found in HIV-1 patients, we hypothesized that HIV-1 infection or HIV-1 proteins can lead to mtDNA damage. Unrepaired mtDNA impairs mitochondrial function, which can lead to oxidative stress and cell death. Investigations of mechanisms of mtDNA damage ...[more]

PMID: 33506104

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Project description:The neuropathological abnormalities of human immunodeficiency virus (HIV)-1 patients abusing illicit drugs suggest extensive interactions between the two agents, thereby leading to increased rate of progression to neurodegeneration. The role of HIV-1 transactivating protein, Tat has been elucidated in mediating neuronal damage via apoptosis, a hallmark of HIV-associated dementia (HAD), however the underlying mechanisms involved in enhanced neurodegeneration by illicit drugs remain elusive. In this study, we demonstrated that morphine enhances HIV-Tat induced toxicity in human neurons and neuroblastoma cells. Enhanced toxicity by Tat and morphine was accompanied by increased numbers of TUNEL positive apoptotic neurons, elevated caspase-3 levels and decreased ratio of anti- and pro-apoptotic proteins, Bcl2/Bax. Tat and morphine together elicited high levels of reactive oxygen species that were NADPH dependent. Significant alterations in mitochondrial membrane homeostasis were also observed with co-exposure of these agents. Extensive studies of mitogen activated protein kinase (MAPK) signaling pathways revealed the involvement of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways in enhanced toxicity of Tat and morphine. In addition to this, we found that pre-treatment of cells with platelet derived growth factor (PDGF-BB) protected neurons from HIV-Tat and morphine induced damage. PDGF-BB alleviated ROS production, maintained mitochondrial membrane potential, decreased caspase-3 activation and hence protected the cells from undergoing apoptosis. PDGF-BB mediated protection against Tat and morphine involved the phosphatidylinositol-3 kinase (PI3K) pathway, as specific inhibitor of PI3K abrogated the protection conferred by PDGF-BB. This study demonstrates the mechanism of enhanced toxicity in human neurons subjected to co-exposure of HIV protein Tat and morphine, thus implying its importance in HIV positive drug abusers, where damage to the brain is reported to be more severe than non-drug abusers. We have also showed for the first time that PDGF-BB can protect against simultaneous exposure of Tat and morphine, strengthening its role as a neuroprotective agent that could be considered for therapeutic intervention.

Project description:Synergistic impairment of the blood-brain barrier (BBB) induced by methamphetamine (METH) and HIV-Tat protein increases the risk of HIV-associated neurocognitive disorders (HAND) in HIV-positive METH abusers. Studies have shown that oxidative stress plays a vital role in METH- and HIV-Tat-induced damage to the BBB but have not clarified the mechanism. This study uses the human brain microvascular endothelial cell line hCMEC/D3 and tree shrews to investigate whether the transient receptor potential melastatin 2 (TRPM2) channel, a cellular effector of the oxidative stress, might regulate synergistic damage to the BBB caused by METH and HIV-Tat. We showed that METH and HIV-Tat damaged the BBB in vitro, producing abnormal cell morphology, increased apoptosis, reduced protein expression of the tight junctions (TJ) including Junctional adhesion molecule A (JAMA) and Occludin, and a junctional associated protein Zonula occludens 1 (ZO1), and increased the flux of sodium fluorescein (NaF) across the hCMEC/D3 cells monolayer. METH and HIV-Tat co-induced the oxidative stress response, reducing catalase (CAT), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) activity, as well as increased reactive oxygen species (ROS) and malonaldehyde (MDA) level. Pretreatment with n-acetylcysteine amide (NACA) alleviated the oxidative stress response and BBB damage characterized by improving cell morphology, viability, apoptosis levels, TJ protein expression levels, and NaF flux. METH and HIV-Tat co-induced the activation and high protein expression of the TRPM2 channel, however, early intervention using 8-Bromoadenosine-5'-O-diphosphoribose (8-Br-ADPR), an inhibitor of TPRM2 channel, or TRPM2 gene knockdown attenuated the BBB damage. Oxidative stress inhibition reduced the activation and high protein expression of the TRPM2 channel in the in vitro model, which in turn reduced the oxidative stress response. Further, 8-Br-ADPR attenuated the effects of METH and HIV-Tat on the BBB in tree shrews-namely, down-regulated TJ protein expression and increased BBB permeability to Evans blue (EB) and NaF. In summary, the TRPM2 channel can regulate METH- and HIV-Tat-induced oxidative stress and BBB injury, giving the channel potential for developing drug interventions to reduce BBB injury and neuropsychiatric symptoms in HIV-infected METH abusers.

Dataset Information

HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons.

Introduction

Methods

Results

Conclusion

Publications

HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons.

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