Elsevier

Molecular Brain Research

Volume 133, Issue 2, 18 February 2005, Pages 299-306
Molecular Brain Research

Research report
Proteomic analysis of oxidatively modified proteins induced by the mitochondrial toxin 3-nitropropionic acid in human astrocytes expressing the HIV protein tat

https://doi.org/10.1016/j.molbrainres.2004.10.024Get rights and content

Abstract

The human immunodeficiency virus (HIV)-Tat protein has been implicated in the neuropathogenesis of HIV infection. However, its role in modulating astroglial function is poorly understood. Astrocyte infection with HIV has been associated with rapid progression of dementia. Intracellularly expressed Tat is not toxic to astrocytes. In fact, intracellularly expressed Tat offers protection against oxidative stress-related toxins such as the mitochondrial toxin 3-nitroproprionic acid (3-NP). In the current study, human astrocytes expressing Tat (SVGA-Tat) and vector controls (SVGA-pcDNA) were each treated with the irreversible mitochondrial complex II inhibitor 3-NP. Proteomics analysis was utilized to identify changes in protein expression levels. By coupling 2D fingerprinting and identification of proteins by mass spectrometry, actin, heat shock protein 90, and mitochondrial single-stranded DNA binding protein were identified as proteins with increased expression, while lactate dehydrogenase had decreased protein expression levels in SVGA-Tat cells treated with 3-NP compared to SVGA-pcDNA cells treated with 3-NP. Oxidative damage can lead to several events including loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, ultimately leading to neuronal death. Identification of specific proteins protected from oxidation is a crucial step in understanding the interaction of Tat with astrocytes. In the current study, proteomics also was used to identify proteins that were specifically oxidized in SVGA-pcDNA cells treated with 3-NP compared to SVGA-Tat cells treated with 3-NP. We found β-actin, calreticulin precursor protein, and synovial sarcoma X breakpoint 5 isoform A to have increased oxidation in control SVGA-pcDNA cells treated with 3-NP compared to SVGA-Tat cells treated with 3-NP. These results are discussed with reference to potential involvement of these proteins in HIV dementia and protection of astrocytes against oxidative stress by the HIV virus, a prerequisite for survival of a viral host cell.

Introduction

It is estimated that one-third of adults infected with human immunodeficiency virus (HIV-1) develop dementia (HIVD) [19]. The pathological features associated with human immunodeficiency virus dementia (HIVD) include microglial cell activation, astrocytosis, decreased synaptic and dendritic density, and selective neuronal loss [30]. In patients with HIV-1 infection, significant neuronal loss and dysfunction occur even though neurons are rarely infected [31], [38]. The most commonly infected cell types in brain are microglia, macrophages and to some extent astrocytes, although limited viral replication is produced in astrocytes [22], [23], [31], [38], [46]. In cell cultures, HIV-1 infection of astrocytes results in an initial productive but non-cytopathogenic infection that diminishes to a viral persistence or latent state [33]. The major barrier to HIV-1 infection of primary astrocytes is at virus entry. Astrocytes have no intrinsic intracellular restriction to efficient HIV-1 replication [6]. Astrocytes may serve as a reservoir for the virus inducing neuronal damage by releasing cellular and viral products or loss of neuronal support functions.

In HIV-infected astrocytes, the regulatory gene tat is over expressed [36] and mRNA levels for Tat are elevated in brain extracts from individuals with HIV-1 dementia [57]. The HIV-1 protein Tat transactivates viral and cellular gene expression, is actively secreted into the extra cellular environment mainly from astrocytes, microglia and macrophages, and is taken up by neighboring uninfected cells such as neurons [11]. The HIV-1 protein Tat released from astrocytes reportedly produces trimming of neurites, mitochondrial dysfunction and cell death in neurons [11]. Intracellular Tat is not toxic to astrocytes. In fact, Tat produced in astrocytic cell lines was able to protect astrocytes from cellular injury induced by 3-nitropropionic acid (3-NP), a mitochondrial toxin; whereas, HeLa cells expressing Tat were not protected [11]. This finding demonstrates that Tat is a neurotoxin at distant sites while protecting the environment where it is produced.

The mechanism in which Tat is capable of protecting astrocytes from cellular death is unknown. In this study, we applied proteomics to identify proteins that change expression levels and proteins that are protected from oxidation when Tat is expressed in human astrocytes, (SVGA-Tat) cells, compared to SVGA cells expressing only the vector (SVGA-pcDNA), after both were treated with the mitochondrial toxin and oxidative stress inducer 3-nitroproprionic acid. 3-NP is used since it is known to cause mitochondrial dysfunction and oxidative stress in the brain [24], [25], and Tat exposed to neurons results in mitochondrial dysfunction and oxidative stress [11], [43]. The proteomics-identified proteins provide insight into potential mechanisms by which astrocytes may act as a reservoir for the HIV virus.

Section snippets

Sample collection

SVGA-Tat- and vector (SVGA-pcDNA)-expressing cells were constructed as previously described [11]. All cell lines were maintained in DMEM media with 5% fetal serum albumin and 1% antibiotic/antimycotic in an incubator at 37 °C and 5% CO2. Cells were collected in isolation buffer containing protease inhibitors: 4 μg/mL leupeptin, 4 μg/mL pepstatin A, 5 μg/mL aprotinin, 20 μg/mL type II-s soybean trypsin inhibitor, 0.2 mM PMSF, 2 mM EDTA, 2 mM EGTA, 20 mM HEPES at pH 7.4, sonicated for 5 s to

Results

In the current study, we tested the hypothesis that inhibition of mitochondrial complex II by 3-NP, a process associated with oxidative stress in rat brain [24], [25], in astrocytes expressing Tat would protect proteins from oxidation. We utilized proteomics to investigate the effect that 3-NP has on proteins in SVGA cells expressing the HIV protein Tat. Proteomic analysis permits identification of differences in protein expression levels and oxidation levels of proteins as measured by protein

Discussion

Significant neuronal loss and dysfunction occurs in HIV dementia even though neurons are rarely infected. Astrocytes may serve as a reservoir for the virus inducing neuronal damage by releasing cellular and viral products or loss of neuronal support functions. We recently demonstrated that astrocytes expressing Tat were protected against oxidative insults including 3-NP, while other cell types expressing Tat experienced cell death [11]. In SVGA-Tat cells treated with 3-NP, a mitochondrial

Acknowledgements

This work was supported in part by grants from NIH (MH64409; AG-10836; AG-05119) to D.A.B. and (RO1 NS39253; P20 RR15592) to A.N.

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