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