Please use this identifier to cite or link to this item: http://hdl.handle.net/2307/5971
Title: Investigating the oxidative/nitrosative stress response induced by HIV-1Tat protein in glial and neuronal cells
Other Titles: Studio della risposta allo stress ossidativo/nitrosativo indotto dalla proteina TAT del virus HIV in cellule gliali e neuronali
Authors: Mastrantonio, Roberta
metadata.dc.contributor.advisor: Persichini, Tiziana
Keywords: Oxidative stress
HIV
Neurodegeneration
HAND
TAT
Issue Date: 16-Feb-2016
Publisher: Università degli studi Roma Tre
Abstract: In the last years, evidence has been accumulated suggesting that oxidative stress plays a major role in the HIV associated neuropathogenesis. Oxidative stress is defined as an imbalance between the pro-oxidant and the anti-oxidant systems, with the shift towards the pro-oxidant system. To balance ROS levels and counteract their toxic effects, cells employ several enzymatic and non-enzymatic antioxidant systems. The transcription factor Nrf2, is an important regulator of cell survival and adaptive mechanisms, in conditions of elevated oxidative stress, translocates into the nucleus, binds to the promoter regions of many phase II detoxifying and antioxidant genes, the antioxidant-response elements. As previously demonstrated in our lab, the neurotoxic effects of the HIV protein Tat are associated with the stimulation of the NMDA receptors that in turn induce an increased spermine oxidase activity and a consequent ROS accumulation. Since in many cell types ROS are also able to induce an antioxidant response, we analyzed the effect of Tat-induced spermine oxidase activation on the Nrf2/ARE pathway in SH-SY5Y human neuroblastoma cells. We found out that Tat was able to induce Nrf2 activation, and this effect was reverted to control levels by chlorexidine, a strong competitive inhibitor of spermine oxidase. Next we evaluated, by RT-qPCR analysis, the expression of some ARE genes in Tat-treated cells. The results indicate a significant up-regulation of all the genes analyzed (HO-1, SOD 1, SOD 2, NQO1 and CAT) at 4h post-treatment. This effect was reverted to control levels by chlorexidine pre-treatment. Since Tat-induced spermine oxidase activation is mediated by the stimulation of NMDAR, we analyzed Nrf2 activation and ARE genes expression in Tat-stimulated cells pretreated with MK801, a specific NMDAR antagonist. We found out that MK801 completely prevented Tat-induced Nrf2 activation and ARE genes expression thus indicating the involvement of NMDAR in this pathway. The results strongly suggest a role for this receptor and for spermine oxidase in Tat-induced antioxidant response in human neuronal cells. Since in neurons the activation of NMDAR leads to enhancement of NO production by the calcium-dependent neuronal NOS, and NO is an endogenous inducer of Nrf2-dependent phase-2 enzymes both in vitro and in vivo, we also analyzed the role of NO in Nrf2-ARE pathway activation in Tat-stimulated neuronal cells. The results showed that L-NAME, a specific NOS inhibitor, was able to significantly reduce the nuclear translocation of Nrf2 induced by Tat. Next, we studied the involvement of NO in Tat-induced SMO activation. Our data indicate that the pre-treatment of SH-SY5Y cells with L-NAME significantly reduced Tat-induced spermine oxidase activation. Therefore, we wondered whether Tat could directly affect the enzyme activity by S-nitrosylation. To this aim we carried out biotin-switch assay on protein extracts of SH-SY5Y cells treated with Tat and found out that, although the treatment with NO donors induced SMO S-nitrosylation, the treatment with Tat was not able to induce this protein modification. Thus, further studies are needed to deeply understand the effects of NO in Tat-elicited SMO activation that could be due to modifications of other targets. Moreover, since oxidative stress leads to protein misfolding and aggregation, we evaluated the induction of p62-mediated selective autophagy in SH-SY5Y cells treated with Tat. We found out that Tat was able to induce a 2/3-fold increase of p62 expression at 8h, 16, 24h post-treatment. Conversely we didn’t observe any induction of LC3 in the same experimental conditions. Next, we evaluated the presence of Tat in both cytosol and nucleus compartments at different time points and we found out that Tat was present in the cytosol at 4h and 8h post-treatment. Tat levels strongly decreased at 16h and 24h when p62 protein increased, thus suggesting a role for p62 in Tat degradation. Based on this consideration, we evaluated Tat/p62 interaction by co-immunoprecipitation experiments and we found that p62 partially co-precipitated with Tat. Besides the study on neuronal cells we analyzed also the effect of HIV proteins on astroglial cells. Astrocytes play a critical role in mediating neuronal toxicity or neuronal rescue. In neurodegeneration associated with HIV-1, chronic inflammation and oxidative stress play a crucial role and these conditions are often related. As an example, excessive amount of NO, as produced by inducible NO synthase upon the exposure of activated astrocytes to cytokines and/or viral proteins, is assumed to contribute to neuronal dysfunction associated to HIV infection. We observed that gp120 and Tat are able to induce a cPLA2-dependent arachidonic acid production, this response being critical for allowing activation of the transcriptional factor NF-kB and subsequent iNOS and interleukin-1β transcription in astroglial cells. Tat and gp120 effects were evaluated in the absence and presence of a cPLA2 inhibitor and/or arachidonic acid. The results demonstrate that treatment of cells with these two HIV proteins was able to activate NF-κB, this activation being inhibited by pre-treatment with cPLA2 inhibitor and restored by pre-treatment with arachidonic acid. Since NF-kB is involved in the transcription of a variety of pro-inflammatory genes, including iNOS and IL1β, we have analyzed the role of the cPLA2-AA pathway in the regulation of iNOS and IL1β transcription. Tat and gp120 induced in a dose-dependent manner both iNOS and IL1β mRNA levels. The pre-treatment with the cPLA2 inhibitor restored mRNA levels of iNOS and IL1β to control levels. Altogether, these results suggest that HIV proteins induce an early arachidonic acid production that seems to act as an upstream proinflammatory effector. Moreover, we analyzed the effect of Tat on the antioxidant response of astroglial cells. In particular, we demonstrated that Tat was able to induce Nrf2 and Nrf2-driven gene expression in U373 cells. The activation of Nrf2 was also evaluated in U373 cells transfected with Tat. Also in this model system, endogenously produced Tat was able to induce an antioxidant response as indicated by Nrf2 nuclear translocation and ARE gene expression (GCLC, GPX, SOD1, SOD2, CAT, NQO1). In particular, we found out increased levels of SystemXc, an amino acid transporter that transports cystine into the cell in exchange for glutamate. SystemXc plays a crucial role in the regulation of extracellular glutamate and the maintenance of glutathione levels therefore it is involved in both excitotoxicity and antioxidant response. Based on the above considerations, we performed co-culture experiments to evaluate neuronal viability and Nrf2/ARE pathway activation in the presence of stably transfected astrocytes expressing Tat. Here, we reported a 20% reduction of viability of SY5Y cells co-cultured with Tat-expressing astrocytes. In addition, Tat also led to Golgi dispersal in neuronal cells. Besides this detrimental actions, we also demonstrated the induction of an antioxidant response in neuronal cells as elicited by astrocyte-released Tat, this effect being due to Nrf2 activation. It should be reminded that Tat-induced ROS/RNS generation may play crucial role in the canonical pathway of Nrf2 activation since they can directly modify the stress sensor protein Keap1. In summary, our findings provide evidence of an antioxidant response activation and may help our understanding of the mechanism by which Nrf2 can mediate protection against neurodegenerative diseases associated with HIV infection.
URI: http://hdl.handle.net/2307/5971
Access Rights: info:eu-repo/semantics/openAccess
Appears in Collections:Dipartimento di Scienze
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