Dexamethasone improves redox state in ataxia telangiectasia cells by promoting an NRF2-mediated antioxidant response

partially_open10noAtaxia telangiectasia (A-T) is a rare incurable neurodegenerative disease caused by biallelic mutations in the gene for ataxia-telangiectasia mutated (ATM). The lack of a functional ATM kinase leads to a pleiotropic phenotype, and oxidative stress is considered to have a crucial role in the complex physiopathology. Recently, steroids have been shown to reduce the neurological symptoms of the disease, although the molecular mechanism of this effect is largely unknown. In the present study, we have demonstrated that dexamethasone treatment of A-T lymphoblastoid cells increases the content of two of the most abundant antioxidants [glutathione (GSH) and NADPH] by up to 30%. Dexamethasone promoted the nuclear accumulation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 to drive expression of antioxidant pathways involved in GSH synthesis and NADPH production. The latter effect was via glucose 6-phosphate dehydrogenase activation, as confirmed by increased enzyme activity and enhancement of the pentose phosphate pathway rate. This evidence indicates that glucocorticoids are able to potentiate antioxidant defenses to counteract oxidative stress in ataxia telangiectasia, and also reveals an unexpected role for dexamethasone in redox homeostasis and cellular antioxidant activity.openBiagiotti, Sara; Menotta, Michele; Orazi, Sara; Spapperi, Chiara; Brundu, Serena; Fraternale, Alessandra; Bianchi, Marzia; Rossi, Luigia; Chessa, Luciana; Magnani, MauroBiagiotti, Sara; Menotta, Michele; Orazi, Sara; Spapperi, Chiara; Brundu, Serena; Fraternale, Alessandra; Bianchi, Marzia; Rossi, Luigia; Chessa, Luciana; Magnani, Maur

Proteomics and transcriptomics analyses of ataxia telangiectasia cells treated with Dexamethasone

Ataxia telangiectasia (A-T) is an incurable and rare hereditary syndrome. In recent times, treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this condition, but the molecular mechanism of action of these analogues remains unknown. Hence, the aim of this study was to gain insight into the molecular mechanism of action of glucocorticoid analogues in the treatment of A-T by investigating the role of Dexamethasone (Dexa) in A-T lymphoblastoid cell lines. We used 2DE and tandem MS to identify proteins that were influenced by the drug in A-T cells but not in healthy cells. Thirty-four proteins were defined out of a total of 746±63. Transcriptome analysis was performed by microarray and showed the differential expression of 599 A-T and 362 wild type (WT) genes and a healthy un-matching between protein abundance and the corresponding gene expression variation. The proteomic and transcriptomic profiles allowed the network pathway analysis to pinpoint the biological and molecular functions affected by Dexamethasone in Dexa-treated cells. The present integrated study provides evidence of the molecular mechanism of action of Dexamethasone in an A-T cellular model but also the broader effects of the drug in other tested cell lines

Western blot.

<p>Representative images of western blots performed in all tested LCLs, subsequently quantified as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195388#pone.0195388.g004" target="_blank">Fig 4</a>.</p

ATM splicing variants as biomarkers for low dose dexamethasone treatment of A-T

Ataxia Telangiectasia (AT) is a rare incurable genetic disease, caused by biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this syndrome. Nevertheless, the molecular mechanism underlying the glucocorticoid action in AT patients is not yet understood. Recently, we have demonstrated that Dexamethasone treatment may partly restore ATM activity in AT lymphoblastoid cells by a new ATM transcript, namely ATMdexa1

Western blot analysis of all tested LCLs.

<p>The protein abundance of selected targets in sample AT129RM is in agreement with the 2DE outcome (paired t-test p<0.05) except for 14.3.3 ζ/δ (see text). Only the AT50RM sample behaved in a similar manner to the AT129RM sample, despite the ATK13RM and ATK36RM cell lines. The W-N graphic reports the whole lane normalization data of the WB experiments.</p

Veen diagram.

<p>The splicing and expression outputs were compared and plotted to show differences about spliced and altered expression genes between WT and AT. Only small amounts of gene symbols were shared in all tested comparisons.</p

HLC outcome obtained by microarray expression profile of A-T samples.

<p>A total of 675 differentially expressed transcripts allowed us to classify AT129RM and AT50RM as similar to each other, while the other A-T samples behaved differently. The same behaviour pattern was inferred by western blot analysis.</p

Proteins regulated by Dexa inferred by the 2DE experiments.

<p>All comparisons scores. The bold text column shows the protein ratio (“<b>U</b>” upregulated, “<b>D</b>” downregulated and “<b>=“</b> as unvaried) of the indicated comparison, while the plain text reports the gene expression ratio (U upregulated, D downregulated and “<b>=“</b> as unvaried), the variance analysis p value and the FDR p value.</p