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Advanced Glycation End-products in neuronal death: role of RAGE and Nrf-2
therein induce free radical damage which compromises steroid hormone synthesis. Keywords: Oxidative Stress, Steroidogenesis, Cholesterol Hydroperoxide, Lipid Trafficking doi:10.1016/j.freeradbiomed.2012.08.163 [0409] PGC-1α regulates TLS activity: Role in oxidative stress gene expression C. Sánchez Ramos2, A. Tierrez1 ,2, O. Fabregat4, B. Wild1 ,2, A. Arduini3, M. Monsalve*1 1 Instituto de Investigaciones Biomédicas, Spain, 2Centro Nacional de Investigaciones Cardiovasculares, Spain, 3 Universidad de Valencia, Spain, 4Hospital General de Valencia, Spain Translocated in liposarcoma (TLS) is a poorly characterized multifunctional protein involved in the genotoxic response. TLS regulates gene expression at several steps, including splicing and mRNA transport, possibly connecting transcriptional and posttranscriptional events. Aims: In this study we aimed to idenfity molecular targets and regulatory partners of TLS. Results and Innovation: Here we report that TLS transcriptionally regulates the expression of oxidative stress protection genes. This regulation requires interaction with the transcriptional coactivator peroxisome proliferator activated receptor gammacoactivator 1alpha (PGC-1alpha), a master regulator of mitochondrial function that coordinately induces the expression of genes involved in detoxification of mitochondrial reactive oxygen species (ROS). Microarray gene expression analysis showed that TLS transcriptional activity is impaired in the absence of PGC-1alpha, and is thus largely dependent on PGC1alpha. Conclusion: These results suggest the existence of a regulatory circuit linking the control of ROS detoxification to the coordinated cross-talk between oxidative metabolism and the cellular response to genomic DNA damage. Keywords: PGC-1alpha, TLS/FUS, Mitochondria, DNA damage doi:10.1016/j.freeradbiomed.2012.08.164
C. Vida, E. Gonzalez, V. Heras, I. Corpas, M. De la Fuente* Complutense University of Madrid, Spain In a murine model of premature ageing, characterised by a poor capacity to adaptively react to stress, adult ICR (CD-1) and BALB/c prematurely ageing mice (PAM) show higher emotional and anxiety-like behaviour than non-prematurely ageing mice (NPAM) of the same sex and chronological age. Moreover, PAM show a premature immunosenescence and oxidative stress as well as a shorter lifespan than NPAM. In old BALB/c PAM higher levels of immunosenescence than in NPAM appear, but the oxidative status of their immune cells has not been studied yet. The aim of the present work was to analyse several parameters of oxidation as well as antioxidant defences in peritoneal leucocytes from old BALB/c PAM and NPAM. Old female BALB/c mice (21 month-old) were used. They were classified as NPAM or PAM according to their performance in the T-maze behavioural test at the adult age of 6 months. Peritoneal leucocytes were obtained and several redox parameters such as extracellular superoxide anion levels, intracellular ROS generation (in response to PMA), activity and expression of xanthine oxidase (XO), as well as the total glutathione (GSH) levels and the activities of catalase and superoxide dismutase (SODt, ZnCuSOD and MnSOD) were analysed. The results showed that PAM have in their leucocytes a significant increase in both extracellular superoxide anion (p<0.05) and intracellular ROS levels (p<0.05), as well as in the XO activity and expression (p<0.05) with respect to NPAM. Moreover, the levels of GSH (p<0.01) as well as the catalase (p<0.01) and ZnCuSOD (p<0.05) activities were lower in PAM than in their NPAM counterparts. Thus, the immune cells from old PAM show an oxidative stress situation, which may be the cause of the higher immunosenescence and of the consequent shorter life span of these animals in comparison to NPAM. Support: MCINN (BFU2011-30336), UCM-ResearchGroup (910379) and REFICEF (RD06/0013/0003). Keywords: Aging, Prematurely aging mice, oxidative stress, Peritoneal leucocytes doi:10.1016/j.freeradbiomed.2012.08.165 [0457]
[0432]
Advanced Glycation End-products death: role of RAGE and Nrf-2
in
neuronal
Oxidative stress in leucocytes from old mice with premature ageing
S. Piras*, A.L. Furfaro, M. Passalacqua, C. Domenicotti, N. Traverso, M.A. Pronzato, U.M. Marinari, M. Nitti et al
S79
Department of Experimental Medicine, University of Genoa, Genoa, Italy
F. Hariton*, M. Xue, N. Rabbani, P.J. Thornalley University of Warwick, UK
Intracellular redox state modulation can influence cell proliferation, differentiation or death. Our previous studies have demonstrated that retinoic acid (RA)induced differentiation of SHSY-5Y neuroblastoma cells is driven by NADPH oxidase-dependent ROS production. Moreover, we showed that differentiated cells are more sensitive to Advanced Glycation Endproducts (AGEs) in comparison to undifferentiated ones, basically resistant to glycoxidative stress. Considering the role of AGEs in neurodegeneration, aging and Alzheimer's disease, it is worth further clarifying the molecular mechanisms driving cell responses to glycoxidative stress. Using the same undifferentiated and differentiated cells exposed to AGEs, here we analysed RAGE (receptor for AGEs) expression, as the receptor mainly involved in pathophysiological effects of glycated products, and Nrf-2 activation, as key transcription factor in antioxidant cell responses.
Introduction: Transcription factor Nrf2 regulates the expression of a battery of genes via interaction with promoter antioxidant response elements (AREs) providing protection against oxidative stress. Sulforaphane (SFN) is an Nrf2 activator derived from Brassica vegetables. In this study we investigated changes induced by SFN of ARE-linked and functional marker gene expression in human fetal lung embryo MRC-5 fibroblasts in vitro.
No changes in RAGE mRNA expression were observed after cell differentiation, but, checking subcellular localisation by confocal microscopy, it was clearly shown the increased expression of RAGE on cell membrane in RA- treated cells. The analysis of Nrf-2 showed that cell differentiation increases total Nrf-2 expression level, in order to balance RA-induced ROS generation. Moreover, while in AGE-treated undifferentiated cells Nrf-2 nuclear localisation was increased in comparison to control cells, in differentiated cells exposed to AGEs Nrf-2 nuclear shuttling was impaired. This evidence was confirmed by the analysis of two Nrf-2-dependent genes: only AGE-treated undifferentiated cells were able to induce HO-1 and GCLM mRNA expression in comparison to untreated cells, while AGE-treated differentiated cells were not able to do the same, even if total expression level of these two enzymes was increased after cell differentiation. We believe that both the expression of RAGE on cell membrane and the Nrf-2 nuclear shuttling could be two crucial points modulated by retinoic acid able to increase the neuronal sensitivity to AGEs (Grants: PRIN2009M8FKBB_002 and Genoa University)
Methods: MRC-5 cells, passage 4, were cultured with and without 1 µM SFN. The median growth inhibitory concentration of SFN was 2.60±0.03 µM (n = 21). Cells were collected at 4, 8, 12, 16, 24, 36, 48 and 72 h posttreatment, RNA extracted and digital mRNA profiling performed (Nanostring method). Results: Treatment of MRC-5 with SFN produced changes in mRNA of ARE-linked gens (pathway classified below) but not functional marker genes: Conventional ARE response markers: quinone reductase increased 2-fold in the initial 36 h and remained at this level thereafter, whereas glutathione transferase P1 was unchanged. Oxidative stress Haem oxygenase increased 3-fold in the initial 4 h and declined to control levels by 24 h. γ-Glutamylcysteine ligase (modulatory subunit), glutathione reductase and increased 2-fold in the initial 12 h and reverted to control levels by 48 h. Thioredoxin and thioredoxin reductase increased similarly but remained increased out to 72 h. Metabolic stress Glucose-6-phosphate dehydrogenase, transketolase and transaldolase increased by 40 - 50% in the initial 24 h and remained increased to 72 h. Proteasomal proteolysis and autophagy Proteasome subunits A1 and B5 were little changed whereas autophagy adaptor p62 was increased 2-fold after 12 h and declined thereafter. Anti-glycation defence Aldoketo reductase 1C1 was 2-fold higher than control from 12 72 h post-treatment. Senescence-linked genes betagalactosidase and cyclin-dependent kinase inhibitors 2A/P16INK4a and 1/p21 were little changed. Conclusions: Activation of Nrf2 protects against multiple stresses in MRC-5 fibroblasts in vitro.
Keywords: RAGE, Nrf-2, glycoxidation, neuronal death doi:10.1016/j.freeradbiomed.2012.08.166
Keywords: Nrf2, Fibroblast, Senescence, Sulforaphane doi:10.1016/j.freeradbiomed.2012.08.167
[0478] Protective response of Nrf2 transactivational activity in MRC-5 fibroblasts in vitro
S80
[0484] Ageing biology using Saccharomyces cerevisiae as experimental model – the role of Sod1
C. Vida, E. Gonzalez, V. Heras, I. Corpas, M. De la Fuente* Complutense University of Madrid, Spain In a murine model of premature ageing, characterised by a poor capacity to adaptively react to stress, adult ICR (CD-1) and BALB/c prematurely ageing mice (PAM) show higher emotional and anxiety-like behaviour than non-prematurely ageing mice (NPAM) of the same sex and chronological age. Moreover, PAM show a premature immunosenescence and oxidative stress as well as a shorter lifespan than NPAM. In old BALB/c PAM higher levels of immunosenescence than in NPAM appear, but the oxidative status of their immune cells has not been studied yet. The aim of the present work was to analyse several parameters of oxidation as well as antioxidant defences in peritoneal leucocytes from old BALB/c PAM and NPAM. Old female BALB/c mice (21 month-old) were used. They were classified as NPAM or PAM according to their performance in the T-maze behavioural test at the adult age of 6 months. Peritoneal leucocytes were obtained and several redox parameters such as extracellular superoxide anion levels, intracellular ROS generation (in response to PMA), activity and expression of xanthine oxidase (XO), as well as the total glutathione (GSH) levels and the activities of catalase and superoxide dismutase (SODt, ZnCuSOD and MnSOD) were analysed. The results showed that PAM have in their leucocytes a significant increase in both extracellular superoxide anion (p<0.05) and intracellular ROS levels (p<0.05), as well as in the XO activity and expression (p<0.05) with respect to NPAM. Moreover, the levels of GSH (p<0.01) as well as the catalase (p<0.01) and ZnCuSOD (p<0.05) activities were lower in PAM than in their NPAM counterparts. Thus, the immune cells from old PAM show an oxidative stress situation, which may be the cause of the higher immunosenescence and of the consequent shorter life span of these animals in comparison to NPAM. Support: MCINN (BFU2011-30336), UCM-ResearchGroup (910379) and REFICEF (RD06/0013/0003). Keywords: Aging, Prematurely aging mice, oxidative stress, Peritoneal leucocytes doi:10.1016/j.freeradbiomed.2012.08.165 [0457]
[0432]
Advanced Glycation End-products death: role of RAGE and Nrf-2
in
neuronal
Oxidative stress in leucocytes from old mice with premature ageing
S. Piras*, A.L. Furfaro, M. Passalacqua, C. Domenicotti, N. Traverso, M.A. Pronzato, U.M. Marinari, M. Nitti et al
S79
Department of Experimental Medicine, University of Genoa, Genoa, Italy
F. Hariton*, M. Xue, N. Rabbani, P.J. Thornalley University of Warwick, UK
Intracellular redox state modulation can influence cell proliferation, differentiation or death. Our previous studies have demonstrated that retinoic acid (RA)induced differentiation of SHSY-5Y neuroblastoma cells is driven by NADPH oxidase-dependent ROS production. Moreover, we showed that differentiated cells are more sensitive to Advanced Glycation Endproducts (AGEs) in comparison to undifferentiated ones, basically resistant to glycoxidative stress. Considering the role of AGEs in neurodegeneration, aging and Alzheimer's disease, it is worth further clarifying the molecular mechanisms driving cell responses to glycoxidative stress. Using the same undifferentiated and differentiated cells exposed to AGEs, here we analysed RAGE (receptor for AGEs) expression, as the receptor mainly involved in pathophysiological effects of glycated products, and Nrf-2 activation, as key transcription factor in antioxidant cell responses.
Introduction: Transcription factor Nrf2 regulates the expression of a battery of genes via interaction with promoter antioxidant response elements (AREs) providing protection against oxidative stress. Sulforaphane (SFN) is an Nrf2 activator derived from Brassica vegetables. In this study we investigated changes induced by SFN of ARE-linked and functional marker gene expression in human fetal lung embryo MRC-5 fibroblasts in vitro.
No changes in RAGE mRNA expression were observed after cell differentiation, but, checking subcellular localisation by confocal microscopy, it was clearly shown the increased expression of RAGE on cell membrane in RA- treated cells. The analysis of Nrf-2 showed that cell differentiation increases total Nrf-2 expression level, in order to balance RA-induced ROS generation. Moreover, while in AGE-treated undifferentiated cells Nrf-2 nuclear localisation was increased in comparison to control cells, in differentiated cells exposed to AGEs Nrf-2 nuclear shuttling was impaired. This evidence was confirmed by the analysis of two Nrf-2-dependent genes: only AGE-treated undifferentiated cells were able to induce HO-1 and GCLM mRNA expression in comparison to untreated cells, while AGE-treated differentiated cells were not able to do the same, even if total expression level of these two enzymes was increased after cell differentiation. We believe that both the expression of RAGE on cell membrane and the Nrf-2 nuclear shuttling could be two crucial points modulated by retinoic acid able to increase the neuronal sensitivity to AGEs (Grants: PRIN2009M8FKBB_002 and Genoa University)
Methods: MRC-5 cells, passage 4, were cultured with and without 1 µM SFN. The median growth inhibitory concentration of SFN was 2.60±0.03 µM (n = 21). Cells were collected at 4, 8, 12, 16, 24, 36, 48 and 72 h posttreatment, RNA extracted and digital mRNA profiling performed (Nanostring method). Results: Treatment of MRC-5 with SFN produced changes in mRNA of ARE-linked gens (pathway classified below) but not functional marker genes: Conventional ARE response markers: quinone reductase increased 2-fold in the initial 36 h and remained at this level thereafter, whereas glutathione transferase P1 was unchanged. Oxidative stress Haem oxygenase increased 3-fold in the initial 4 h and declined to control levels by 24 h. γ-Glutamylcysteine ligase (modulatory subunit), glutathione reductase and increased 2-fold in the initial 12 h and reverted to control levels by 48 h. Thioredoxin and thioredoxin reductase increased similarly but remained increased out to 72 h. Metabolic stress Glucose-6-phosphate dehydrogenase, transketolase and transaldolase increased by 40 - 50% in the initial 24 h and remained increased to 72 h. Proteasomal proteolysis and autophagy Proteasome subunits A1 and B5 were little changed whereas autophagy adaptor p62 was increased 2-fold after 12 h and declined thereafter. Anti-glycation defence Aldoketo reductase 1C1 was 2-fold higher than control from 12 72 h post-treatment. Senescence-linked genes betagalactosidase and cyclin-dependent kinase inhibitors 2A/P16INK4a and 1/p21 were little changed. Conclusions: Activation of Nrf2 protects against multiple stresses in MRC-5 fibroblasts in vitro.
Keywords: RAGE, Nrf-2, glycoxidation, neuronal death doi:10.1016/j.freeradbiomed.2012.08.166
Keywords: Nrf2, Fibroblast, Senescence, Sulforaphane doi:10.1016/j.freeradbiomed.2012.08.167
[0478] Protective response of Nrf2 transactivational activity in MRC-5 fibroblasts in vitro
S80
[0484] Ageing biology using Saccharomyces cerevisiae as experimental model – the role of Sod1