Search

Search Results

• 1. [Article] The aging endoplasmic reticulum

  Hallmarks of aging include the accumulation of aberrant proteins and a lower resistance to stresses. Because the endoplasmic reticulum (ER) functions to fold proteins and possesses unique stress sensing ...

  Citation × Citation Citation Abstract Copy Title: The aging endoplasmic reticulum Author: Dixon, Brian M. Hallmarks of aging include the accumulation of aberrant proteins and a lower resistance to stresses. Because the endoplasmic reticulum (ER) functions to fold proteins and possesses unique stress sensing pathways, the central hypothesis of this dissertation is that the ER is a target of cellular aging and significantly underlies these age-related phenotypes. This hypothesis was tested using isolated microsomes from young (3-6 mo) and old (24-26 mo) rats and a novel primary hepatocyte cell culture model that maintains the aging phenotype. The ER luminal oxidation/reduction (redox) environment, exemplified by the glutathione redox ratio (GSH:GSSG), is vital for both ER stress signaling and the regulation of ER oxidoreductase activity. Therefore, the hepatic microsomal GSH:GSSG ratio from young and old rats was determined. Our results showed that the GSH:GSSG ratio in young rats was 3.8:1 and that, in contrast to the age-related loss of GSH redox in whole liver, microsomal GSH:GSSG increased to 6.4:1 in old rats. This increase is sufficient to convert ER oxidoreductases from folding proteins to enzymes involved in protein unfolding. These results show that the ER is under an increased stress with age, yet no activation of ER stress response pathways was detected. Using the aging cell culture model noted above to examine the loss of ER stress response with age, we show that following induction of an ER stress by tunicamycin, ER-mediated "pro-apoptotic" pathways were in intact with age, but induction of "pro-survival" genes were significantly delayed. Taken together, these show that aging leads to: 1) a reductive shift in ER thiol redox, and 2) an attenuation of ER stress response. Lastly, previous work showed that (R)-alpha-lipoic acid (R-LA) restores GSH levels and redox status in aged rat livers. To determine whether R-LA also reversed the age-related changes in ER GSH redox status, young and old rats were supplemented with R-LA. Results showed that R-LA reversed both the age-related increase in the ER GSH redox state and induced protective ER stress response pathways in a manner similar to young animals. Thus, R-LA may be an effective therapeutic to maintain ER homeostatic mechanisms with age. Title: The aging endoplasmic reticulum Author: Dixon, Brian M. Hallmarks of aging include the accumulation of aberrant proteins and a lower resistance to stresses. Because the endoplasmic reticulum (ER) functions to fold proteins and possesses unique stress sensing pathways, the central hypothesis of this dissertation is that the ER is a target of cellular aging and significantly underlies these age-related phenotypes. This hypothesis was tested using isolated microsomes from young (3-6 mo) and old (24-26 mo) rats and a novel primary hepatocyte cell culture model that maintains the aging phenotype. The ER luminal oxidation/reduction (redox) environment, exemplified by the glutathione redox ratio (GSH:GSSG), is vital for both ER stress signaling and the regulation of ER oxidoreductase activity. Therefore, the hepatic microsomal GSH:GSSG ratio from young and old rats was determined. Our results showed that the GSH:GSSG ratio in young rats was 3.8:1 and that, in contrast to the age-related loss of GSH redox in whole liver, microsomal GSH:GSSG increased to 6.4:1 in old rats. This increase is sufficient to convert ER oxidoreductases from folding proteins to enzymes involved in protein unfolding. These results show that the ER is under an increased stress with age, yet no activation of ER stress response pathways was detected. Using the aging cell culture model noted above to examine the loss of ER stress response with age, we show that following induction of an ER stress by tunicamycin, ER-mediated "pro-apoptotic" pathways were in intact with age, but induction of "pro-survival" genes were significantly delayed. Taken together, these show that aging leads to: 1) a reductive shift in ER thiol redox, and 2) an attenuation of ER stress response. Lastly, previous work showed that (R)-alpha-lipoic acid (R-LA) restores GSH levels and redox status in aged rat livers. To determine whether R-LA also reversed the age-related changes in ER GSH redox status, young and old rats were supplemented with R-LA. Results showed that R-LA reversed both the age-related increase in the ER GSH redox state and induced protective ER stress response pathways in a manner similar to young animals. Thus, R-LA may be an effective therapeutic to maintain ER homeostatic mechanisms with age. Close

• 2. [Article] Age-related loss of Nrf2, a novel mechanism for the potential attenuation of xenobiotic detoxification capacity

  Nuclear Factor, Erythroid Derived 2, Like 2 (NFE2L2 or Nrf2) is the primary transcription factor in cellular defense against oxidative and xenobiotic stresses in higher eukaryotes. This basic leucine zipper ...

  Citation × Citation Citation Abstract Copy Title: Age-related loss of Nrf2, a novel mechanism for the potential attenuation of xenobiotic detoxification capacity Author: Smith, Eric Jonathan Nuclear Factor, Erythroid Derived 2, Like 2 (NFE2L2 or Nrf2) is the primary transcription factor in cellular defense against oxidative and xenobiotic stresses in higher eukaryotes. This basic leucine zipper transcription factor regulates over 200 antioxidant, detoxification, and lipid metabolizing genes by binding to the Antioxidant Response Element (ARE; a cis-acting element) as a transcription enhancer. Our group, and others, have identified that the nuclear steady state levels of this highly inducible transcription factor declines with age which coincides with decreased expression of many ARE regulated genes. To elucidate the mechanism(s) involved in lowered Nrf2 levels we used hepatocytes isolated from young and old rats as a relevant cellular model that maintains the aging phenotype with respect to Nrf2 levels. Our results show that steady state Nrf2 levels decline by ~40% with age (N=4, p<0.05), which led us to investigate the multifaceted regulation of Nrf2 homeostasis. Specifically, analysis of Nrf2 mRNA levels, and the rate of Nrf2 protein turnover and translation were investigated. Surprisingly, Keap1-mediated degradation of Nrf2, its primary negative regulator, did not account for the age-related Nrf2 decline, nor did mRNA levels change with age. Rather, Nrf2 protein synthesis declines 5.3-fold with age (N=3, p<0.05). Furthermore, we identify that rno-miR-146a, a key inflammation regulating miRNA, both inhibits Nrf2 translation and increases significantly with age. Taken together, our data suggest that the age-related loss of Nrf2 stems from the loss of its translation and may be mediated through miR regulation. Our lab previously demonstrated in rats, that the age-related loss of Nrf2 consequentially attenuates synthesis and steady state levels of liver glutathione (GSH) synthesis and manifests as a diminished level of GSH. This antioxidant is the most abundant non-protein thiol in most cells and is pivotal for the detoxification of many xenobiotics. The rate-limiting enzyme in GSH synthesis, γ-glutamyl-cysteine ligase (Gcl), is composed of a catalytic (Gclc), and a modulatory (Gclm) subunit. In mice and humans, it is established that Nrf2, through the ARE, regulates Gclc expression. However, this had not been established in rats. From a bioinformatics analysis of the 5’ upstream sequence of the rat Gclc gene, we identified 3 putative AREs (ARE1, ARE3, and ARE4), and one cis-element containing the core but not the flanking nucleotides of the ARE (ARE2). Luciferase reporter plasmids containing each individual ARE were transfected into H4IIE rat hepatoma cells to test for ARE activity. Only one, designated “ARE4”, produced appreciable luciferase activity and was dependent on Nrf2 expression, suggesting that this ARE is a “true” enhancer element. Chromatin immunoprecipitation (ChIP) and qPCR identified that Nrf2 binds ARE4 but not ARE1-3. Further ChIP analysis identified that known partner transcription factors of Nrf2 (small maf, c-Jun and c-Fos) were also bound to ARE4. Taken together, our data demonstrates that Nrf2 regulates, in part, the expression of the rat Gclc gene through ARE4. Having identified the Gclc-AREs in the rat we sought to describe the molecular consequence of the age-related decrement of Nrf2 in hepatocytes isolated from young and old rats. ChIPs and reporter assays (ARE1-4) were used to investigate known ARE-binding transcription factors and the ARE mediated enhancement of Gclc transcription. We identified a decline in Nrf2 occupancy of ARE4 (40%) and a loss of transcriptional activity (70%; P < 0.05), consistent with the loss in Gclc levels. In addition, hepatic chromatin from old rat samples demonstrated ARE4 enrichment of the transcriptional repressor Bach1, and a loss of the histone acetyltransferase CREB-Binding Protein (CBP). These results demonstrate that the active Gclc transcriptional complex is remodeled into a repressive motif in the liver of old rats. Furthermore, ChIPs identified that Nrf2 binds ARE2 in liver lysate isolated from old rats but not young. In agreement with this finding, results from luciferase reporter assays suggest that ARE2 facilitates an Nrf2-dependent enhancement of Gclc expression in the old. Thus, a promoter switching mechanism may occur with age. In summary, we have identified key mechanisms of how the age-related decrement in Nrf2 protein levels is controlled and also gathered preliminary evidence that a pro-inflammatory state may contribute the loss of Nrf2 synthesis via increased miR levels. Finally, we have characterized the consequences of the loss of Nrf2 to GSH synthesis as an example of age-related impairment of cellular stress response. Title: Age-related loss of Nrf2, a novel mechanism for the potential attenuation of xenobiotic detoxification capacity Author: Smith, Eric Jonathan Nuclear Factor, Erythroid Derived 2, Like 2 (NFE2L2 or Nrf2) is the primary transcription factor in cellular defense against oxidative and xenobiotic stresses in higher eukaryotes. This basic leucine zipper transcription factor regulates over 200 antioxidant, detoxification, and lipid metabolizing genes by binding to the Antioxidant Response Element (ARE; a cis-acting element) as a transcription enhancer. Our group, and others, have identified that the nuclear steady state levels of this highly inducible transcription factor declines with age which coincides with decreased expression of many ARE regulated genes. To elucidate the mechanism(s) involved in lowered Nrf2 levels we used hepatocytes isolated from young and old rats as a relevant cellular model that maintains the aging phenotype with respect to Nrf2 levels. Our results show that steady state Nrf2 levels decline by ~40% with age (N=4, p<0.05), which led us to investigate the multifaceted regulation of Nrf2 homeostasis. Specifically, analysis of Nrf2 mRNA levels, and the rate of Nrf2 protein turnover and translation were investigated. Surprisingly, Keap1-mediated degradation of Nrf2, its primary negative regulator, did not account for the age-related Nrf2 decline, nor did mRNA levels change with age. Rather, Nrf2 protein synthesis declines 5.3-fold with age (N=3, p<0.05). Furthermore, we identify that rno-miR-146a, a key inflammation regulating miRNA, both inhibits Nrf2 translation and increases significantly with age. Taken together, our data suggest that the age-related loss of Nrf2 stems from the loss of its translation and may be mediated through miR regulation. Our lab previously demonstrated in rats, that the age-related loss of Nrf2 consequentially attenuates synthesis and steady state levels of liver glutathione (GSH) synthesis and manifests as a diminished level of GSH. This antioxidant is the most abundant non-protein thiol in most cells and is pivotal for the detoxification of many xenobiotics. The rate-limiting enzyme in GSH synthesis, γ-glutamyl-cysteine ligase (Gcl), is composed of a catalytic (Gclc), and a modulatory (Gclm) subunit. In mice and humans, it is established that Nrf2, through the ARE, regulates Gclc expression. However, this had not been established in rats. From a bioinformatics analysis of the 5’ upstream sequence of the rat Gclc gene, we identified 3 putative AREs (ARE1, ARE3, and ARE4), and one cis-element containing the core but not the flanking nucleotides of the ARE (ARE2). Luciferase reporter plasmids containing each individual ARE were transfected into H4IIE rat hepatoma cells to test for ARE activity. Only one, designated “ARE4”, produced appreciable luciferase activity and was dependent on Nrf2 expression, suggesting that this ARE is a “true” enhancer element. Chromatin immunoprecipitation (ChIP) and qPCR identified that Nrf2 binds ARE4 but not ARE1-3. Further ChIP analysis identified that known partner transcription factors of Nrf2 (small maf, c-Jun and c-Fos) were also bound to ARE4. Taken together, our data demonstrates that Nrf2 regulates, in part, the expression of the rat Gclc gene through ARE4. Having identified the Gclc-AREs in the rat we sought to describe the molecular consequence of the age-related decrement of Nrf2 in hepatocytes isolated from young and old rats. ChIPs and reporter assays (ARE1-4) were used to investigate known ARE-binding transcription factors and the ARE mediated enhancement of Gclc transcription. We identified a decline in Nrf2 occupancy of ARE4 (40%) and a loss of transcriptional activity (70%; P < 0.05), consistent with the loss in Gclc levels. In addition, hepatic chromatin from old rat samples demonstrated ARE4 enrichment of the transcriptional repressor Bach1, and a loss of the histone acetyltransferase CREB-Binding Protein (CBP). These results demonstrate that the active Gclc transcriptional complex is remodeled into a repressive motif in the liver of old rats. Furthermore, ChIPs identified that Nrf2 binds ARE2 in liver lysate isolated from old rats but not young. In agreement with this finding, results from luciferase reporter assays suggest that ARE2 facilitates an Nrf2-dependent enhancement of Gclc expression in the old. Thus, a promoter switching mechanism may occur with age. In summary, we have identified key mechanisms of how the age-related decrement in Nrf2 protein levels is controlled and also gathered preliminary evidence that a pro-inflammatory state may contribute the loss of Nrf2 synthesis via increased miR levels. Finally, we have characterized the consequences of the loss of Nrf2 to GSH synthesis as an example of age-related impairment of cellular stress response. Close

• 3. [Article] Age-related alterations in transcriptional regulation of hepatic glutathione synthesis : remediation by R-alpha-lipoic acid

  Glutathione (GSH) is the predominant low molecular weight thiol antioxidant in liver tissue. GSH plays an important role in maintaining the intracellular thiol redox ratio as well as detoxification of ...

  Citation × Citation Citation Abstract Copy Title: Age-related alterations in transcriptional regulation of hepatic glutathione synthesis : remediation by R-alpha-lipoic acid Author: Shenvi, Swapna V. Glutathione (GSH) is the predominant low molecular weight thiol antioxidant in liver tissue. GSH plays an important role in maintaining the intracellular thiol redox ratio as well as detoxification of electrophiles and xenobiotics. Aging leads to a significant decline (35%; P ≤ 0.05) in hepatocellular GSH levels. Using young (2-4 months corresponding to an adolescent human) and old (24-28 months corresponding to a 60-75 year old person) male Fischer 344 rats, we determined that the age-related loss of GSH levels were due to lower activity (53 + 6%; P ≤ 0.05) and levels of the rate-limiting enzyme, γ-glutamate cysteine ligase (GCL). GCL is composed of a catalytic subunit (GCLC) and also a modulatory subunit (GCLM) that affects the KM of its substrate. Since GCLC levels are regulated by transcription, we sought to elucidate its precise transcriptional mechanism and whether aging alters the transcriptome of the enzyme subunit. A cis-acting DNA sequence called the antioxidant response element (ARE) has been previously implicated in the transcriptional regulation of Phase II enzymes, including GCLC and GCLM. Computer-based analysis of the promoter region of Gclc revealed the presence of three putative AREs and a single cis element (ARE-like) containing the core but not the flanking nucleotides of the ARE. Results from experiments where H4IIE rat hepatoma cells were transfected with luciferase reporter constructs containing individual Gclc ARE elements revealed that only the ARE element 3.9 kb upstream of the transcriptional start site (ARE3) possessed basal transcriptional activity. Electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments on liver tissue and primary hepatocytes in culture showed that NF-E2-related factor 2 (Nrf2) was the predominant transcription factor bound to ARE3 and was partnered with small maf proteins, c-Jun, c-Fos and the histone acetyltransferase CREB-Binding Protein (CBP). In aging, nuclear steady-state levels of Nrf2 showed a profound 51 + 7% (P ≤ 0.0001) decline leading to lower Nrf2-ARE3 binding (40%) and transcriptional activity (70 + 10%; P ≤ 0.05), consistent with the loss in GCLC levels. Concomitantly, the transcriptional repressor Bach1 was enriched at the ARE3 site and was accompanied by a loss of CBP. These results show that a negative remodeling of the active Gclc transcriptional complex occurs in the liver of old rats. Furthermore, Nrf2 was detected at the ARE-like site, which was not transcriptionally active in hepatocytes from young rats. Thus, a promoter switching mechanism may occur with age. In previously published reports, we demonstrated that administration of the dithiol compound R-alpha-lipoic acid (LA; 40 mg/kg body weight; intraperitoneal injection) to old rats reversed the age-related decline in hepatic glutathione levels. LA admininstration both to old rats and to hepatocytes in primary cell culture (100 μM) replenished nuclear Nrf2 levels lost during aging. Additionally, LA increased Nrf2 enrichment and activity of both the ARE3 and ARE-like promoters albeit, to a greater extent at the ARE-like promoter (60 + 10%; P ≤ 0.05). This was accompanied by reversal of the age-related decline in GCLC expression, protein levels and GCL activity. Thus, LA maintains hepatic GSH status during aging by permitting normal ARE-mediated GCLC expression, suggesting that it would be a good therapeutic agent to restore GSH-dependent detoxification systems during aging. Title: Age-related alterations in transcriptional regulation of hepatic glutathione synthesis : remediation by R-alpha-lipoic acid Author: Shenvi, Swapna V. Glutathione (GSH) is the predominant low molecular weight thiol antioxidant in liver tissue. GSH plays an important role in maintaining the intracellular thiol redox ratio as well as detoxification of electrophiles and xenobiotics. Aging leads to a significant decline (35%; P ≤ 0.05) in hepatocellular GSH levels. Using young (2-4 months corresponding to an adolescent human) and old (24-28 months corresponding to a 60-75 year old person) male Fischer 344 rats, we determined that the age-related loss of GSH levels were due to lower activity (53 + 6%; P ≤ 0.05) and levels of the rate-limiting enzyme, γ-glutamate cysteine ligase (GCL). GCL is composed of a catalytic subunit (GCLC) and also a modulatory subunit (GCLM) that affects the KM of its substrate. Since GCLC levels are regulated by transcription, we sought to elucidate its precise transcriptional mechanism and whether aging alters the transcriptome of the enzyme subunit. A cis-acting DNA sequence called the antioxidant response element (ARE) has been previously implicated in the transcriptional regulation of Phase II enzymes, including GCLC and GCLM. Computer-based analysis of the promoter region of Gclc revealed the presence of three putative AREs and a single cis element (ARE-like) containing the core but not the flanking nucleotides of the ARE. Results from experiments where H4IIE rat hepatoma cells were transfected with luciferase reporter constructs containing individual Gclc ARE elements revealed that only the ARE element 3.9 kb upstream of the transcriptional start site (ARE3) possessed basal transcriptional activity. Electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments on liver tissue and primary hepatocytes in culture showed that NF-E2-related factor 2 (Nrf2) was the predominant transcription factor bound to ARE3 and was partnered with small maf proteins, c-Jun, c-Fos and the histone acetyltransferase CREB-Binding Protein (CBP). In aging, nuclear steady-state levels of Nrf2 showed a profound 51 + 7% (P ≤ 0.0001) decline leading to lower Nrf2-ARE3 binding (40%) and transcriptional activity (70 + 10%; P ≤ 0.05), consistent with the loss in GCLC levels. Concomitantly, the transcriptional repressor Bach1 was enriched at the ARE3 site and was accompanied by a loss of CBP. These results show that a negative remodeling of the active Gclc transcriptional complex occurs in the liver of old rats. Furthermore, Nrf2 was detected at the ARE-like site, which was not transcriptionally active in hepatocytes from young rats. Thus, a promoter switching mechanism may occur with age. In previously published reports, we demonstrated that administration of the dithiol compound R-alpha-lipoic acid (LA; 40 mg/kg body weight; intraperitoneal injection) to old rats reversed the age-related decline in hepatic glutathione levels. LA admininstration both to old rats and to hepatocytes in primary cell culture (100 μM) replenished nuclear Nrf2 levels lost during aging. Additionally, LA increased Nrf2 enrichment and activity of both the ARE3 and ARE-like promoters albeit, to a greater extent at the ARE-like promoter (60 + 10%; P ≤ 0.05). This was accompanied by reversal of the age-related decline in GCLC expression, protein levels and GCL activity. Thus, LA maintains hepatic GSH status during aging by permitting normal ARE-mediated GCLC expression, suggesting that it would be a good therapeutic agent to restore GSH-dependent detoxification systems during aging. Close

• 4. [Article] Age-related decrease in resilience against acute redox cycling agents: Critical role of declining GSH-dependent detoxification capacity

  Citation × Citation Citation Abstract Copy Title: Age-related decrease in resilience against acute redox cycling agents: Critical role of declining GSH-dependent detoxification capacity Author: Thomas, Nicholas, O. Title: Age-related decrease in resilience against acute redox cycling agents: Critical role of declining GSH-dependent detoxification capacity Author: Thomas, Nicholas, O. Close