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S6. Oxidative Stress and Neuronal Function
S6. Oxidative Stress and Neuronal Function
Following brain inflammatory stimuli, astrocytes actively synthesize nitric oxide and peroxynitrite. These nitrogenderived species trigger a repertoire of biochemical effects, including alteration of mitochondrial function and redox status both in the astrocytes and neighboring neurons. Furthermore, under such nitrosative stress astrocytes show remarkable resistance in spite of having their mitochondria impaired, whereas the neighboring neurons show vulnerability. Here, we discuss recent evidence strongly suggesting that nitrogen-derived species modulate key regulatory steps of glucose metabolism. These involve up-regulation of high-affinity glucose transporter, stimulation of glycolysis at 6-phosphofructo-1-kinase, and activation of pentose-phosphate pathway at glucose-6phosphate dehydrogenase. We conclude that the orchestrated stimulation of glucose-metabolising pathways by nitric oxide would be a transient attempt of certain neural cells to compensate for the impaired energy status and oxidised glutathione, and thus emerge from an otherwise neuropathological outcome.
hippocampal neurons prior to excitotoxic glutamate (200 AM) exposure attenuated the rise in [Ca2+]i while significantly potentiating the rise in [Ca2+]m. Although enhanced Ca2+ levels can be beneficial for cellular signaling, excessively high [Ca2+]m leads to apoptosis and one might presume that potentiation of the glutamate-induced rise in [Ca2+]m would exacerbate toxicity. In this study, using isolated rat brain mitochondrial, we showed that estrogen pretreatment attenuates the Ca2+-induced decline in respiratory activity. This protection against Ca2+ overload was due in part to an increase in the basal respiratory activity of estrogen-treated brain mitochondia. These data indicate that in addition to altering the set point for [Ca2+]m sequestration there is a compensatory regulation of mitochondrial Ca2+ load tolerability in response to estrogen. Apoptosis during neurodegeneration is preceded by release of cytochrome c from the mitochondrial intermembrane space. Translocation of cytochrome C is tightly regulated by the expression and localization of the Bcl-2 family of proteins, many of which are regulated by estrogen. We examined the effect of E2 treatment on glutamateand hA-induced expression and translocation of cytochrome c and the Bcl-2 family proteins, in which E2 prevents the reciprocal mitochondrial/cytosolic translocation of cytochrome c and Bax. Together, these data suggest a mechanism of estrogen neuroprotection involving an increase in Ca2+ sequestration by mitochondria coupled with an increase in mitochondrial Ca2+ load tolerability and an increase in mitochondrial respiratory function. Supported by: National Institutes of Mental Health, National Institute of Aging, the K.T. and E.L. Norris Foundation, the L.K. Whittier Foundation, and the John D. French Foundation.
S6-32
S6-33
ESTRADIOL-MEDIATED REGULATION OF MITOCHONDRIAL FUNCTION: STRATEGIES FOR PREVENTION OF NEURODEGENERATION
DIFFERENTIAL SUSCEPTIBILITY OF ASTROCYTES AND NEURONS TO OXIDATIVE STRESS
S6-31 REGULATION OF GLUCOSE METABOLISM BY NITROSATIVE STRESS IN NEURAL CELLS J. P. Bolan˜os,1 P. Cidad,1 P. Garcı´a-Nogales,2 M. Delgado-Esteban,1,3 E. Ferna´ndez,1 and A. Almeida1,4 1 Departamento de Bioquı´mica y Biologı´a Molecular, Universidad de Salamanca, 2DI&A Functional Genomics, Aventis Pharma Deutschland GmbH, Industriepark Ho¨chst-G879, 65926 Frankfurt am Main, Germany, 3Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain, 4Unidad de Investigacio´n, Hospital Universitario de Salamanca, Salamanca, Spain
1
R. Dı´az-Brinton 1 Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, The USC Neuroscience Program, Los Angeles, CA, USA
Estrogens are neuroprotective against neurodegenerative insults, including glutamate excitotoxicity and Alzheimer’s disease-related beta amyloid toxicity. Each of these insults are associated with calcium dysregulation and increased oxidative damage. Previous studies from our laboratory have shown that these neuroprotective effects of estradiol are dependent upon mitochondrial calcium sequestration. As abnormalities in mitochondrial function central to many neurodegenerative pathologies, we thought to further investigate estrogen regulation of mitochondrial function. Used Fura4F and RhodFF to simultaneously monitor cytosolic ([Ca2+]i) and mitochondrial ([Ca2+]m) Ca2+, respectively, we showed that estrogen treatment of primary rat
S16
S. J. R. Heales1 1
Institute of Neurology and National Hospital, Queen Square, London, UK
Exposure of astrocytes and neurons to reactive nitrogen species (RNS) reveals a differential susceptibility. Thus, neurons exhibit mitochondrial damage, at the level of the electron transport chain (ETC) and subsequent cell death. In contrast, astrocytes, under comparable conditions appear relatively resistant. An important factor influencing cellular susceptibility may relate to the availability of the antioxidant, glutathione (GSH) which can nullify the deleterious effects of some RNS. Whilst astrocytes and neurons, when cultured alone, differ in their GSH content, i.e. with astrocytes having approximately double the GSH content of neurons, this is not the case when these two cell types are cultured together. Under such conditions up-regulation of neuronal GSH metabolism occurs as result of the trafficking of GSH precursors
SFRR 2004
from astrocytes to the neuronal cells. Furthermore, astrocytic GSH metabolism, appears to be enhanced in the presence of nitric oxide. Thus, under nitrosative stress conditions an optimal supply of GSH precursors may be maintained, at least in the short term, for utilisation by neuronal cells in close proximity. Failure of one or more of the components of this GSH trafficking system may conceivably be an important factor in those neurodegenerative conditions associated with oxidative stress and loss of ETC activity.
calcium release, producing calcium-dependent increase of ERK and CREB phosphorylation, requisite sequential steps for neuronal plasticity. We propose that activation of excitable cells RyR channels by endogenous ROS/RNS enhances calcium release and calcium-dependent gene expression under conditions of normal ROS/RNS generation or may cause cell death following excess calcium release in pathological oxidative stress conditions. Supported by FONDAP 1501006.
S6-35 S6-34 REDOX MODIFICATION OF RYANODINE RECEPTOR/CALCIUM RELEASE CHANNELS ENHANCES CALCIUM RELEASE IN VESICLES AND STIMULATES ERK AND CREB PHOSPHORYLATION IN NEURONAL CELLS IN CULTURE C. Hidalgo, P. Aracena, G. Sa´nchez,1 U. Kemmerling,1 P. Donoso,1 and M. A. Carrasco1 1
1
1 CEMC, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile
Calcium release mediated by ryanodine receptor channels (RyR channels), which are highly sensitive to redox modifications, is essential for skeletal and cardiac muscle contraction and neuronal plasticity. We have found that Snitrosoglutathione (GSNO) and glutathione disulfide (GSSG), and the NO donor NOR-3, exert different effects on calcium release kinetics mediated by skeletal RyR channels. GSSG significantly reduces the inhibitory effect of Mg2+ on release kinetics without altering Ca2+ activation, NOR-3 enhances Ca2+ activation without altering Mg2+ inhibition and GSNO produces both effects. Incubation with [35S]-GSNO at pCa 5 promoted 35S incorporation into 2.5 cysteine residues per channel monomer. These findings indicate that GSNO, through S-glutathionylation of specific cysteine residues, modulates channel inhibition by Mg2+, whereas NOR-3 induces S-nitrosylation of different cysteines that modulate channel activation by Ca2+. To test the cellular effects of RyR channel redox modification, we added 100 – 200 uM H2O2 to N2a or hippocampal neurons in culture. We found that H2O2 enhances significantly both ERK and CREB phosphorylation in zero external calcium; this stimulation was inhibited by selectively blocking RyR channels with 50 uM ryanodine. These results suggest that redox modification of neuronal RyR channels stimulates
BRAIN CORTEX NITRIC OXIDE SYNTHASE AND MITOCHONDRIAL FUNCTION DURING RAT GROWTH J. Bustamante,1 S. Lores Arnaiz,1 D. Korotkov,1 N. Garcı´a Rosolen,1 and A. Boveris1 1
Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
Mitochondria were isolated from 1, 3, 7 and 14 months old rats, oxygen uptake and mitochondrial nitric oxide synthase (mtNOS) activity were compared. Western blot followed by Northern blot analysis were conducted to analyze protein and mRNA expression. Succinate-dependent state 3 respiration rates were 34% and 78% higher in 7 and 14 months rats respectively as compared with 1 month old rats. Slight increase was observed in state 4 respiration rates, but no significant differences were found between the different ages. Malate-glutamate supported state 3 respiration rates were 39% and 85% higher in 7 and 14 months rats respectively compared with 1 month old rats. mtNOS measured as NO production was 0.80 F 0.01 nmol/min.mg protein in 1 month rats, decreasing by 19%, 40% and 71% in 3, 7, and 14 months rats respectively. Western blot analysis of NOS mitochondrial fractions indicated that it reacts against nNOS antibodies (amino and carboxy terminus), and that the expression was higher in 1 month than in 14 months old rats. Northern blot indicated that nNOS brain mRNA levels increased in 1 month as compared with 14 months old rats. Interestingly eNOS brain cortex mRNA levels were higher in the older animals, while iNOS brain cortex mRNA levels were minimal. Our results indicate that during rat growth (from 1 to 14 months) the activity and expression of brain cortex mtNOS decreases associated with an increase in mitochondrial respiration.
SFRR 2004
S17
Following brain inflammatory stimuli, astrocytes actively synthesize nitric oxide and peroxynitrite. These nitrogenderived species trigger a repertoire of biochemical effects, including alteration of mitochondrial function and redox status both in the astrocytes and neighboring neurons. Furthermore, under such nitrosative stress astrocytes show remarkable resistance in spite of having their mitochondria impaired, whereas the neighboring neurons show vulnerability. Here, we discuss recent evidence strongly suggesting that nitrogen-derived species modulate key regulatory steps of glucose metabolism. These involve up-regulation of high-affinity glucose transporter, stimulation of glycolysis at 6-phosphofructo-1-kinase, and activation of pentose-phosphate pathway at glucose-6phosphate dehydrogenase. We conclude that the orchestrated stimulation of glucose-metabolising pathways by nitric oxide would be a transient attempt of certain neural cells to compensate for the impaired energy status and oxidised glutathione, and thus emerge from an otherwise neuropathological outcome.
hippocampal neurons prior to excitotoxic glutamate (200 AM) exposure attenuated the rise in [Ca2+]i while significantly potentiating the rise in [Ca2+]m. Although enhanced Ca2+ levels can be beneficial for cellular signaling, excessively high [Ca2+]m leads to apoptosis and one might presume that potentiation of the glutamate-induced rise in [Ca2+]m would exacerbate toxicity. In this study, using isolated rat brain mitochondrial, we showed that estrogen pretreatment attenuates the Ca2+-induced decline in respiratory activity. This protection against Ca2+ overload was due in part to an increase in the basal respiratory activity of estrogen-treated brain mitochondia. These data indicate that in addition to altering the set point for [Ca2+]m sequestration there is a compensatory regulation of mitochondrial Ca2+ load tolerability in response to estrogen. Apoptosis during neurodegeneration is preceded by release of cytochrome c from the mitochondrial intermembrane space. Translocation of cytochrome C is tightly regulated by the expression and localization of the Bcl-2 family of proteins, many of which are regulated by estrogen. We examined the effect of E2 treatment on glutamateand hA-induced expression and translocation of cytochrome c and the Bcl-2 family proteins, in which E2 prevents the reciprocal mitochondrial/cytosolic translocation of cytochrome c and Bax. Together, these data suggest a mechanism of estrogen neuroprotection involving an increase in Ca2+ sequestration by mitochondria coupled with an increase in mitochondrial Ca2+ load tolerability and an increase in mitochondrial respiratory function. Supported by: National Institutes of Mental Health, National Institute of Aging, the K.T. and E.L. Norris Foundation, the L.K. Whittier Foundation, and the John D. French Foundation.
S6-32
S6-33
ESTRADIOL-MEDIATED REGULATION OF MITOCHONDRIAL FUNCTION: STRATEGIES FOR PREVENTION OF NEURODEGENERATION
DIFFERENTIAL SUSCEPTIBILITY OF ASTROCYTES AND NEURONS TO OXIDATIVE STRESS
S6-31 REGULATION OF GLUCOSE METABOLISM BY NITROSATIVE STRESS IN NEURAL CELLS J. P. Bolan˜os,1 P. Cidad,1 P. Garcı´a-Nogales,2 M. Delgado-Esteban,1,3 E. Ferna´ndez,1 and A. Almeida1,4 1 Departamento de Bioquı´mica y Biologı´a Molecular, Universidad de Salamanca, 2DI&A Functional Genomics, Aventis Pharma Deutschland GmbH, Industriepark Ho¨chst-G879, 65926 Frankfurt am Main, Germany, 3Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain, 4Unidad de Investigacio´n, Hospital Universitario de Salamanca, Salamanca, Spain
1
R. Dı´az-Brinton 1 Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, The USC Neuroscience Program, Los Angeles, CA, USA
Estrogens are neuroprotective against neurodegenerative insults, including glutamate excitotoxicity and Alzheimer’s disease-related beta amyloid toxicity. Each of these insults are associated with calcium dysregulation and increased oxidative damage. Previous studies from our laboratory have shown that these neuroprotective effects of estradiol are dependent upon mitochondrial calcium sequestration. As abnormalities in mitochondrial function central to many neurodegenerative pathologies, we thought to further investigate estrogen regulation of mitochondrial function. Used Fura4F and RhodFF to simultaneously monitor cytosolic ([Ca2+]i) and mitochondrial ([Ca2+]m) Ca2+, respectively, we showed that estrogen treatment of primary rat
S16
S. J. R. Heales1 1
Institute of Neurology and National Hospital, Queen Square, London, UK
Exposure of astrocytes and neurons to reactive nitrogen species (RNS) reveals a differential susceptibility. Thus, neurons exhibit mitochondrial damage, at the level of the electron transport chain (ETC) and subsequent cell death. In contrast, astrocytes, under comparable conditions appear relatively resistant. An important factor influencing cellular susceptibility may relate to the availability of the antioxidant, glutathione (GSH) which can nullify the deleterious effects of some RNS. Whilst astrocytes and neurons, when cultured alone, differ in their GSH content, i.e. with astrocytes having approximately double the GSH content of neurons, this is not the case when these two cell types are cultured together. Under such conditions up-regulation of neuronal GSH metabolism occurs as result of the trafficking of GSH precursors
SFRR 2004
from astrocytes to the neuronal cells. Furthermore, astrocytic GSH metabolism, appears to be enhanced in the presence of nitric oxide. Thus, under nitrosative stress conditions an optimal supply of GSH precursors may be maintained, at least in the short term, for utilisation by neuronal cells in close proximity. Failure of one or more of the components of this GSH trafficking system may conceivably be an important factor in those neurodegenerative conditions associated with oxidative stress and loss of ETC activity.
calcium release, producing calcium-dependent increase of ERK and CREB phosphorylation, requisite sequential steps for neuronal plasticity. We propose that activation of excitable cells RyR channels by endogenous ROS/RNS enhances calcium release and calcium-dependent gene expression under conditions of normal ROS/RNS generation or may cause cell death following excess calcium release in pathological oxidative stress conditions. Supported by FONDAP 1501006.
S6-35 S6-34 REDOX MODIFICATION OF RYANODINE RECEPTOR/CALCIUM RELEASE CHANNELS ENHANCES CALCIUM RELEASE IN VESICLES AND STIMULATES ERK AND CREB PHOSPHORYLATION IN NEURONAL CELLS IN CULTURE C. Hidalgo, P. Aracena, G. Sa´nchez,1 U. Kemmerling,1 P. Donoso,1 and M. A. Carrasco1 1
1
1 CEMC, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile
Calcium release mediated by ryanodine receptor channels (RyR channels), which are highly sensitive to redox modifications, is essential for skeletal and cardiac muscle contraction and neuronal plasticity. We have found that Snitrosoglutathione (GSNO) and glutathione disulfide (GSSG), and the NO donor NOR-3, exert different effects on calcium release kinetics mediated by skeletal RyR channels. GSSG significantly reduces the inhibitory effect of Mg2+ on release kinetics without altering Ca2+ activation, NOR-3 enhances Ca2+ activation without altering Mg2+ inhibition and GSNO produces both effects. Incubation with [35S]-GSNO at pCa 5 promoted 35S incorporation into 2.5 cysteine residues per channel monomer. These findings indicate that GSNO, through S-glutathionylation of specific cysteine residues, modulates channel inhibition by Mg2+, whereas NOR-3 induces S-nitrosylation of different cysteines that modulate channel activation by Ca2+. To test the cellular effects of RyR channel redox modification, we added 100 – 200 uM H2O2 to N2a or hippocampal neurons in culture. We found that H2O2 enhances significantly both ERK and CREB phosphorylation in zero external calcium; this stimulation was inhibited by selectively blocking RyR channels with 50 uM ryanodine. These results suggest that redox modification of neuronal RyR channels stimulates
BRAIN CORTEX NITRIC OXIDE SYNTHASE AND MITOCHONDRIAL FUNCTION DURING RAT GROWTH J. Bustamante,1 S. Lores Arnaiz,1 D. Korotkov,1 N. Garcı´a Rosolen,1 and A. Boveris1 1
Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina
Mitochondria were isolated from 1, 3, 7 and 14 months old rats, oxygen uptake and mitochondrial nitric oxide synthase (mtNOS) activity were compared. Western blot followed by Northern blot analysis were conducted to analyze protein and mRNA expression. Succinate-dependent state 3 respiration rates were 34% and 78% higher in 7 and 14 months rats respectively as compared with 1 month old rats. Slight increase was observed in state 4 respiration rates, but no significant differences were found between the different ages. Malate-glutamate supported state 3 respiration rates were 39% and 85% higher in 7 and 14 months rats respectively compared with 1 month old rats. mtNOS measured as NO production was 0.80 F 0.01 nmol/min.mg protein in 1 month rats, decreasing by 19%, 40% and 71% in 3, 7, and 14 months rats respectively. Western blot analysis of NOS mitochondrial fractions indicated that it reacts against nNOS antibodies (amino and carboxy terminus), and that the expression was higher in 1 month than in 14 months old rats. Northern blot indicated that nNOS brain mRNA levels increased in 1 month as compared with 14 months old rats. Interestingly eNOS brain cortex mRNA levels were higher in the older animals, while iNOS brain cortex mRNA levels were minimal. Our results indicate that during rat growth (from 1 to 14 months) the activity and expression of brain cortex mtNOS decreases associated with an increase in mitochondrial respiration.
SFRR 2004
S17