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Differences in Distribution and Reduction Rate of BBB Permeable Nitroxides by in Vivo EPR Imaging Method
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Miho C Emoto , Hideo Sato-Akaba2, Hiroshi Hirata3, and Hirotada G Fujii1 1 2 Sapporo Medical University, Japan, Osaka University, Japan, 3 Hokkaido University, Japan EPR imaging using nitroxides is a powerful non-invasive method for visualizing the redox status produced by free radicals. Two blood–brain barrier (BBB)-permeable nitroxides, 3-hydroxymethyl2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP) and 3methoxycarbonyl-2,2,5,5-tetramethyl-pirrolidine-1-yloxy (MCP), were used for EPR imaging of small rodent brains, but information about their in vivo properties has not been compared in detail. In this study, the detailed distributions and reduction rate of HMP and MCP in mouse heads were examined using our improved EPR imaging system. EPR imaging experiments using faster data acquisition technique were performed to investigate the time courses for localization of nitroxides in mouse heads. In obtained 2D slice-selected EPR images, HMP was mainly located outside the brain immediately after injection. However, MCP showed remarkable localization within the brain. HMP gradually spread to brain and distributed throughout all regions of the mouse head. To examine the effect of blood flow on reduction rate of nitroxides, blood flow was stopped by KCl injection after administration of nitroxides. The reduction rate of HMP without flow was significantly slow compared with flow. On the contrary, there were no significant differences in reduction rate of MCP with or without flow. These results suggest that MCP reduction occurs with intracellular reductant such as GSH and ascorbic acid within the brain.
doi:10.1016/j.freeradbiomed.2012.10.195
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Rodrigo Franco , Laura Zavala-Flores1, Humberto Rodriguez-Rocha1, and Aracely Garcia-Garcia1 1 University of Nebraska-Lincoln Dopaminergic cell death in Parkinson’s disease (PD) is associated with oxidative stress. However, the exact signaling cascades by which redox signaling regulates cell death progression are unclear. Glutaredoxins (Grxs) are oxidoreductases that use the reducing power of GSH to regulate protein function and activity by means of protein glutathionylation. In this work, we aimed to study the role of Grxs in dopaminergic cell death induced by experimental PD models. Dopaminergic cell death induced by the pesticide paraquat and the dopamine analog 6-hydroxydopamine was significantly inhibited by overexpression of the cytosolic isoform Grx1. Paraquat induced a decrease in the levels of glutathionylated proteins (PSSG) in dopaminergic cells in vitro and in vivo. Interestingly, overexpression of Grx1 enhanced PSSG formation upon paraquat stimulation. Grx1-induced protein glutathionylation significantly reduced the degradation of cellular substrates such as actin binding proteins (Flightless-1) and scaffolding signaling proteins (REPS2) during cell death. These results demonstrate that under oxidative stress Grx1 acts as a glutathionylating enzyme, and that increased PSSG formation protects cellular substrates from degradation during cell death progression. In contrast to the
protective role of Grx1, dopaminergic cell death induced by complex I inhibition with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) was significantly decreased in the mitochondrial isoform Grx2 knock-out mice. Accordingly, overexpression of Grx2 enhanced dopaminergic cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and paraquat in vitro. Overall these results demonstrate the distinct role of Grx1 and Grx2 in regulating protein glutathionylation and dopaminergic cell death in experimental PD models.
doi:10.1016/j.freeradbiomed.2012.10.196
142 0LWRFKRQGULDO3HUR[LUHGR[LQ3URWHFWV'RSDPLQHUJLF &HOOV$JDLQVW3DUNLQVRQLDQ1HXURWR[LQV,QGHSHQGHQW IURP+\GURJHQ3HUR[LGH6LJQDOLQJ 1
Aracely Garcia-Garcia , Humberto Rodriguez-Rocha1, and Rodrigo Franco1 1 University of Nebraska-Lincoln Oxidative stress plays a major role in dopaminergic degeneration during the pathogenesis of Parkinson’s disease (PD). Peroxiredoxins (Prx) are thioredoxin (Trx)-dependent thiol peroxidases expressed at low levels in the dopaminergic neurons of the substantia nigra, being peroxiredoxin 5 the isoform with the lowest expression, which might be associated with the increased sensitivity of dopaminergic cells to environmental/mitochondrial insults. We aimed to determine the protective role of peroxiredoxin 5 against experimental models of PD. Generation of hydrogen peroxide (H2O2) was monitored using the genetically encoded fluorescent sensor Hyper targeted to the cytosol and mitochondrial matrix. Dopaminergic cell death induced by the mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium + (MPP ) and rotenone, but not by the pesticide paraquat or the pro-oxidant dopamine analog 6-hydroxydopamine (6-OHDA), was preceded by an increase in H2O2 formation. However, neither overexpression of catalase nor mitochondrial-targeted catalase reduced dopaminergic cell death. Peroxiredoxin 5 is found in distinct cellular compartments including the cytosol and mitochondria, and has the ability to reduce alkyl hydroperoxides or peroxynitrite (ONOO ) with high rate constants, whereas its reaction with H2O2 is more modest. Dopaminergic cell death induced by parkinsonian neurotoxins was inhibited by overexpression of peroxiredoxin 5 in the mitochondrial matrix. Proteomic analyses revealed an increase in the protein levels of Į-enolase and microtubule-associated protein 1B (MAP1B) involved in metabolic homeostasis and autophagy, respectively. Conversely, knock-down of endogenous peroxiredoxin 5 decreased Į-enolase and MAP1B levels, and sensitized cells to paraquat and 6-OHDA. Inhibition of thioredoxin-reductase (TrxR) activity with auranofin overcomes the protective effects of peroxiredoxin 5. These results suggest that mitochondrial peroxiredoxin 5 regulates metabolic/autophagic pathways and protects dopaminergic cells against parkinsonian neurotoxins independent from H2O2 signaling, suggesting a role for mitochondrial peroxynitrite in dopaminergic cell death.
doi:10.1016/j.freeradbiomed.2012.10.197
SFRBM 2012
S65
Miho C Emoto , Hideo Sato-Akaba2, Hiroshi Hirata3, and Hirotada G Fujii1 1 2 Sapporo Medical University, Japan, Osaka University, Japan, 3 Hokkaido University, Japan EPR imaging using nitroxides is a powerful non-invasive method for visualizing the redox status produced by free radicals. Two blood–brain barrier (BBB)-permeable nitroxides, 3-hydroxymethyl2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP) and 3methoxycarbonyl-2,2,5,5-tetramethyl-pirrolidine-1-yloxy (MCP), were used for EPR imaging of small rodent brains, but information about their in vivo properties has not been compared in detail. In this study, the detailed distributions and reduction rate of HMP and MCP in mouse heads were examined using our improved EPR imaging system. EPR imaging experiments using faster data acquisition technique were performed to investigate the time courses for localization of nitroxides in mouse heads. In obtained 2D slice-selected EPR images, HMP was mainly located outside the brain immediately after injection. However, MCP showed remarkable localization within the brain. HMP gradually spread to brain and distributed throughout all regions of the mouse head. To examine the effect of blood flow on reduction rate of nitroxides, blood flow was stopped by KCl injection after administration of nitroxides. The reduction rate of HMP without flow was significantly slow compared with flow. On the contrary, there were no significant differences in reduction rate of MCP with or without flow. These results suggest that MCP reduction occurs with intracellular reductant such as GSH and ascorbic acid within the brain.
doi:10.1016/j.freeradbiomed.2012.10.195
141 'LVWLQFW5ROHRI*OXWDUHGR[LQDQG5HJXODWLQJ 3URWHLQ*OXWDWKLRQ\ODWLRQDQG'RSDPLQHUJLF&HOO 'HDWK 1
Rodrigo Franco , Laura Zavala-Flores1, Humberto Rodriguez-Rocha1, and Aracely Garcia-Garcia1 1 University of Nebraska-Lincoln Dopaminergic cell death in Parkinson’s disease (PD) is associated with oxidative stress. However, the exact signaling cascades by which redox signaling regulates cell death progression are unclear. Glutaredoxins (Grxs) are oxidoreductases that use the reducing power of GSH to regulate protein function and activity by means of protein glutathionylation. In this work, we aimed to study the role of Grxs in dopaminergic cell death induced by experimental PD models. Dopaminergic cell death induced by the pesticide paraquat and the dopamine analog 6-hydroxydopamine was significantly inhibited by overexpression of the cytosolic isoform Grx1. Paraquat induced a decrease in the levels of glutathionylated proteins (PSSG) in dopaminergic cells in vitro and in vivo. Interestingly, overexpression of Grx1 enhanced PSSG formation upon paraquat stimulation. Grx1-induced protein glutathionylation significantly reduced the degradation of cellular substrates such as actin binding proteins (Flightless-1) and scaffolding signaling proteins (REPS2) during cell death. These results demonstrate that under oxidative stress Grx1 acts as a glutathionylating enzyme, and that increased PSSG formation protects cellular substrates from degradation during cell death progression. In contrast to the
protective role of Grx1, dopaminergic cell death induced by complex I inhibition with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) was significantly decreased in the mitochondrial isoform Grx2 knock-out mice. Accordingly, overexpression of Grx2 enhanced dopaminergic cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and paraquat in vitro. Overall these results demonstrate the distinct role of Grx1 and Grx2 in regulating protein glutathionylation and dopaminergic cell death in experimental PD models.
doi:10.1016/j.freeradbiomed.2012.10.196
142 0LWRFKRQGULDO3HUR[LUHGR[LQ3URWHFWV'RSDPLQHUJLF &HOOV$JDLQVW3DUNLQVRQLDQ1HXURWR[LQV,QGHSHQGHQW IURP+\GURJHQ3HUR[LGH6LJQDOLQJ 1
Aracely Garcia-Garcia , Humberto Rodriguez-Rocha1, and Rodrigo Franco1 1 University of Nebraska-Lincoln Oxidative stress plays a major role in dopaminergic degeneration during the pathogenesis of Parkinson’s disease (PD). Peroxiredoxins (Prx) are thioredoxin (Trx)-dependent thiol peroxidases expressed at low levels in the dopaminergic neurons of the substantia nigra, being peroxiredoxin 5 the isoform with the lowest expression, which might be associated with the increased sensitivity of dopaminergic cells to environmental/mitochondrial insults. We aimed to determine the protective role of peroxiredoxin 5 against experimental models of PD. Generation of hydrogen peroxide (H2O2) was monitored using the genetically encoded fluorescent sensor Hyper targeted to the cytosol and mitochondrial matrix. Dopaminergic cell death induced by the mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium + (MPP ) and rotenone, but not by the pesticide paraquat or the pro-oxidant dopamine analog 6-hydroxydopamine (6-OHDA), was preceded by an increase in H2O2 formation. However, neither overexpression of catalase nor mitochondrial-targeted catalase reduced dopaminergic cell death. Peroxiredoxin 5 is found in distinct cellular compartments including the cytosol and mitochondria, and has the ability to reduce alkyl hydroperoxides or peroxynitrite (ONOO ) with high rate constants, whereas its reaction with H2O2 is more modest. Dopaminergic cell death induced by parkinsonian neurotoxins was inhibited by overexpression of peroxiredoxin 5 in the mitochondrial matrix. Proteomic analyses revealed an increase in the protein levels of Į-enolase and microtubule-associated protein 1B (MAP1B) involved in metabolic homeostasis and autophagy, respectively. Conversely, knock-down of endogenous peroxiredoxin 5 decreased Į-enolase and MAP1B levels, and sensitized cells to paraquat and 6-OHDA. Inhibition of thioredoxin-reductase (TrxR) activity with auranofin overcomes the protective effects of peroxiredoxin 5. These results suggest that mitochondrial peroxiredoxin 5 regulates metabolic/autophagic pathways and protects dopaminergic cells against parkinsonian neurotoxins independent from H2O2 signaling, suggesting a role for mitochondrial peroxynitrite in dopaminergic cell death.
doi:10.1016/j.freeradbiomed.2012.10.197
SFRBM 2012
S65