Resistance to cerebral ischemic injury in UCP2 knockout mice: evidence for a role of UCP2 as a regulator of mitochondrial glutathione levels

“Primary antibodies to Mn SOD (Calbiochem, San Diego, CA, USA, 1/200), cytochrome c antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1/200) or cytochrome oxidase (COX) subunit IV (Molecular Probes, Eugene, OR, USA) were used.”

“The UCP2 gene is expressed in most tissues (Fleury et al. 1997; Gimeno et al. 1997; Pecqueur et al. 2001) including brain (Richard et al.”

“In vitro studies also suggest thatUCP2may lead to an increase in proton conductance through interaction with superoxide (Echtay et al. 2002).”

“Consistent with the recent marked progress in this field, our data support the role of UCP2 in oxidative pathophys- iology (Arsenijevic et al. 2000; Blanc et al. 2003) and further contribute to the understanding of the physiological function of UCP2.”

“It now appears that ROS production in mammalian cells is mainly a toxic by-product of the mitochondrial electron transport chain ( Raha and Robinson 2000 ).”

“ROS have been implicated in the pathogenesis of ischemic brain injury (Chan 1996).”

“Changes in mitochondrial levels of GSH have been described in pathological states associated with oxidative stress (particularly obesity, HIV, diabetes, cancer, inflammatory and infectious diseases ( Zaidan and Sims 1996; Szaleczky et al. 1999; Carretero et al. 2000; Perl and Banki 2000; Yang et al. 2000; Abou-Seif an…”

“Moreover, UCP2 has been implicated in the control of mitochondrial ROS levels (Negre-Salvayre et al. 1997; Lee et al. 1999; Arsenijevic et al. 2000; Yang et al. 2000; Li et al. 2001; Pecqueur et al. 2001) possibly by decreasing mitochondrial membrane potential (Skulachev 1998).”

“This response involves a consistent increase in Mn SOD, which may contribute to the reduction of ischemic injury and reduced levels of lipid peroxidation in UCP2–/– mice (Keller et al. 1998; Murakami et al. 1998; Fujimura et al. 1999; Guégan et al. 1999).”

“The UCP’s closest structural relatives dicarboxylate and 2oxoglutarate carriers (Ricquier and Bouillaud 2000) are thought to be involved in the mitochondrial uptake of the anti-oxidant GSH (Chen and Lash 1998).”

“Moreover, UCP2 has been implicated in the control of mitochondrial ROS levels (Negre-Salvayre et al. 1997; Lee et al. 1999; Arsenijevic et al. 2000; Yang et al. 2000; Li et al. 2001; Pecqueur et al. 2001) possibly by decreasing mitochondrial membrane potential (Skulachev 1998).”

“A role of intracellular GSH in protection against free radical processes induced by cerebral ischemia has also been evidenced (Love 1999).”

“In particular, our data draw attention to the status of UCP2 in the substantia nigra of patients with Parkinson’s disease where subnormal levels of GSH have been recorded (Jenner et al. 1992).”

“The UCP2 gene is expressed in most tissues (Fleury et al. 1997; Gimeno et al. 1997; Pecqueur et al. 2001) including brain (Richard et al. 1998).”

“UCP2 is induced in underfeeding (Pecqueur et al. 2001) and under pathological conditions (obesity, anorexia/ cachexia, diabetes, cardiovascular diseases, neurodegeneration, HIV, infection, inflammation) where excessive ROS production occurs, further supporting a link between UCP2 and oxidative stress (Chavin et al.”

“This response involves a consistent increase in Mn SOD, which may contribute to the reduction of ischemic injury and reduced levels of lipid peroxidation in UCP2–/– mice (Keller et al. 1998; Murakami et al. 1998; Fujimura et al. 1999; Guégan et al. 1999).”

“…cardiovascular diseases, neurodegenera- tion, HIV, infection, inflammation) where excessive ROS production occurs, further supporting a link between UCP2 and oxidative stress (Chavin et al. 1999; Cortez-Pinto et al. 1999; Rashid et al. 1999; Li et al. 2001; Pecqueur et al. 2001; Zhang et al. 2001).”

“Uncoupling protein 2 (UCP2) (Fleury et al. 1997; Boss et al. 2000; Ricquier and Bouillaud 2000) a homologue of the brown adipose tissue-specific proton transporter UCP1, belongs to the mitochondrial anion carrier family that are present in the inner mitochondrial membrane (el Moualij et al.”

“Moreover, UCP2 has been implicated in the control of mitochondrial ROS levels (Negre-Salvayre et al. 1997; Lee et al. 1999; Arsenijevic et al. 2000; Yang et al. 2000; Li et al. 2001; Pecqueur et al. 2001) possibly by decreasing mitochondrial membrane potential (Skulachev 1998).”

“Moreover, GSH has been shown to alter ROS formation in mitochondria and to counteract endogenous oxidative stress produced at the ubiquinone site of the electron transport chain (Garcia-Ruiz et al. 1995).”

“It has been suggested that interaction of superoxide with UCPs may be critical for decreasing mitochondrial ROS levels in vitro (Echtay et al. 2001, 2002).”

“In particular, previous data strongly suggest a key role of the mitochondrial manganese SOD (Mn SOD) in neuro- protection following focal ischemia (Keller et al. 1998; Murakami et al. 1998; Fujimura et al. 1999; Guégan et al. 1999).”

“The post-ischemic decrease in mitochondrial GSH is observed in WT but not in UCP2–/– mice The role of GSH against ROS and lipid peroxidation is well documented (Wendel and Feuerstein 1981).”

“2001) and under pathological conditions (obesity, anorexia/ cachexia, diabetes, cardiovascular diseases, neurodegeneration, HIV, infection, inflammation) where excessive ROS production occurs, further supporting a link between UCP2 and oxidative stress (Chavin et al. 1999; Cortez-Pinto et al. 1999; Rashid et al. 1999;…”

“…previous reports have demonstrated that most biochemical and morphological markers used in the present study are only marginally affected in sham-operated animals compared with animals with cerebral ischemia (Mizui et al. 1992; Suzuki et al. 1999; Candelario-Jalil et al. 2001; Pratico et al. 2002).”

“Moreover, UCP2 has been implicated in the control of mitochondrial ROS levels (Negre-Salvayre et al. 1997; Lee et al. 1999; Arsenijevic et al. 2000; Yang et al. 2000; Li et al. 2001; Pecqueur et al. 2001) possibly by decreasing mitochondrial membrane potential (Skulachev 1998).”

“UCP2 is induced in underfeeding (Pecqueur et al. 2001) and under pathological conditions (obesity, anorexia/ cachexia, diabetes, cardiovascular diseases, neurodegenera- tion, HIV, infection, inflammation) where excessive ROS production occurs, further supporting a link between UCP2 and oxidative stress (Chavin et al.…”

“Moreover, UCP2 has been implicated in the control of mitochondrial ROS levels (Negre-Salvayre et al. 1997; Lee et al. 1999; Arsenijevic et al. 2000; Yang et al. 2000; Li et al. 2001; Pecqueur et al. 2001) possibly by decreasing mitochondrial membrane potential (Skulachev 1998).”

“Endogenous GSH levels have been shown to correlate with the severity of the ischemic insult (Mizui et al. 1992).”

“…infectious diseases (Zaidan and Sims 1996; Szaleczky et al. 1999; Carretero et al. 2000; Perl and Banki 2000; Yang et al. 2000; Abou-Seif and Youssef 2001; Dobashi et al. 2001; Soltys et al. 2001) indicating that the regulatory role of UCP2 may be of vital importance in the progression of disease.”

“…infectious diseases (Zaidan and Sims 1996; Szaleczky et al. 1999; Carretero et al. 2000; Perl and Banki 2000; Yang et al. 2000; Abou-Seif and Youssef 2001; Dobashi et al. 2001; Soltys et al. 2001) indicating that the regulatory role of UCP2 may be of vital importance in the progression of disease.”

“…states associated with oxidative stress (par- ticularly obesity, HIV, diabetes, cancer, inflammatory and infectious diseases (Zaidan and Sims 1996; Szaleczky et al. 1999; Carretero et al. 2000; Perl and Banki 2000; Yang et al. 2000; Abou-Seif and Youssef 2001; Dobashi et al. 2001; Soltys et al.…”

“In line with our results, previous data have reported that microglial cells have a prominent GSH system for the defence against the reactive compounds they generate (Hirrlinger et al. 2000; Ong et al. 2000).”

“Previously, a tight associ- ation between changes in the redox state (i.e. superoxide ion level) and compensating increases in GSH has been found in macrophages (Sato et al. 2001).”

“In particular, depletion of mitochondrial rather than cytosolic GSH is crucial to elicit cell damage (Reed 1993; Shan et al. 1993; Armstrong et al. 2001).”

“…obesity, HIV, diabetes, cancer, inflammatory and infectious diseases (Zaidan and Sims 1996; Szaleczky et al. 1999; Carretero et al. 2000; Perl and Banki 2000; Yang et al. 2000; Abou-Seif and Youssef 2001; Dobashi et al. 2001; Soltys et al. 2001) indicating that the regulatory role…”

“ischemia (Mizui et al. 1992; Suzuki et al. 1999; Candelario-Jalil et al. 2001; Pratico et al. 2002).”

“Translocation of cytochrome c to the cytosol is prevented in UCP2 mice The cytosolic release of cytochrome c from the mitochondrial fraction represents a critical step of the cell death induced by ischemia (Guégan et al. 1999; Touzani et al. 2001).”

“Surgical preparation, volume of the infarct We performed permanent occlusion of the middle cerebral artery (MCAO) as previously described (De Bilbao et al. 2000) in 2-month-old UCP2–/–malemice (Arsenijevic et al. 2000) and their wild-type (WT) co-littermates (n = Ucp2 ¼ 6 and nWT ¼ 5).”

“The infarcted area of each section was calculated by the substraction of healthy tissue areas of the contralateral to the ipsilateral side of the section (Guégan et al. 1998) using a computer-assisted image analyzing system (Software >Morphometry?, Samba 2005 TITN, Alcatel, Grenoble, France).”

“In line with our results, previous data have reported that microglial cells have a prominent GSH system for the defence against the reactive compounds they generate (Hirrlinger et al. 2000; Ong et al. 2000 ).”

“Changes in mitochondrial levels of GSH have been described in pathological states associated with oxidative stress (particularly obesity, HIV, diabetes, cancer, inflammatory and infectious diseases (Zaidan and Sims 1996; Szaleczky et al. 1999; Carretero et al. 2000; Perl and Banki 2000; Yang et al. 2000; Abou-Seif and…”

“Changes in mitochondrial levels of GSH have been described in pathological states associated with oxidative stress (particularly obesity, HIV, diabetes, cancer, inflammatory and infectious diseases (Zaidan and Sims 1996; Szaleczky et al. 1999; Carretero et al. 2000; Perl and Banki 2000; Yang et al. 2000; Abou-Seif and…”

“ischemia (Mizui et al. 1992; Suzuki et al. 1999; Candelario-Jalil et al. 2001; Pratico et al. 2002).”

“Translocation of cytochrome c to the cytosol is prevented in UCP2 mice The cytosolic release of cytochrome c from the mitochondrial fraction represents a critical step of the cell death induced by ischemia (Guégan et al. 1999; Touzani et al. 2001).”

“In particular, depletion of mitochondrial rather than cytosolic GSH is crucial to elicit cell damage (Reed 1993; Shan et al. 1993; Armstrong et al. 2001).”

“Mitochondrial permeability and membrane potential, which may be altered by the uncoupling activity of UCP2 (Ricquier and Bouillaud 2000), can also be influenced by GSH levels (Masini et al. 1992).”

“…pathological states associated with oxidative stress (par- ticularly obesity, HIV, diabetes, cancer, inflammatory and infectious diseases (Zaidan and Sims 1996; Szaleczky et al. 1999; Carretero et al. 2000; Perl and Banki 2000; Yang et al. 2000; Abou-Seif and Youssef 2001; Dobashi et…”

“2b) mice (Durmaz et al. 1999).”

“We then proceed for gel electrophoresis and immunodetection as previously described (Llames et al. 2000).”

“Consistent with these latter findings, knockout of UCP2 was found to be protective in a model of cerebral ischemic injury (110).”

“Interestingly, a role for UCP2 in the transport of mGSH has been described in neurons, suggesting that the transport of protons back into the matrix by UCP2 may favor the movement of GSH (de Bilbao et al., 2004).”

“interconnected [133,134].”