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Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1.

Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT, Surmeier DJ - Nature (2010)

Bottom Line: Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery.The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling.The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

ABSTRACT
Parkinson's disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson's disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson's disease.

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Loss of DJ-1 attenuated UCP-dependent flickering in mitochondrial membrane potential(a) Left, mito-roGFP measurements in a SNc dopaminergic neuron (as in Figure 1) before and after application of genipin (red trace). Right, box plots summarizing mean mito-roGFP measurements following isradipine application (green box) (n=9) or genipin (red box) (n=6) to SNc dopaminergic neurons; isradipine significantly decreased oxidation, whereas genipin increased oxidation (P<0.05). Also show is a box plot of mito-roGFP measurements from VTA dopaminergic neurons following genipin; genipin had no effect on these measurements (n=5, P>0.05). (b) Left, TMRM fluorescence measurement from a wild-type SNc dopaminergic neuron (black trace) and a DJ-1 knockout (red trace). Right, box plots of mean frequency and amplitude data from wild-type (n=21) and DJ-1 knockout neurons (n=7); both amplitude and frequency of flickering were decreased in DJ-1 knockouts (P<0.05). (c) Quantitative PCR analysis of UCP expression in DJ-1 knockout mice: UCP2 mRNA abundance (relative to GAPDH) was not significantly altered in VTA and SNc (P>0.05, n=9). To the right, bar graphs plot the abundance of UCP4 and UCP5 mRNAs, normalized by that of UCP2 in each sample. The relative abundance of UCP4 and UCP5 mRNA was decreased in the SNc of DJ-1 knockout mice (P<0.05, n=9). UCP4 mRNA abundance was higher in VTA from DJ-1 knockout mice (P<0.05, n=9), while UCP5 mRNA was unchanged. (d) Schematic summary of the results presented linking calcium entry through L-type channels during pacemaking with elevated mitochondrial oxidant stress and opening of UCPs. The model also proposes that oxidized DJ-1 translocates to the nucleus and increases the transcription of UCP4 and UCP5, leading to increased UCP protein in the IMM.
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Figure 4: Loss of DJ-1 attenuated UCP-dependent flickering in mitochondrial membrane potential(a) Left, mito-roGFP measurements in a SNc dopaminergic neuron (as in Figure 1) before and after application of genipin (red trace). Right, box plots summarizing mean mito-roGFP measurements following isradipine application (green box) (n=9) or genipin (red box) (n=6) to SNc dopaminergic neurons; isradipine significantly decreased oxidation, whereas genipin increased oxidation (P<0.05). Also show is a box plot of mito-roGFP measurements from VTA dopaminergic neurons following genipin; genipin had no effect on these measurements (n=5, P>0.05). (b) Left, TMRM fluorescence measurement from a wild-type SNc dopaminergic neuron (black trace) and a DJ-1 knockout (red trace). Right, box plots of mean frequency and amplitude data from wild-type (n=21) and DJ-1 knockout neurons (n=7); both amplitude and frequency of flickering were decreased in DJ-1 knockouts (P<0.05). (c) Quantitative PCR analysis of UCP expression in DJ-1 knockout mice: UCP2 mRNA abundance (relative to GAPDH) was not significantly altered in VTA and SNc (P>0.05, n=9). To the right, bar graphs plot the abundance of UCP4 and UCP5 mRNAs, normalized by that of UCP2 in each sample. The relative abundance of UCP4 and UCP5 mRNA was decreased in the SNc of DJ-1 knockout mice (P<0.05, n=9). UCP4 mRNA abundance was higher in VTA from DJ-1 knockout mice (P<0.05, n=9), while UCP5 mRNA was unchanged. (d) Schematic summary of the results presented linking calcium entry through L-type channels during pacemaking with elevated mitochondrial oxidant stress and opening of UCPs. The model also proposes that oxidized DJ-1 translocates to the nucleus and increases the transcription of UCP4 and UCP5, leading to increased UCP protein in the IMM.

Mentions: Uncoupling proteins (UCPs) are another class of mitochondrial ion channel whose open probability is increased by superoxide 13. UCP opening decreases the IMM potential modestly 14, making it a plausible mediator of the drop in IMM potential inferred from the TMRM measurements. Five UCPs have been cloned, three of which are robustly expressed in the SNc (UCP2,4,5) 15. Application of the UCP antagonist genipin 16 significantly decreased the frequency and amplitude of mitochondrial flickering (Fig. 3e), as did dialyzing neurons with adenosine triphosphate (Supplementary Fig. 3), providing support for UCP mediation 17. The modest depolarization brought about by UCP activation is thought to diminish superoxide generation without significantly compromising ATP production, creating a protective, negative feedback system to complement enzymatic defenses against ROS 17,18. If this were the case, blocking UCPs with genipin should cause superoxide levels and oxidation of mitochondrial proteins to rise. Indeed, genipin significantly elevated oxidation of mito-roGFP in SNc dopaminergic neurons, whereas it had no effect on mitochondrial oxidation in VTA dopaminergic neurons, where the UCP defense was not engaged (Fig. 4a).


Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1.

Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT, Surmeier DJ - Nature (2010)

Loss of DJ-1 attenuated UCP-dependent flickering in mitochondrial membrane potential(a) Left, mito-roGFP measurements in a SNc dopaminergic neuron (as in Figure 1) before and after application of genipin (red trace). Right, box plots summarizing mean mito-roGFP measurements following isradipine application (green box) (n=9) or genipin (red box) (n=6) to SNc dopaminergic neurons; isradipine significantly decreased oxidation, whereas genipin increased oxidation (P<0.05). Also show is a box plot of mito-roGFP measurements from VTA dopaminergic neurons following genipin; genipin had no effect on these measurements (n=5, P>0.05). (b) Left, TMRM fluorescence measurement from a wild-type SNc dopaminergic neuron (black trace) and a DJ-1 knockout (red trace). Right, box plots of mean frequency and amplitude data from wild-type (n=21) and DJ-1 knockout neurons (n=7); both amplitude and frequency of flickering were decreased in DJ-1 knockouts (P<0.05). (c) Quantitative PCR analysis of UCP expression in DJ-1 knockout mice: UCP2 mRNA abundance (relative to GAPDH) was not significantly altered in VTA and SNc (P>0.05, n=9). To the right, bar graphs plot the abundance of UCP4 and UCP5 mRNAs, normalized by that of UCP2 in each sample. The relative abundance of UCP4 and UCP5 mRNA was decreased in the SNc of DJ-1 knockout mice (P<0.05, n=9). UCP4 mRNA abundance was higher in VTA from DJ-1 knockout mice (P<0.05, n=9), while UCP5 mRNA was unchanged. (d) Schematic summary of the results presented linking calcium entry through L-type channels during pacemaking with elevated mitochondrial oxidant stress and opening of UCPs. The model also proposes that oxidized DJ-1 translocates to the nucleus and increases the transcription of UCP4 and UCP5, leading to increased UCP protein in the IMM.
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Figure 4: Loss of DJ-1 attenuated UCP-dependent flickering in mitochondrial membrane potential(a) Left, mito-roGFP measurements in a SNc dopaminergic neuron (as in Figure 1) before and after application of genipin (red trace). Right, box plots summarizing mean mito-roGFP measurements following isradipine application (green box) (n=9) or genipin (red box) (n=6) to SNc dopaminergic neurons; isradipine significantly decreased oxidation, whereas genipin increased oxidation (P<0.05). Also show is a box plot of mito-roGFP measurements from VTA dopaminergic neurons following genipin; genipin had no effect on these measurements (n=5, P>0.05). (b) Left, TMRM fluorescence measurement from a wild-type SNc dopaminergic neuron (black trace) and a DJ-1 knockout (red trace). Right, box plots of mean frequency and amplitude data from wild-type (n=21) and DJ-1 knockout neurons (n=7); both amplitude and frequency of flickering were decreased in DJ-1 knockouts (P<0.05). (c) Quantitative PCR analysis of UCP expression in DJ-1 knockout mice: UCP2 mRNA abundance (relative to GAPDH) was not significantly altered in VTA and SNc (P>0.05, n=9). To the right, bar graphs plot the abundance of UCP4 and UCP5 mRNAs, normalized by that of UCP2 in each sample. The relative abundance of UCP4 and UCP5 mRNA was decreased in the SNc of DJ-1 knockout mice (P<0.05, n=9). UCP4 mRNA abundance was higher in VTA from DJ-1 knockout mice (P<0.05, n=9), while UCP5 mRNA was unchanged. (d) Schematic summary of the results presented linking calcium entry through L-type channels during pacemaking with elevated mitochondrial oxidant stress and opening of UCPs. The model also proposes that oxidized DJ-1 translocates to the nucleus and increases the transcription of UCP4 and UCP5, leading to increased UCP protein in the IMM.
Mentions: Uncoupling proteins (UCPs) are another class of mitochondrial ion channel whose open probability is increased by superoxide 13. UCP opening decreases the IMM potential modestly 14, making it a plausible mediator of the drop in IMM potential inferred from the TMRM measurements. Five UCPs have been cloned, three of which are robustly expressed in the SNc (UCP2,4,5) 15. Application of the UCP antagonist genipin 16 significantly decreased the frequency and amplitude of mitochondrial flickering (Fig. 3e), as did dialyzing neurons with adenosine triphosphate (Supplementary Fig. 3), providing support for UCP mediation 17. The modest depolarization brought about by UCP activation is thought to diminish superoxide generation without significantly compromising ATP production, creating a protective, negative feedback system to complement enzymatic defenses against ROS 17,18. If this were the case, blocking UCPs with genipin should cause superoxide levels and oxidation of mitochondrial proteins to rise. Indeed, genipin significantly elevated oxidation of mito-roGFP in SNc dopaminergic neurons, whereas it had no effect on mitochondrial oxidation in VTA dopaminergic neurons, where the UCP defense was not engaged (Fig. 4a).

Bottom Line: Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery.The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling.The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

ABSTRACT
Parkinson's disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson's disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson's disease.

Show MeSH
Related in: MedlinePlus