Limits...
DJ-1 dopaminergic neuronal cells exhibit defects in mitochondrial function and structure: involvement of mitochondrial complex I assembly.

Heo JY, Park JH, Kim SJ, Seo KS, Han JS, Lee SH, Kim JM, Park JI, Park SK, Lim K, Hwang BD, Shong M, Kweon GR - PLoS ONE (2012)

Bottom Line: DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function.On the basis of these experiments, we concluded that DJ-1 cells have a defect in the assembly of complex I.It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chungnam National University School of Medicine, Daejeon, Korea.

ABSTRACT
DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O(2) consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease.

Show MeSH

Related in: MedlinePlus

DJ-1 gene deletion decreases mitochondrial respiratory chain function in dopaminergic neuronal cells.(A-a, b): The O2 consumption rate (OCR) was measured in SN4741 cells and DJ-1  cells in at least three independent experiments by using an XF analyzer. For validation of the measured O2 consumption rate, we used the 2 µg/ml oligomycin, 5 µM CCCP, and 1 µM rotenone sequentially. Each time point represents the mean (±SD), and compensated OCR data without background levels are shown in (b) with bar graphs. ***, p<0.001 (B): Mitochondrial membrane potential was investigated by rhodamine 123 staining and quantified by FACS analysis. ***, p<0.001 (C): The cell pellet was solubilized by digitonin and the mitochondrial ATP production rate was measured by a luminometer. To confirm that the calculated luminescence values represented ATP content, oligomycin was used to inhibit ATP production. *, p<0.05; **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3293835&req=5

pone-0032629-g002: DJ-1 gene deletion decreases mitochondrial respiratory chain function in dopaminergic neuronal cells.(A-a, b): The O2 consumption rate (OCR) was measured in SN4741 cells and DJ-1 cells in at least three independent experiments by using an XF analyzer. For validation of the measured O2 consumption rate, we used the 2 µg/ml oligomycin, 5 µM CCCP, and 1 µM rotenone sequentially. Each time point represents the mean (±SD), and compensated OCR data without background levels are shown in (b) with bar graphs. ***, p<0.001 (B): Mitochondrial membrane potential was investigated by rhodamine 123 staining and quantified by FACS analysis. ***, p<0.001 (C): The cell pellet was solubilized by digitonin and the mitochondrial ATP production rate was measured by a luminometer. To confirm that the calculated luminescence values represented ATP content, oligomycin was used to inhibit ATP production. *, p<0.05; **, p<0.01.

Mentions: Basal oxygen consumption was significantly lower in DJ-1 cells compared to SN4741 cells, whereas no difference in oxygen consumption was observed following treatment of the cells with oligomycin (an inhibitor of ATP synthase), which serves as a negative control (Figure 2A-a). Oxygen consumption was also lower in DJ-1 cells compared to SN4741 cells after treatment with CCCP, which uncouples proton pumping from ATP synthesis and represents a positive control for oxygen consumption (Figure 2A-a). To normalize the level of oxygen consumption, basal oxygen consumption was compared in cells treated with rotenone (which disrupts electron transfer from complex I to ubiquinone) to give a non-mitochondrial oxygen consumption value: overall, basal respiration was about 23% lower in DJ-1 cells compared to SN4741 cells (Figure 2A-b).


DJ-1 dopaminergic neuronal cells exhibit defects in mitochondrial function and structure: involvement of mitochondrial complex I assembly.

Heo JY, Park JH, Kim SJ, Seo KS, Han JS, Lee SH, Kim JM, Park JI, Park SK, Lim K, Hwang BD, Shong M, Kweon GR - PLoS ONE (2012)

DJ-1 gene deletion decreases mitochondrial respiratory chain function in dopaminergic neuronal cells.(A-a, b): The O2 consumption rate (OCR) was measured in SN4741 cells and DJ-1  cells in at least three independent experiments by using an XF analyzer. For validation of the measured O2 consumption rate, we used the 2 µg/ml oligomycin, 5 µM CCCP, and 1 µM rotenone sequentially. Each time point represents the mean (±SD), and compensated OCR data without background levels are shown in (b) with bar graphs. ***, p<0.001 (B): Mitochondrial membrane potential was investigated by rhodamine 123 staining and quantified by FACS analysis. ***, p<0.001 (C): The cell pellet was solubilized by digitonin and the mitochondrial ATP production rate was measured by a luminometer. To confirm that the calculated luminescence values represented ATP content, oligomycin was used to inhibit ATP production. *, p<0.05; **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293835&req=5

pone-0032629-g002: DJ-1 gene deletion decreases mitochondrial respiratory chain function in dopaminergic neuronal cells.(A-a, b): The O2 consumption rate (OCR) was measured in SN4741 cells and DJ-1 cells in at least three independent experiments by using an XF analyzer. For validation of the measured O2 consumption rate, we used the 2 µg/ml oligomycin, 5 µM CCCP, and 1 µM rotenone sequentially. Each time point represents the mean (±SD), and compensated OCR data without background levels are shown in (b) with bar graphs. ***, p<0.001 (B): Mitochondrial membrane potential was investigated by rhodamine 123 staining and quantified by FACS analysis. ***, p<0.001 (C): The cell pellet was solubilized by digitonin and the mitochondrial ATP production rate was measured by a luminometer. To confirm that the calculated luminescence values represented ATP content, oligomycin was used to inhibit ATP production. *, p<0.05; **, p<0.01.
Mentions: Basal oxygen consumption was significantly lower in DJ-1 cells compared to SN4741 cells, whereas no difference in oxygen consumption was observed following treatment of the cells with oligomycin (an inhibitor of ATP synthase), which serves as a negative control (Figure 2A-a). Oxygen consumption was also lower in DJ-1 cells compared to SN4741 cells after treatment with CCCP, which uncouples proton pumping from ATP synthesis and represents a positive control for oxygen consumption (Figure 2A-a). To normalize the level of oxygen consumption, basal oxygen consumption was compared in cells treated with rotenone (which disrupts electron transfer from complex I to ubiquinone) to give a non-mitochondrial oxygen consumption value: overall, basal respiration was about 23% lower in DJ-1 cells compared to SN4741 cells (Figure 2A-b).

Bottom Line: DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function.On the basis of these experiments, we concluded that DJ-1 cells have a defect in the assembly of complex I.It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chungnam National University School of Medicine, Daejeon, Korea.

ABSTRACT
DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O(2) consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease.

Show MeSH
Related in: MedlinePlus