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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.

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Fissional/fragmental changes of mitochondrial structure in DJ-1  cells.(A): To identify mitochondrial morphologic changes, SN4741 cells and DJ-1  cells were stained with MitoTracker red and co-stained with antibodies to COX4, which is a subunit of complex IV. The nucleus was identified by DAPI staining. The cells were sequentially observed by confocal microscopy at a magnification of ×400. (B-a): Cells were fixed, cryosectioned, and observed by transmission electron microscopy (TEM). DJ-1  cells exhibit smaller mitochondria, which is apparent at a higher magnification (inset, ×50,000). The original magnification was ×15,000. (B-b): Mitochondrial area was analyzed by Image J software. A minimum of 20 TEM slides was evaluated to characterize mitochondrial dynamics. We divided the section equally between the largest area and the smallest area of mitochondria and measured the number of mitochondria in that section. Compared to SN4741 cells, most of the DJ-1  cells had smaller mitochondria. (C-a, b): Mitochondrial mass was evaluated by MitoTracker green staining and quantified by FACS analysis. DJ-1  cells showed a left shift in the curve, indicating reduced mitochondrial mass. Significant differences were consistently observed in three independent experiments. ***, p<0.001 (D): Rotenone (10 nM), an inhibitor of complex I, did not induce changes in mitochondrial complex native protein as assessed by BN-PAGE analysis. HSP60 was used as a loading control. *** p<0.001.
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pone-0032629-g003: Fissional/fragmental changes of mitochondrial structure in DJ-1 cells.(A): To identify mitochondrial morphologic changes, SN4741 cells and DJ-1 cells were stained with MitoTracker red and co-stained with antibodies to COX4, which is a subunit of complex IV. The nucleus was identified by DAPI staining. The cells were sequentially observed by confocal microscopy at a magnification of ×400. (B-a): Cells were fixed, cryosectioned, and observed by transmission electron microscopy (TEM). DJ-1 cells exhibit smaller mitochondria, which is apparent at a higher magnification (inset, ×50,000). The original magnification was ×15,000. (B-b): Mitochondrial area was analyzed by Image J software. A minimum of 20 TEM slides was evaluated to characterize mitochondrial dynamics. We divided the section equally between the largest area and the smallest area of mitochondria and measured the number of mitochondria in that section. Compared to SN4741 cells, most of the DJ-1 cells had smaller mitochondria. (C-a, b): Mitochondrial mass was evaluated by MitoTracker green staining and quantified by FACS analysis. DJ-1 cells showed a left shift in the curve, indicating reduced mitochondrial mass. Significant differences were consistently observed in three independent experiments. ***, p<0.001 (D): Rotenone (10 nM), an inhibitor of complex I, did not induce changes in mitochondrial complex native protein as assessed by BN-PAGE analysis. HSP60 was used as a loading control. *** p<0.001.

Mentions: Mitochondrial defects may be involved in the early stages of PD pathogenesis [20]. In addition to mitochondrial functional defects, disruption of the fission/fusion machinery is considered an important factor in PD pathogenesis, leading to decreased mitochondrial energy production, increased oxidative stress, and impaired calcium homeostasis [21]. Therefore, we examined mitochondrial morphology in DJ-1 cells by confocal microscopy. We found the mitochondria to be smaller in DJ-1 cells than in SN4741 cells (Figure 3A). Furthermore, electron microscopy revealed that most DJ-1 cells had smaller mitochondrial areas than SN4741 cells (Figure 3B-a,b). However, obvious defects of the mitochondrial matrix or mitochondrial swelling were not observed. In addition to the smaller mitochondrial size, the numbers of mitochondria were reduced in DJ-1 cells although DJ-1 cells were bigger than SN4741 cells (Figure 4B-a,c). We also checked the viable mitochondrial mass by staining cells with MitoTracker green dye and analyzing them by FACS. The viable mitochondrial mass was lower in DJ-1 cells compared to SN4741 cells, indicating that both mitochondrial size and number were reduced in the cells (Figure 3C).


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)

Fissional/fragmental changes of mitochondrial structure in DJ-1  cells.(A): To identify mitochondrial morphologic changes, SN4741 cells and DJ-1  cells were stained with MitoTracker red and co-stained with antibodies to COX4, which is a subunit of complex IV. The nucleus was identified by DAPI staining. The cells were sequentially observed by confocal microscopy at a magnification of ×400. (B-a): Cells were fixed, cryosectioned, and observed by transmission electron microscopy (TEM). DJ-1  cells exhibit smaller mitochondria, which is apparent at a higher magnification (inset, ×50,000). The original magnification was ×15,000. (B-b): Mitochondrial area was analyzed by Image J software. A minimum of 20 TEM slides was evaluated to characterize mitochondrial dynamics. We divided the section equally between the largest area and the smallest area of mitochondria and measured the number of mitochondria in that section. Compared to SN4741 cells, most of the DJ-1  cells had smaller mitochondria. (C-a, b): Mitochondrial mass was evaluated by MitoTracker green staining and quantified by FACS analysis. DJ-1  cells showed a left shift in the curve, indicating reduced mitochondrial mass. Significant differences were consistently observed in three independent experiments. ***, p<0.001 (D): Rotenone (10 nM), an inhibitor of complex I, did not induce changes in mitochondrial complex native protein as assessed by BN-PAGE analysis. HSP60 was used as a loading control. *** p<0.001.
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Related In: Results  -  Collection

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pone-0032629-g003: Fissional/fragmental changes of mitochondrial structure in DJ-1 cells.(A): To identify mitochondrial morphologic changes, SN4741 cells and DJ-1 cells were stained with MitoTracker red and co-stained with antibodies to COX4, which is a subunit of complex IV. The nucleus was identified by DAPI staining. The cells were sequentially observed by confocal microscopy at a magnification of ×400. (B-a): Cells were fixed, cryosectioned, and observed by transmission electron microscopy (TEM). DJ-1 cells exhibit smaller mitochondria, which is apparent at a higher magnification (inset, ×50,000). The original magnification was ×15,000. (B-b): Mitochondrial area was analyzed by Image J software. A minimum of 20 TEM slides was evaluated to characterize mitochondrial dynamics. We divided the section equally between the largest area and the smallest area of mitochondria and measured the number of mitochondria in that section. Compared to SN4741 cells, most of the DJ-1 cells had smaller mitochondria. (C-a, b): Mitochondrial mass was evaluated by MitoTracker green staining and quantified by FACS analysis. DJ-1 cells showed a left shift in the curve, indicating reduced mitochondrial mass. Significant differences were consistently observed in three independent experiments. ***, p<0.001 (D): Rotenone (10 nM), an inhibitor of complex I, did not induce changes in mitochondrial complex native protein as assessed by BN-PAGE analysis. HSP60 was used as a loading control. *** p<0.001.
Mentions: Mitochondrial defects may be involved in the early stages of PD pathogenesis [20]. In addition to mitochondrial functional defects, disruption of the fission/fusion machinery is considered an important factor in PD pathogenesis, leading to decreased mitochondrial energy production, increased oxidative stress, and impaired calcium homeostasis [21]. Therefore, we examined mitochondrial morphology in DJ-1 cells by confocal microscopy. We found the mitochondria to be smaller in DJ-1 cells than in SN4741 cells (Figure 3A). Furthermore, electron microscopy revealed that most DJ-1 cells had smaller mitochondrial areas than SN4741 cells (Figure 3B-a,b). However, obvious defects of the mitochondrial matrix or mitochondrial swelling were not observed. In addition to the smaller mitochondrial size, the numbers of mitochondria were reduced in DJ-1 cells although DJ-1 cells were bigger than SN4741 cells (Figure 4B-a,c). We also checked the viable mitochondrial mass by staining cells with MitoTracker green dye and analyzing them by FACS. The viable mitochondrial mass was lower in DJ-1 cells compared to SN4741 cells, indicating that both mitochondrial size and number were reduced in the cells (Figure 3C).

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