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Impaired respiratory function in MELAS-induced pluripotent stem cells with high heteroplasmy levels.

Kodaira M, Hatakeyama H, Yuasa S, Seki T, Egashira T, Tohyama S, Kuroda Y, Tanaka A, Okata S, Hashimoto H, Kusumoto D, Kunitomi A, Takei M, Kashimura S, Suzuki T, Yozu G, Shimojima M, Motoda C, Hayashiji N, Saito Y, Goto Y, Fukuda K - FEBS Open Bio (2015)

Bottom Line: We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243A>G heteroplasmy.MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels.The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPSC-derived fibroblasts with low heteroplasmy levels showed normal complex I activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Keio University School of Medicine, Tokyo, Japan.

ABSTRACT
Mitochondrial diseases are heterogeneous disorders, caused by mitochondrial dysfunction. Mitochondria are not regulated solely by nuclear genomic DNA but by mitochondrial DNA. It is difficult to develop effective therapies for mitochondrial disease because of the lack of mitochondrial disease models. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major mitochondrial diseases. The aim of this study was to generate MELAS-specific induced pluripotent stem cells (iPSCs) and to demonstrate that MELAS-iPSCs can be models for mitochondrial disease. We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243A>G heteroplasmy. MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels. The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPSC-derived fibroblasts with low heteroplasmy levels showed normal complex I activity. Our data indicate that MELAS-iPSCs can be models for MELAS but we should carefully select MELAS-iPSCs with appropriate heteroplasmy levels and respiratory functions for mitochondrial disease modeling.

No MeSH data available.


Related in: MedlinePlus

Enzymatic activity analysis for mitochondrial respiratory complexes in healthy-fibroblasts, donor MELAS-fibroblasts and 4 MELAS-iPSC-derived fibroblasts, normalized against citrate synthase (CS) activity (A) and complex II (B).
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f0020: Enzymatic activity analysis for mitochondrial respiratory complexes in healthy-fibroblasts, donor MELAS-fibroblasts and 4 MELAS-iPSC-derived fibroblasts, normalized against citrate synthase (CS) activity (A) and complex II (B).

Mentions: To further characterize the potential for disease modeling, we examined the enzymatic activities of mitochondrial respiratory complexes I, II, III, and IV in MELAS-iPSC-differentiated fibroblasts from the four MELAS-iPSC lines (K1, H1, C11, and M2) and in primary skin fibroblasts derived from a donor patient, and compared them with skin fibroblasts of ten healthy persons (normalized as 100%). Enzymatic activities for mitochondrial respiratory complexes were analyzed as described elsewhere [14] with modifications. Mitochondrial heteroplasmy levels at the time of enzymatic analysis in the donor and iPSC-derived fibroblasts (K1, H1, C11, and M2) were 75%, 64%, 70%, 81%, and 100%, respectively. To normalize for content of mitochondria, each enzymatic activity was normalized against citrate synthase (CS) activity (Fig. 4A) and complex II (Fig. 4B) [16]. The complex II is regulated not by mtDNA but by nuclear DNA. The difference in the complex II would not be directly affected by mtDNA mutation. The normalization by complex II can minimize the effect of mitochondrial DNA mutation. The enzymatic activities of complex I were decreased in MELAS-iPSC lines H1, C11, and M2, and in the MELAS-donor fibroblasts (Fig. 4A and B). However, no decrease in enzymatic activity was observed for complexes II, III, and IV in the MELAS-iPSC-derived fibroblasts, despite the possibility that these complexes might be induced by the overall tRNA functional defect. In MELAS patients, complex I deficiency is the most common feature of mitochondrial enzymatic activity. A defect in the taurine modification of mutant tRNALeu(UUR) causes reduction in mitochondrial translation of the ND6 gene, which encodes a component of respiratory chain complex I. On the contrary, complexes II, III, and IV are not affected in MELAS patients [17]. Complexes II and IV normalized against CS activity were increased in M2 (Fig. 4A), which would be due to functional complementation for the decreased activity of complex I, but complex IV normalized against complex II didn’t show significant difference (Fig. 4B). These results indicate that the pathogenic threshold levels of m.3243A>G were recapitulated in MELAS-iPSC-derived fibroblasts from a donor patient.


Impaired respiratory function in MELAS-induced pluripotent stem cells with high heteroplasmy levels.

Kodaira M, Hatakeyama H, Yuasa S, Seki T, Egashira T, Tohyama S, Kuroda Y, Tanaka A, Okata S, Hashimoto H, Kusumoto D, Kunitomi A, Takei M, Kashimura S, Suzuki T, Yozu G, Shimojima M, Motoda C, Hayashiji N, Saito Y, Goto Y, Fukuda K - FEBS Open Bio (2015)

Enzymatic activity analysis for mitochondrial respiratory complexes in healthy-fibroblasts, donor MELAS-fibroblasts and 4 MELAS-iPSC-derived fibroblasts, normalized against citrate synthase (CS) activity (A) and complex II (B).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383791&req=5

f0020: Enzymatic activity analysis for mitochondrial respiratory complexes in healthy-fibroblasts, donor MELAS-fibroblasts and 4 MELAS-iPSC-derived fibroblasts, normalized against citrate synthase (CS) activity (A) and complex II (B).
Mentions: To further characterize the potential for disease modeling, we examined the enzymatic activities of mitochondrial respiratory complexes I, II, III, and IV in MELAS-iPSC-differentiated fibroblasts from the four MELAS-iPSC lines (K1, H1, C11, and M2) and in primary skin fibroblasts derived from a donor patient, and compared them with skin fibroblasts of ten healthy persons (normalized as 100%). Enzymatic activities for mitochondrial respiratory complexes were analyzed as described elsewhere [14] with modifications. Mitochondrial heteroplasmy levels at the time of enzymatic analysis in the donor and iPSC-derived fibroblasts (K1, H1, C11, and M2) were 75%, 64%, 70%, 81%, and 100%, respectively. To normalize for content of mitochondria, each enzymatic activity was normalized against citrate synthase (CS) activity (Fig. 4A) and complex II (Fig. 4B) [16]. The complex II is regulated not by mtDNA but by nuclear DNA. The difference in the complex II would not be directly affected by mtDNA mutation. The normalization by complex II can minimize the effect of mitochondrial DNA mutation. The enzymatic activities of complex I were decreased in MELAS-iPSC lines H1, C11, and M2, and in the MELAS-donor fibroblasts (Fig. 4A and B). However, no decrease in enzymatic activity was observed for complexes II, III, and IV in the MELAS-iPSC-derived fibroblasts, despite the possibility that these complexes might be induced by the overall tRNA functional defect. In MELAS patients, complex I deficiency is the most common feature of mitochondrial enzymatic activity. A defect in the taurine modification of mutant tRNALeu(UUR) causes reduction in mitochondrial translation of the ND6 gene, which encodes a component of respiratory chain complex I. On the contrary, complexes II, III, and IV are not affected in MELAS patients [17]. Complexes II and IV normalized against CS activity were increased in M2 (Fig. 4A), which would be due to functional complementation for the decreased activity of complex I, but complex IV normalized against complex II didn’t show significant difference (Fig. 4B). These results indicate that the pathogenic threshold levels of m.3243A>G were recapitulated in MELAS-iPSC-derived fibroblasts from a donor patient.

Bottom Line: We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243A>G heteroplasmy.MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels.The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPSC-derived fibroblasts with low heteroplasmy levels showed normal complex I activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Keio University School of Medicine, Tokyo, Japan.

ABSTRACT
Mitochondrial diseases are heterogeneous disorders, caused by mitochondrial dysfunction. Mitochondria are not regulated solely by nuclear genomic DNA but by mitochondrial DNA. It is difficult to develop effective therapies for mitochondrial disease because of the lack of mitochondrial disease models. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the major mitochondrial diseases. The aim of this study was to generate MELAS-specific induced pluripotent stem cells (iPSCs) and to demonstrate that MELAS-iPSCs can be models for mitochondrial disease. We successfully established iPSCs from the primary MELAS-fibroblasts carrying 77.7% of m.3243A>G heteroplasmy. MELAS-iPSC lines ranged from 3.6% to 99.4% of m.3243A>G heteroplasmy levels. The enzymatic activities of mitochondrial respiratory complexes indicated that MELAS-iPSC-derived fibroblasts with high heteroplasmy levels showed a deficiency of complex I activity but MELAS-iPSC-derived fibroblasts with low heteroplasmy levels showed normal complex I activity. Our data indicate that MELAS-iPSCs can be models for MELAS but we should carefully select MELAS-iPSCs with appropriate heteroplasmy levels and respiratory functions for mitochondrial disease modeling.

No MeSH data available.


Related in: MedlinePlus