Creating Cell Model 2.0 Using Patient Samples Carrying a Pathogenic Mitochondrial DNA Mutation: iPSC Approach for LHON.

Leber's Hereditary Optic Neuropathy is the most prevalent mitochondrial neurological disease caused by mutations in mitochondrial DNA encoded respiratory complex I subunits. Although the genetic origin for Leber's hereditary optic neuropathy was identified about 30 years ago, the underlying pathogenesis is still unclear primarily due to the lack of a relevant system or cell model. Current models are limited to lymphoblasts, fibroblasts, or cybrid cell lines. As the disease phenotype is limited to retinal ganglion cells, induced pluripotent stem cells will serve as an excellent model for studying this tissue-specific disease, elucidating its underlying molecular mechanisms, and identifying novel therapeutic targets. Here, we describe a detailed protocol for the generation of retinal ganglion cells, and also cardiomyocytes for proof of iPSC pluripotency.

Aging-associated mitochondrial DNA mutations alter oxidative phosphorylation machinery and cause mitochondrial dysfunctions.

Our previous study generated a series of cybrids containing mitochondria of synaptosomes from mice at different ages. The following functional analysis on these cybrids revealed an age-dependent decline of mitochondrial function. To understand the underlying mechanisms that contribute to the age-related mitochondrial dysfunction, we focused on three cybrids carrying mitochondria derived from synaptosomes of the old mice that exhibited severe respiratory deficiencies. In particular, we started with a comprehensive analysis of mitochondrial genome by high resolution, high sensitive deep sequencing method. Compared with young control, we detected a significant accumulation of heteroplasmic mtDNA mutations. These mutations included six alterations in main control region that has been shown to regulate overall gene-expression, and four alterations in protein coding region, two of which led to significant changes in complex I subunit ND5 and complex III subunit CytB. Interestingly, a reduced mtDNA-encoded protein synthesis was associated with the changes in the main control region. Likewise, mutations in ND5 and CytB were associated with defects in assembly of respiratory complexes. Altogether, the identified age-dependent accumulation of mtDNA mutations in mouse brain likely contributes to the decline in mitochondrial function.

Comparative bioenergetic study of neuronal and muscle mitochondria during aging.

Mitochondrial respiratory chain defects have been associated with various diseases and with normal aging, particularly in tissues with high energy demands, including brain and skeletal muscle. Tissue-specific manifestation of mitochondrial DNA (mtDNA) mutations and mitochondrial dysfunction are hallmarks of mitochondrial diseases although the underlying mechanisms are largely unclear. Previously, we and others have established approaches for transferring mtDNA from muscle and synaptosomes of mice at various ages to cell cultures. In this study, we carried out a comprehensive bioenergetic analysis of cells bearing mitochondria derived from young, middle-aged, and old mouse skeletal muscles and synaptosomes. Significant age-associated alterations in oxidative phosphorylation and regulation during aging were observed in cybrids carrying mitochondria from both skeletal muscle and synaptosomes. Our results also revealed that loss of oxidative phosphorylation capacity may occur at various ages in muscle and brain. These findings indicate the existence of a tissue-specific regulatory mechanism for oxidative phosphorylation.

Generation and bioenergetic analysis of cybrids containing mitochondrial DNA from mouse skeletal muscle during aging.

Mitochondrial respiratory chain defects have been associated with various diseases and normal aging, particularly in tissues with high energy demands including skeletal muscle. Muscle-specific mitochondrial DNA (mtDNA) mutations have also been reported to accumulate with aging. Our understanding of the molecular processes mediating altered mitochondrial gene expression to dysfunction associated with mtDNA mutations in muscle would be greatly enhanced by our ability to transfer muscle mtDNA to established cell lines. Here, we report the successful generation of mouse cybrids carrying skeletal muscle mtDNA. Using this novel approach, we performed bioenergetic analysis of cells bearing mtDNA derived from young and old mouse skeletal muscles. A significant decrease in oxidative phosphorylation coupling and regulation capacity has been observed with cybrids carrying mtDNA from skeletal muscle of old mice. Our results also revealed decrease growth capacity and cell viability associated with the mtDNA derived from muscle of old mice. These findings indicate that a decline in mitochondrial function associated with compromised mtDNA quality during aging leads to a decrease in both the capacity and regulation of oxidative phosphorylation.

An assembled complex IV maintains the stability and activity of complex I in mammalian mitochondria.

In the mammalian mitochondrial electron transfer system, the majority of electrons enter at complex I, go through complexes III and IV, and are finally delivered to oxygen. Previously we generated several mouse cell lines with suppressed expression of the nuclearly encoded subunit 4 of complex IV. This led to a loss of assembly of complex IV and its defective function. Interestingly, we found that the level of assembled complex I and its activity were also significantly reduced, whereas levels and activity of complex III were normal or up-regulated. The structural and functional dependence of complex I on complex IV was verified using a human cell line carrying a nonsense mutation in the mitochondrially encoded complex IV subunit 1 gene. Our work documents that, although there is no direct electron transfer between them, an assembled complex IV helps to maintain complex I in mammalian cells.

Nuclear suppression of mitochondrial defects in cells without the ND6 subunit.

Previously, we characterized a mouse cell line, 4A, carrying a mitochondrial DNA mutation in the subunit for respiratory complex I, NADH dehydrogenase, in the ND6 gene. This mutation abolished the complex I assembly and disrupted the respiratory function of complex I. We now report here that a galactose-resistant clone, 4AR, was isolated from the cells carrying the ND6 mutation. 4AR still contained the homoplasmic mutation, and apparently there was no ND6 protein synthesis, whereas the assembly of other complex I subunits into complex I was recovered. Furthermore, the respiratory activity and mitochondrial membrane potential were fully recovered. To investigate the genetic origin of this compensation, the mitochondrial DNA (mtDNA) from 4AR was transferred to a new nuclear background. The transmitochondrial lines failed to grow in galactose medium. We further transferred mtDNA with a nonsense mutation at the ND5 gene to the 4AR nuclear background, and a suppression for mitochondrial deficiency was observed. Our results suggest that change(s) in the expression of a certain nucleus-encoded factor(s) can compensate for the absence of the ND6 or ND5 subunit.

Restoration of mitochondrial function in cells with complex I deficiency.

The mammalian mitochondrial NADH dehydrogenase (complex I) is the major entry point for the electron transport chain. It is the largest and most complicated respiratory complex consisting of at least 46 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). Deficiency in complex I function has been associated with various human diseases including neurodegenerative diseases and the aging process. To explore ways to restore mitochondrial function in complex I-deficient cells, various cell models with mutations in genes encoding subunits for complex I have been established. In this paper, we discuss various approaches to recover mitochondrial activity, the complex I activity in particular, in cultured cells.

Genetic and functional analysis of mitochondrial DNA-encoded complex I genes.

Mammalian mitochondrial NADH dehydrogenase (complex I) is a multimeric complex consisting of at least 45 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). The function of these subunits is largely unknown. We have established an efficient method to isolate and characterize cells carrying mutations in various mtDNA-encoded complex I genes. With this method, 15 mouse cell lines with deficiencies in complex I-dependent respiration were obtained, and two near-homoplasmic mutations in mouse ND5 and ND6 genes were isolated. Furthermore, by generating a series of cell lines with the same nuclear background but different content of an mtDNA nonsense mutation, we analyzed the genetic and functional thresholds in mouse mitochondria. We found that in wild-type cells, about 40% of ND5 mRNA is in excess of that required to support a normal rate of ND5 subunit synthesis. However, there is no indication of compensatory upsurge in either transcription or translation with the increase in the proportion of mutant ND5 genes. Interestingly, the highest ND5 protein synthesis rate was just sufficient to support the maximum complex I-dependent respiration rate, suggesting a tight regulation at the translational level. In another line of research, we showed that the mitochondrial NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1), although consisting of a single subunit, can completely restore respiratory NADH dehydrogenase activity in mutant human cells that lack the essential mtDNA-encoded subunit ND4. In particular, in these transfected cells, the yeast enzyme becomes integrated into the human respiratory chain and fully restores the capacity of the cells to grow in galactose medium.

Revisiting the mouse mitochondrial DNA sequence.

The existence of reliable mtDNA reference sequences for each species is of great relevance in a variety of fields, from phylogenetic and population genetics studies to pathogenetic determination of mtDNA variants in humans or in animal models of mtDNA-linked diseases. We present compelling evidence for the existence of sequencing errors on the current mouse mtDNA reference sequence. This includes the deletion of a full codon in two genes, the substitution of one amino acid on five occasions and also the involvement of tRNA and rRNA genes. The conclusions are supported by: (i) the re-sequencing of the original cell line used by Bibb and Clayton, the LA9 cell line, (ii) the sequencing of a second L-derivative clone (L929), and (iii) the comparison with 12 other mtDNA sequences from live mice, 10 of them maternally related with the mouse from which the L cells were generated. Two of the latest sequences are reported for the first time in this study (Balb/cJ and C57BL/6J). In addition, we found that both the LA9 and L929 mtDNAs also contain private clone polymorphic variants that, at least in the case of L929, promote functional impairment of the oxidative phosphorylation system. Consequently, the mtDNA of the strain used for the mouse genome project (C57BL/6J) is proposed as the new standard for the mouse mtDNA sequence.