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Leber’s hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mitochondrial DNA (mtDNA) mutations. LHON-linked ND6 14484T > C (p.M64V) mutation affected structural components of complex I but its pathophysiology is poorly understood. The structural analysis of complex I revealed that the M64 forms a nonpolar interaction Y59 in the ND6, Y59 in the ND6 interacts with E34 of ND4L, and L60 of ND6 interacts with the Y114 of ND1. These suggested that the m.14484T > C mutation may perturb the structure and function of complex I. Mutant cybrids constructed by transferring mitochondria from lymphoblastoid cell lines of one Chinese LHON family into mtDNA-less (ρo) cells revealed decreases in the levels of ND6, ND1 and ND4L. The m.14484T > C mutation may affect mitochondrial mRNA homeostasis, supported by reduced levels of SLIRP and SUPV3L1 involved in mRNA degradation and increasing expression of ND6, ND1 and ND4L genes. These alterations yielded decreased activity of complex I, respiratory deficiency, diminished mitochondrial ATP production and reduced membrane potential, and increased production of reactive oxygen species in the mutant cybrids. Furthermore, the m.14484T > C mutation promoted apoptosis, evidenced by elevating Annexin V-positive cells, release of cytochrome c into cytosol, levels in apoptotic proteins BAX, caspases 3, 7, 9 and decreasing levels in anti-apoptotic protein Bcl-xL in the mutant cybrids. Moreover, the cybrids bearing the m.14484T > C mutation exhibited the reduced levels of autophagy protein LC3, increased levels of substrate P62 and impaired PINK1/Parkin-dependent mitophagy. Our findings highlighted the critical role of m.14484T > C mutation in the pathogenesis of LHON.

Sensorineural hearing loss often results from damaged or deficient inner ear hair cells. Mitochondrial 12S rRNA 1555A>G mutation has been associated with hearing loss in many families. The m.1555A>G mutation is a primary factor underlying the development of hearing loss and TRMU allele (c.28G>T, p.Ala10Sser) encoding tRNA thiouridylase interact with m.1555A>G mutation to cause hearing loss. However, the tissue specificity of mitochondrial dysfunction remains elusive and there is no highly effective therapy for mitochondrial deafness. We report here the generation of induced pluripotent stem cells (iPSCs) from lymphoblastoid cell lines derived from members of an Arab-Israeli family (asymptomatic individual carrying only m.1555A>G mutation, symptomatic individual bearing both m.1555A>G and c.28G>T mutations, and control subject). The c.28G>T mutation in iPSC lines from a hearing-impaired subject was corrected by CRISPR/Cas9. These iPSCs were differentiated into otic epithelial progenitor (OEP) cells and subsequent inner ear hair cell (HC)-like cells. The iPSCs bearing m.1555A>G mutation exhibited mildly deficient differentiation into OEP and resultant HC-like cells displayed mild defects in morphology and electrophysiological properties. Strikingly, those HC-like cells harboring m.1555A>G and TRMU c.28G>T mutations displayed greater defects in the development, morphology and functions than those in cells bearing only m.1555A>G mutation. Transcriptome analysis of patients-derived HC-like cells revealed altered expressions of genes vital for mechanotransduction of hair cells. Genetic correction of TRMU c.28G>T mutation yielded morphologic and functional recovery of patient derived HC-like cells. These findings provide new insights into pathophysiology of maternally inherited hearing loss and a step toward therapeutic interventions for this disease.

Leber’s hereditary optic neuropathy (LHON) is a maternally transmitted eye disease due to the degeneration of retinal ganglion cells (RGCs). Mitochondrial 11778G > A mutation is the most common LHON-associated mitochondrial DNA (mtDNA) mutation. Our recent studies demonstrated some LHON families manifested by synergic interaction between m.11778G > A mutation and YARS2 allele (c.572G > T, p.Gly191Val) encoding mitochondrial tyrosyl-tRNA synthetase. However, the RGC-specific effects of LHON-associated mtDNA mutations remain elusive and there is no highly effective therapy for LHON. Here, we generated patients-derived induced pluripotent stem cells (iPSCs) from fibroblasts derived from a Chinese LHON family (both m.11778G > A and c.572G > T mutations, only m.11778G > A mutation, and control subject). The c.572G > T mutation in iPSC lines from a syndromic individual was corrected by CRISPR/Cas9. Those iPSCs were differentiated into neural progenitor cells and subsequently induced RGC-like cells using a stepwise differentiation procedure. Those RGC-like cells derived from symptomatic individual harboring both m.11778G > A and c.572G > T mutations exhibited greater defects in neuronal differentiation, morphology including reduced area of soma, numbers of neurites and shortened length of axons, electrophysiological properties than those in cells bearing only m.11778G > A mutation. Furthermore, these RGC-like cells revealed more drastic reductions in oxygen consumption rates, levels of mitochondrial ATP and increasing productions of reactive oxygen species than those in other cell models. These mitochondrial dysfunctions promoted the apoptotic process for RGC degenerations. Correction of YARS2 c.572G > T mutation rescued deficiencies of patient-derived RGC-like cells. These findings provide new insights into pathophysiology of LHON arising from RGC-specific mitochondrial dysfunctions and step toward therapeutic intervention for this disease.

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited eye disease that results from degeneration of retinal ganglion cells (RGC). Mitochondrial ND4 11778G > A mutation, which affects structural components of complex I, is the most prevalent LHON-associated mitochondrial DNA (mtDNA) mutation worldwide. The m.11778G > A mutation is the primary contributor underlying the development of LHON and X-linked PRICKLE3 allele (c.157C > T, p.Arg53Trp) linked to biogenesis of ATPase interacts with m.11778G > A mutation to cause LHON. However, the lack of appropriate cell and animal models of LHON has been significant obstacles for deep elucidation of disease pathophysiology, specifically the tissue-specific effects. Using RGC-like cells differentiated from induced pluripotent stem cells (iPSCs) from members of one Chinese family (asymptomatic subjects carrying only m.11778G > A mutation or PRICKLE3 p.Arg53Trp mutation, symptomatic individuals bearing both m.11778G > A and PRICKLE3 p.Arg53Trp mutations and control lacking these mutations), we demonstrated the deleterious effects of mitochondrial dysfunctions on the morphology and functions of RGCs. Notably, iPSCs bearing only m.11778G > A or p.Arg53Trp mutation exhibited mild defects in differentiation to RGC-like cells. The RGC-like cells carrying only m.11778G > A or p.Arg53Trp mutation displayed mild defects in RGC morphology, including the area of soma and numbers of neurites, electrophysiological properties, ATP contents and apoptosis. Strikingly, those RGC-like cells derived from symptomatic individuals harboring both m.11778G > A and p.Arg53Trp mutations displayed greater defects in the development, morphology and functions than those in cells bearing single mutation. These findings provide new insights into pathophysiology of LHON arising from RGC deficiencies caused by synergy between m.11778G > A and PRICKLE3 p.Arg53Trp mutation.

Mitochondrial DNA (mtDNA) mutations have been associated with Leber’s hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we investigated the pathophysiology of a LHON susceptibility allele (m.3394T>C, p.30Y>H) in the Mitochondrial (MT)-ND1 gene. The incidence of m.3394T>C mutation was 2.7% in the cohort of 1741 probands with LHON. Extremely low penetrances of LHON were observed in 26 pedigrees carrying only m.3394T>C mutation, while 21 families bearing m.3394T>C, together with m.11778G>A or m.14484T>C mutation, exhibited higher penetrance of LHON than those in families carrying single mtDNA mutation(s). The m.3394T>C mutation disrupted the specific electrostatic interactions between Y30 of p.MT-ND1 with the sidechain of E4 and backbone carbonyl group of M1 of NDUFA1 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 1) of complex I, thereby altering the structure and function of complex I. We demonstrated that these cybrids bearing only m.3394T>C mutation caused mild mitochondrial dysfunctions and those harboring both m.3394T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions than cybrids carrying only m.11778G>A mutation. In particular, the m.3394T>C mutation altered the stability of p.MT-ND1 and complex I assembly. Furthermore, the m.3394T>C mutation decreased the activities of mitochondrial complexes I, diminished mitochondrial ATP levels and membrane potential and increased the production of reactive oxygen species in the cybrids. These m.3394T>C mutation-induced alterations aggravated mitochondrial dysfunctions associated with the m.11778G>A mutation. These resultant biochemical defects contributed to higher penetrance of LHON in these families carrying both mtDNA mutations. Our findings provide new insights into the pathophysiology of LHON arising from the synergy between mitochondrial ND1 and ND4 mutations.

Mutations in mitochondrial DNA (mtDNA) have been associated with Leber's hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we demonstrated that a missense mutation (m.12338T>C, p.1M>T) in the ND5 gene contributed to the pathogenesis of LHON. The m.12338T>C mutation affected the first methionine (Met1) with a threonine and shortened two amino acids of ND5. We therefore hypothesized that the mutated ND5 perturbed the structure and function of complex I. Using the cybrid cell models, generated by fusing mtDNA-less (ρ°) cells with enucleated cells from LHON patients carrying the m.12338T>C mutation and a control subject belonging to the same mtDNA haplogroup, we demonstrated that the m.12338T>C mutation caused the reduction of ND5 polypeptide, perturbed assemble and activity of complex I. Furthermore, the m.12338T>C mutation caused respiratory deficiency, diminished mitochondrial adenosine triphosphate levels and membrane potential and increased the production of reactive oxygen species. The m.12338T>C mutation promoted apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 9, 3, 7 and Poly ADP ribose polymerase in the cybrids carrying the m.12338T>C mutation, as compared with control cybrids. Moreover, we also document the involvement of m.12338T>C mutation in decreased mitophagy, as showed by reduced levels of autophagy protein light chain 3 and accumulation of autophagic substrate p62 in the in mutant cybrids as compared with control cybrids. These data demonstrated the direct link between mitochondrial dysfunction caused by complex I mutation and apoptosis or mitophagy. Our findings may provide new insights into the pathophysiology of LHON.

Coronary heart disease (CHD) is the leading cause of death worldwide. Mitochondrial genetic determinant for the development of CHD remains poorly explored. We report there the clinical, genetic, molecular and biochemical characterization of a four-generation Chinese family with maternally inherited CHD. Thirteen of 32 adult members in this family exhibited variable severity and age-at-onset of CHD. Mutational analysis of their mitochondrial genomes identified the tRNA(Thr) 15927G>A mutation belonging to the Eastern Asian haplogroup B5. The anticipated destabilization of a highly conserved base-pairing (28C-42G) by the 15927G>A mutation affects structure and function of tRNA(Thr). Northern analysis revealed ≈80% decrease in the steady-state level of tRNA(Thr) in the mutant cell lines carrying the 15927G>A mutation. The 15927G>A mutation changed the conformation of tRNA(Thr), as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. In addition, ∼39% reduction in aminoacylated efficiency of tRNA(Thr) was observed in mutant cells derived from this Chinese family. An in vivo mitochondrial protein labeling analysis showed ∼53% reduction in the rate of mitochondrial translation in mutant cells. The impaired mitochondrial protein synthesis leads to defects in overall respiratory capacity or malate/glutamate-promoted respiration or succinate/glycerol-3-phosphate-promoted respiration, or N,N,N',N'-tetramethyl-pphenylenediamine/ascorbate-promoted respiration in mutant cells. An increasing production of reactive oxygen species was observed in the mutant cells carrying the 15927G>A mutation. These results provide the direct evidence that the tRNA(Thr) 15927G>A mutation is associated with CHD. Our findings may provide new insights into pathophysiology and intervention targets of this disorder.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2.

Iron–sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe–S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans.

The pathogenetic mechanism of the human mitochondrial 12S rRNA gene mutation at position 1555, associated with non-syndromic deafness and aminoglycoside-induced deafness, has been investigated in 33 transformants obtained by transferring mitochondria from lymphoblastoid cell lines into human mitochondrial DNA (mtDNA)-less (rho *206) cells. In this nearly constant nuclear background, 15 transformants derived from five symptomatic individuals from a large Arab-Israeli family, carrying this mutation in homoplasmic form, exhibited significant decreases compared with nine control transformants in the rate of growth in a medium containing galactose instead of glucose, as well as in the rates of mitochondrial protein synthesis and of substrate-dependent respiration. Most significantly, these decreases were very similar to those observed in nine transformants derived from three asymptomatic members of the family. This result in transmitochondrial cybrids is in contrast to the differences in the same parameters previously demonstrated between the original lymphoblastoid cell lines derived from the symptomatic and asymptomatic members of the Arab-Israeli family. In addition, the intragroup variability in biochemical dysfunction among the lymphoblastoid cell lines from different symptomatic or asymptomatic or control individuals was significantly reduced in the derived mitochondrial transformants carrying the same nuclear background. These observations provide strong genetic and biochemical evidence in support of the idea that the nuclear background plays a determinant role in the phenotypic manifestation of the non-syndromic deafness associated with the A1555G mutation.

The phenotypic effects of the human mitochondrial 12S rRNA gene mutation at position 1555 associated with maternally inherited non-syndromic deafness and sensitivity to aminoglycoside-induced deafness have been analyzed in 25 lymphoblastoid cell lines derived from members of a large family carrying this mutation in homoplasmic form and from control individuals. A clear decrease in the rates of growth in galactose medium, mitochondrial protein synthesis, total oxygen consumption, and complex I-, complex III- and complex IV-dependent respiration was observed in two groups of nine and 10 mutant cell lines derived, respectively, from symptomatic and asymptomatic members of the family, as compared with six control cell lines. The severity of mitochondrial dysfunction in the mutant cell lines was correlated with the presence or absence of hearing loss in the donor individuals. These observations strongly suggest a role of a nuclear factor(s) in the phenotypic manifestation of the mutation. The approach used here provides a paradigm for the analysis of the nuclear background involvement in other mtDNA-linked disorders, including the putative ones associated with neurodegenerative diseases. Exposure of the cell lines derived from several symptomatic or asymptomatic individuals from the same family to high concentrations of neomycin or paromomycin decreased to a significant, nearly identical extent their rate of growth in glucose-containing medium, as contrasted with the unchanged growth rate of control cell lines or of mtDNA-less cells. These results support the hypothesis that the main target of the antibiotics is the mitochondrial 12S rRNA carrying the 1555 mutation, without any apparent role of the nuclear background.