In Vitro Genetic Transfer of Protein Synthesis and Respiration Defects to Mitochondrial DNA-Less Cells with Myopathy-Patient Mitochondria

Abstract

A severe mitochondrial protein synthesis defect in myoblasts from a patient with mitochondrial myopathy was transferred with myoblast mitochondria into two genetically unrelated mitochondrial DNA (mtDNA)-less human cell lines, pointing to an mtDNA alteration as being responsible and sufficient for causing the disease. The transfer of the defect correlated with marked deficiencies in respiration and cytochrome c oxidase activity of the transformants and the presence in their mitochondria of mtDNA carrying a tRNA(Lys) mutation. Furthermore, apparently complete segregation of the defective genotype and phenotype was observed in the transformants derived from the heterogeneous proband myoblast population, suggesting that the mtDNA heteroplasmy in this population was to a large extent intercellular. The present work thus establishes a direct link between mtDNA alteration and a biochemical defect.

Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys

“…A severe defect in mitochondrial protein synthesis occurs due to the reduced steady-state levels of tRNA Lys and its aminoacylation (Enriquez et al 1995 ), as well as lack of post-transcriptional modifications, as the 5-taurinomethyl-2-thiouridine at the wobble position (Yasukawa et al 2001 ) and the 1-methyladenosine at position m.8348 (Richter et al 2018 ). Ultimately, a defective incorporation of lysine within the mtDNA-encoded proteins results in premature termination of translation and specific truncated protein products (Chomyn et al 1991 ), but also instability of full-length proteins with increased rate of their degradation (Boulet et al 1992 ; Richter et al 2021 ). The final outcome is COX deficiency and reduced respiration rate, mitochondrial membrane potential and ATP synthesis (De la Mata et al 2012 ; Chomyn 1998 ; James et al 1996 ; Antonická et al 1999 ).…”

“…Furthermore, since its identification, the MERRF mutation has also been investigated by using the cybrid cell model, documenting a global impairment in translation of the 13 mtDNA-encoded proteins, characterized by the presence of some aberrant translation products (Chomyn et al 1991 , 1994 ). Further studies deepened the understanding of the pathogenic mechanism showing altered tRNA Lys amino-acylation and premature termination of translation (Enriquez et al 1995 ), as well as a wobble base modification defect (Yasukawa et al 2001 ).…”

Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology

“…42,43 Such cybrids have been powerful tools to dissect mitochondrial disorders, including those caused by the A3243G mutation 44,45 and by others. 46,47 A unique strength of cybrids is that they provide direct evidence for the association of a specific mt-DNA mutation with the disease pathology, 48 excluding the role of the nuclear genome in the biogenesis of mitochondria. We previously used the cybrid technique, starting with a patient cell line heteroplasmic of the A3243G mutation, and isolated a series of clones, ranging in the frequency of the mutation (0, 20, 30, 50, 60, 90, and 100%) (Figure 2B).…”

Clinical Approaches for Mitochondrial Diseases

“…Another prominent mutation in the same gene is m.G8363A, along with the m.A3243G, m.G3255A, and m.T3291C mutations, which have also been reported to cause MERRF with a defects in tRNA leucine [57][58][59]99,102,103]. Mutations in tRNA-coding genes induce the global impairment of mtDNA-encoded proteins rather than affecting certain complexes or pathways [101,104,105]. This can result in the overall dysfunction of mitochondria.…”