Complex I mutations synergize to worsen the phenotypic expression of Leber's hereditary optic neuropathy

Abstract

Leber’s hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease due to the mitochondrial DNA mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation, within the gene for the ND4 subunit of Complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I, respectively. Cellular data obtained using cybrids constructed by transferring mitochondria from the Chinese families into mtDNA-less (ro) cells demonstrated that the mutations perturbed the stability, assembly and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential, and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only m.3866T>C mutation displayed mild mitochondrial dysfunctions while those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted as the synergy with m.11778G>A mutation, aggravating mitochondrial dysfunctions contributing to higher penetrance of LHON in these families carrying both mtDNA mutations.

A deafness-associated tRNA mutation caused pleiotropic effects on the m1G37 modification, processing, stability and aminoacylation of tRNAIle and mitochondrial translation

“…To investigate if the m.4295A>G mutation affected the autophagy, we evaluated the mitophagic states of mutant and control cell lines by flow cytometry using Cyto-ID, a cationic amphilic tracer dye that labeled mitophagic compartments ( 69 , 70 ). Geometric mean intensity was measured for assessment of autophagy in each sample.…”

“…Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with LC3, p62 and with β -actin as a loading control. ( D ) Quantification of autophagy markers LC3 I/II and p62 in mutant and control cell lines were determined as described elsewhere ( 70 ). Three independent determinations were done in each cell line.…”

“…Hence, these alterations in mitochondrial protein synthesis perturbed the stabilities and activities of complex I, III, IV and V in the cells bearing the m.4295A>G mutation. In particular, the defects in the synthesis of ND1, ND2, ND4, ND5 and ND6 resulted in the pronounced effects on the assembly and activity of complex I, as in the cases of mutations in the genes encoding complex I subunits ( 39 , 70 , 83 ). Furthermore, the impairment of mitochondrial translation yielded the respiratory and oxidative phosphorylation deficiencies, including decreased rates in the basal OCR, ATP-linked OCR, reserve capacity and maximal OCR as well as significant decreases in the level of mitochondrial ATP in the cell lines carrying the m.4295A>G mutation, as revealed by those in cell lines carrying the tRNA Asp 7551A>G and tRNA Met 4435A>G mutations ( 31 , 32 ).…”

Leber's Hereditary Optic Neuropathy Arising From the Synergy Between ND1 3635G>A Mutation and Mitochondrial YARS2 Mutations

“…Blue native gel electrophoresis was performed by isolating mitochondrial proteins from various cell lines, as detailed elsewhere. 20,34 Samples containing 30 μg of mitochondrial proteins were separated on 3% to 11% Bis-Tris native polyacrylamide gel. The primary antibodies applied for this experiment were a totally human OXPHOS antibody cocktail with a voltage-dependent anion-selective channel (VDAC) as a loading control.…”

“…ited mild defects in complex I activity. [18][19][20] The incomplete penetrance and gender bias in patients presenting with optic neuropathy suggest a role for the nuclear modifier genes in the phenotypic expression of LHON-associated mtDNA mutations. 21 Our recent study demonstrated that several LHON families were manifested by synergic interaction between the m.11778G>A mutation and mutated modifiers YARS2 encoding mitochondrial tyrosyl-tRNA synthetase and X-linked modifier PRICKLE3 encoding a mitochondrial protein linked to biogenesis of ATPase.…”