Abstract
Leber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of 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. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (ρ°) 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 the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations.
Highlights
Leber's hereditary optic neuropathy (LHON) is the most common maternally transmitted eye disorder, characterized by the degeneration of retinal ganglion cells (RGC) and loss of central vision [1,2,3,4,5,6]
The higher penetrance of LHON in the families carrying both m.11778G.A and m.3866T.C mutations implied that biochemical consequences caused by the m.3866T.C mutation deteriorate the mitochondrial dysfunction associated with m.11778G.A mutation, thereby increasing the penetrance and risk of LHON
Functional consequences of m.3866T.C mutation was further investigated through the cybrid cell lines constructed by transferring mitochondria from lymphoblastoid cell lines derived from an affected matrilineal relative carrying the m.11778G.A and m.3866T.C mutation, individuals bearing only m.3866T.C or m.11778G.A mutation, and a control subject belonging to the same mitochondrial DNA (mtDNA) haplogroup, all into human mtDNA-less cells [19, 34]
Summary
Clinical and genetic evaluation of 10 Chinese families carrying the m.3866T>C mutation. The replacement of hydrophobic isoleucine at position 187 with the hydrophilic threonine by m.3866T.C mutation in ND1 or arginine at position 340 with histidine by m.11778G.A mutation in ND4 may destabilize these interactions inside ND1 or ND4, thereby perturbing the structure and stability of ND1 and ND4 proteins, respectively To experimentally test this hypothesis, we examined the levels of ND1 and ND4 proteins by Western blotting in these mutant cell lines carrying only m.3866T.C mutation, only m.11778G.A mutation, or both m.3866T.C and m.11778G.A mutations, as well as control cell lines lacking these mutations. To examine whether the m.3866T.C or m.11778G.A mutation affected the expression of other subunits of complex I, we measured the levels of NDUFA3, NDUFA8, NDUFA13, ND5, NDUFS2, NDUFB4, NDUFB5, NDUFB8, and NDUFB11 by Western blot analysis among mutant and control cell lines, respectively.
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