The background of mitochondrial DNA haplogroup J increases the sensitivity of Leber's hereditary optic neuropathy cells to 2,5-hexanedione toxicity

Abstract

Leber's hereditary optic neuropathy (LHON) is a maternally inherited blinding disease due to mitochondrial DNA (mtDNA) point mutations in complex I subunit genes, whose incomplete penetrance has been attributed to both genetic and environmental factors. Indeed, the mtDNA background defined as haplogroup J is known to increase the penetrance of the 11778/ND4 and 14484/ND6 mutations. Recently it was also documented that the professional exposure to n-hexane might act as an exogenous trigger for LHON. Therefore, we here investigate the effect of the n-hexane neurotoxic metabolite 2,5-hexanedione (2,5-HD) on cell viability and mitochondrial function of different cell models (cybrids and fibroblasts) carrying the LHON mutations on different mtDNA haplogroups. The viability of control and LHON cybrids and fibroblasts, whose mtDNAs were completely sequenced, was assessed using the MTT assay. Mitochondrial ATP synthesis rate driven by complex I substrates was determined with the luciferine/luciferase method. Incubation with 2,5-HD caused the maximal loss of viability in control and LHON cells. The toxic effect of this compound was similar in control cells irrespective of the mtDNA background. On the contrary, sensitivity to 2,5-HD induced cell death was greatly increased in LHON cells carrying the 11778/ND4 or the 14484/ND6 mutation on haplogroup J, whereas the 11778/ND4 mutation in association with haplogroups U and H significantly improved cell survival. The 11778/ND4 mutation on haplogroup U was also more resistant to inhibition of complex I dependent ATP synthesis by 2,5-HD. In conclusion, this study shows that mtDNA haplogroups modulate the response of LHON cells to 2,5-HD. In particular, haplogroup J makes cells more sensitive to its toxic effect. This is the first evidence that an mtDNA background plays a role by interacting with an environmental factor and that 2,5-HD may be a risk element for visual loss in LHON. This proof of principle has broad implications for other neurodegenerative disorders such as Parkinson's disease.

Figure 1. Effect of 2,5-HD on viability of control and LHON cybrids with mtDNAs belonging to different haplogroups.

Dose-responses of viability of control (A) and LHON (B) cybrids, with the respective mtDNA haplogroups, incubated for 24 hours in DMEM containing the indicated amounts of 2,5-HD. Time-courses of viability of control (C) and LHON (D) cybrids incubated with 12 mg/ml 2,5-HD. (E) Statistical analysis of the same data obtained in control and LHON cybrids, incubated for 24 hours with 12 mg/ml 2,5-HD. Cell viability was determined and statistically analyzed as described in the Methods section. Data are means±SD of at least 3 determinations. *denotes significantly different values (p<0.05) determined by One Way ANOVA followed by the Holm-Sidak method.

Figure 2. Effect of 2,5-HD on ATP synthesis of control and LHON cybrids with mtDNAs belonging to different haplogroups.

Dose-responses of ATP synthesis rate driven by complex I substrates (A) and complex II substrate (B) in digitonin-permeabilized cybrids after addition of the indicated amounts of 2,5-HD. Data are expressed as percentage of the values obtained in untreated samples. Statistical analysis of the same data of ATP synthesis rate obtained in control and LHON cybrids incubated with 10 mg/ml 2,5-HD in the presence of complex I substrates (C) or complex II substrate (D). Data are means±SD of at least 3 determinations. *denotes significantly different values (p<0.05), determined by One Way ANOVA followed by the Holm-Sidak method.

Figure 3. Effect of 2,5-HD on viability of control and LHON fibroblasts with mtDNAs belonging to different haplogroups.

Dose-responses of viability of control (A) and LHON (B) fibroblasts, with the respective mtDNA haplogroups, incubated for 24 hours in DMEM containing the indicated amounts of 2,5-HD. Time-courses of viability of control (C) and LHON (D) fibroblasts incubated with 12 mg/ml 2,5-HD. (E) Statistical analysis of the same data obtained in control and LHON fibroblasts, incubated for 24 hours with 12 mg/ml 2,5-HD. Cell viability was determined and statistically analyzed as described in the Methods section. Data are means±SD of 3 determinations. *denotes significantly different values (p<0.05), determined by One Way ANOVA followed by the Holm-Sidak method.

Figure 4. Viability of control and LHON cells after incubation with 2,5-HD and toluene.

Cybrids (A) and fibroblasts (B) were incubated for 24 hours in DMEM containing 12 mg/ml 2,5-HD alone or in the presence of 5 mg/ml toluene. Cell viability was determined as described in figure 1. Data are means±SD of 5 determinations. *denotes significantly different values (p<0.05) between cells treated with 2,5-HD alone or in the presence of toluene, using the Student's t test.