Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number,oxidative phosphorylation and ROS detoxification pathways

Leber''s hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that modulate LHON penetrance must take into account also the exposure to environmental triggers such as tobacco smoke.Leber''s hereditary optic neuropathy (LHON) is among the most frequent mitochondrial diseases, affecting about 1 in 35 000–60 000 in Europe.^{1, 2} LHON is associated in over 90% of cases with one of three common mitochondrial DNA (mtDNA) point mutations affecting the Complex I subunit genes ND4 (m.11778G>A), ND1 (m.3460G>A) and ND6 (m.14484 T>C), usually occurring in homoplasmic fashion^{3, 4} (100% of mtDNA is mutant). This maternally inherited blinding disorder is caused by selective degeneration of retinal ganglion cells, particularly those originating the small axons composing the papillomacular bundle, which leads to optic atrophy.^{5, 6, 7} Clinically, a subacute loss of central vision develops in weeks/months, mostly affecting young adult men, with a peculiar pattern of fiber depletion⁸ and a relatively predictable natural history of visual function decline.⁹ Exceptions apply to age of onset, with childhood or late cases,^{10, 11} to propensity in recovering vision, more frequent with the m.14484 T>C mutation,¹² and to clinical expression that in a subset of patients may be more widespread.⁴The mtDNA mutations are necessary but not sufficient to cause LHON,¹³ with penetrance being on average about 50% for males and 10% for females. The association of specific mtDNA haplotypes of haplogroup J with the m.14484 T>C and m.11778G>A mutations has been consistently documented in patients of European descent, indicating that mtDNA background modulates to a certain extent disease penetrance.^{14, 15} However, in a prototypical LHON maternal lineage, despite all the individuals carry the homoplasmic mtDNA mutation regardless the haplogroup, only some develop the disease, pointing to further factors that may be genetic and environmental.¹⁶ Thus, male prevalence and incomplete penetrance remain the two investigated and problematic issues in LHON. Both issues have been recently mechanistically related to the efficiency of compensatory mitochondrial biogenesis.^{17, 18} Estrogens ameliorate mitochondrial dysfunction by activating mitochondrial biogenesis, suggesting that females are naturally protected during their fertile period.^{17, 19} Furthermore, by studying different experimental systems (blood cells, skeletal muscle, skin-derived fibroblasts and ocular tissue) we found that the unaffected mutation carriers had a significantly higher mtDNA copy number and mitochondrial mass compared with their affected relatives,¹⁸ indicating that efficiently increasing mitochondrial biogenesis may overcome the pathogenic effect of the mtDNA mutation. Recently, others obtained similar results in different LHON cohorts.²⁰ Notwithstanding, nuclear modifiers remain elusive. In particular, association of LHON with genetic variants was not consistent across different studies.^{18, 21} Similarly inconsistent was the association with chromosome X-linked loci, hypothesized to explain male prevalence.^{22, 23, 24}Several other factors have been implicated in LHON, including exposure to cigarette smoke, alcohol and chemical toxins, head trauma, acute physical illness, psychological stress, antiretroviral and antituberculosis drugs.^{4, 25} These and other environmental factors can have a triggering role in LHON pathogenesis. For example, in vitro exposure to 2,5 exanedione had a toxic effect on LHON cybrid cells, with an increased sensitivity if they harbored a haplogroup J background.²⁶ A major environmental trigger of LHON is cigarette smoke; Sadun et al.²⁷ and Kirkman et al.²⁵ showed that LHON penetrance is significantly increased in smokers, independently of gender and alcohol intake.In the current study, we explored further the effect of cigarette smoking in LHON, showing in white blood cells from patients of large LHON cohorts, and in skin-derived fibroblasts, that cigarette derivatives exert their toxicity by depressing mtDNA copy number and oxidative phosphorylation (OXPHOS). However, unaffected mutation carriers displayed the most efficient capacity for reactive oxygen species (ROS) detoxification, which was not hampered by exposure to cigarette derivatives.