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Telethon Associazione Serena Talarico per i giovani nel mondo and Fondazione Giuseppe Tomasello O.N.L.U.S. Mitocon Onlus Research to Prevent Blindness International Foundation for Optic Nerve Diseases (IFOND) Struggling Within Leber's Poincenot Family Eierman Foundation National Eye Institute Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber's hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber's hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies. Univ Rome, Dept Radiol Oncol & Pathol, Rome, Italy Univ Bologna, Dept Biomed & NeuroMotor Sci DIBINEM, Bologna, Italy Univ Bari, Dept Biosci Biotechnol & Biopharmaceut, Bari, Italy Bellaria Hosp, IRCCS Ist Sci Neurol Bologna, I-40139 Bologna, Italy USC, Keck Sch Med, Dept Ophthalmol, Los Angeles, CA USA USC, Keck Sch Med, Dept Neurosurg, Los Angeles, CA USA Univ Trieste, Dept Reprod Sci Dev & Publ Hlth, Trieste, Italy Univ Trieste, IRCCS Burlo Garofolo Children Hosp, Trieste, Italy Newcastle Univ, Inst Med Genet, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, England Fdn Ist Neurol Carlo Besta IRCCS, Unit Mol Neurogenet, Milan, Italy MRC Mitochondrial Biol Unit, Cambridge, England Fed Univ São Paulo UNIFESP, Dept Ophthalmol, São Paulo, Brazil Univ São Paulo, Inst Psychol, Dept Expt Psychol, São Paulo, Brazil Studio Oculist dAzeglio, Bologna, Italy Osped San Giovanni Evangelista, Tivoli, Italy Azienda Osped San Camillo Forlanini, Rome, Italy Univ Rome, Dipartimento Metodi & Modelli Econ Finanza & Terr, Rome, Italy Univ Rome, Dept Mol Med, Rome, Italy Fed Univ São Paulo UNIFESP, Dept Ophthalmol, São Paulo, Brazil Telethon: GGP06233 Telethon: GGP11182 Telethon: GPP10005 National Eye Institute: EY03040 Web of Science

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.