Rotenone

To model a loss of Complex I activity, as in LHON, we used intravitreal injection of rotenone. This model has previously been described [10 (link), 19 , 20 (link)]. Animals were anesthetized using an intraperitoneal injection of Ketamine (37.5 mg/kg) and Midazolam (Dormitol, 1.25 mg/kg). Bilateral intravitreal injections were performed using a NanoFil 10 μl glass syringe with a 33G needle (WPI). Mice were injected with either 1.5 μL of 10 mM rotenone (MP Biochemicals) in DMSO (Sigma Life science) or 1.5 μL of DMSO only (control). The NAM treated groups underwent one-week of pre-treatment with NAM prior to injection and continuing until mice were euthanized. Mice were euthanized 24 h after intravitreal injection by cervical dislocation and tissues were collected for analysis.

OCR measurements were performed using a Seahorse Biosciences XF96 Extracellular Flux Analyzer. Cells were seeded at 6,000/well in XF96 microplates (Seahorse Biosciences, Santa Clara, CA). After a 24-h incubation, the growth medium was replaced with XF Assay Medium (Seahorse Biosciences) supplemented with 25 mM glucose (Sigma-Aldrich, Darmstadt, Germany). OCR measurements were made over 5-min periods following a 3-min mix period. The following components were sequentially added to the cells: 1 µg/mL oligomycin (Sigma-Aldrich), 300 nM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; Sigma-Aldrich), and 2 µM rotenone (MP Biomedicals, Tokyo, Japan). The spare respiratory capacity and coupling efficiency were calculated according to the Seahorse Bioscience instructions and the basal OCR was normalized to the cell number.

N-acetylcysteine (NAC–A5270, Sigma) and Rotenone (MP Biomedicals - 150154) have been administered to the mice in a concentration of 25 g/L and 10 mg/L respectively, dissolved in mice drinking water starting from the age of 4 weeks. The solutions were freshly prepared and replenished every 3 days. The range of NAC doses was selected on the basis of previous literature^{78 (link)–80 (link)}. As far as Rotenone is concerned, we took as a starting point the work of Pan-Montojo and colleagues^{81 (link)}; here, a dose of 5 mg/kg principally affected the gastrointestinal tract, therefore minimizing systemic toxicity. In total 5 mg/kg correspond to 0.125 mg for an average mouse with 25 grams body weight. Here, we decided to use a slightly lower dosage than what reported by Pan-Montojo and co-workers in light of the fragile phenotype of Ercc1 mutants and of their general sensitivity. We therefore administered 10 mg/mL Rotenone in drinking water, which approximately corresponds to 0.04 mg per mouse per day given that mice (including Ercc1 mutants) drink ~4 mL of water daily (please see http://web.jhu.edu/animalcare/procedures/mouse.html).