OMA1 activity assay was performed by the technique developed recently [25 (link)]. The assay utilizes an OPA1 fluorogenic reporter substrate that contains a fluorogenic MCA moiety on the N-terminus and a DNP quencher moiety on the C-terminus (LifeTein LLC). Briefly, 5 μg of each mitochondrial sample was resolved in the OMA1 activity assay buffer, containing (in mM): 50 Tris-HCl and 40 KCl, pH 7.5. Reaction medium contained 200 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (the zinc chelator) and 5 μM OPA1 fluorogenic reporter substrate. The assay was run with a final volume of 100 μL using black opaque 96 well plates (Costar). Relative fluorescence was recorded at 37°C in 5-min intervals for 30-min, using a fluorescent plate reader (SpectraMax M2e, Molecular Devices equipped with SoftMax Pro v5 software) with excitation/emission of 320/405 nm. OMA1 activation was proportional to the fluorescence released as a result of cleavage of the OPA1 fluorogenic reporter substrate by OMA1. Results are presented in relative fluorescence units.