In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that in vitro potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.

In order to establish a regulatory role for phosphoproteins in receptor-stimulated enzyme secre tion, dispersed rabbit pancreatic acinar cells were stim ulated with the COOH-terminal octapeptide of cholecystokinin (CCK8) in the absence and presence of staurosporine and/or 12-O-tetradecanoylphorbol 13-acetate (TPA) or forskolin. The dose/response curve for the stimulatory effect of CCK8 on amylase secre tion was biphasic, with a mean half-maximal concen tration (EC50) of 21 pM. Staurosporine (1 |uM) did not affect secretion elicited by CCK8 concentrations below 0.1 nM, but reduced the response to CCKS concentra tions above 0.1 nM. As a result, the mean EC50 for CCK8 decreased to 8 pM and its efficacy to 70%. The phorbol ester TPA (0.1 jiM) attenuated secretion evoked by CCK8 concentrations below 0.1 nM and potentiated the response to CCKS concentrations above 0.1 nM. As a result, the mean EC50for CCK8 increased to 0.14 nM and its efficacy to 300%. Staurosporine abolished both the inhibitory and the potentiating effect of TPA, thereby turning the inhibitory effect into a strong potentiating effect. As a result, the mean EC50 for CCK8 decreased to 3 pM, whereas its efficacy increased to 190%. Forskolin (30 |iM) potentiated the response to both the lower and the higher CCK8 con centrations. As a result, the mean EC50 for CCK8 increased to 28 pM and its efficacy to 300%. Staurosporine enhanced the potentiating effect of forskolin at CCK8 concentrations below 0.1 nM, but abolished potentiation at CCKS concentrations above 0.1 nM. As a result, the mean EC50for CCK8 decreased to 1.4 pM, whereas its efficacy increased to 260%. The data presented demonstrate that the apparent sensitiv ity of dispersed pancreatic acinar cells to stimulation of the process of enzyme secretion by CCK8 decreases