Biochemical characterization and validation of a novel cell model for dominant optic atrophy.

Mutations in the OPA1 gene, encoding the mitochondrial dynamin-like GTPase OPA1, are well known to cause Dominant Optic Atrophy (DOA), the most common inherited optic neuropathy. The missense variants, envisaged to exert a dominant-negative effect, are associated with high risk to develop the severe multisystem disorder (DOA “plus”), characterized by extra-ocular features, including sensorineural deafness, ataxia, myopathy, chronic progressive external ophthalmoplegia, and peripheral neuropathy. Primary skin fibroblasts derived from patients bearing OPA1 mutations represent the cell model for studying DOA pathophysiology, although they often reveal a mild phenotype, as a consequence of the autosomal genetic transmission of DOA. Other genetically modified cellular models characterized by a phenotype strikingly different from wild-type, are therefore desirable. In this study we describe a novel cell model obtained from Opa1-/- MEFs, where human OPA1 isoform 1 bearing OPA1 mutations was expressed. Under this setting, all OPA1 protein is mutated, ruling out the effect of the wild-type allele. We present here a detailed molecular and biochemical analysis in parallel of fibroblasts and MEFs bearing three known OPA1 pathogenic mutations (I382M, G439V, R445H) and a novel one (D603H), selected on the basis of their clinical phenotypes, ranging from very mild to more detrimental causing severe syndromic forms. The results indicate that MEFs bearing OPA1 mutations are a model useful to predict the pathogenicity of new mutations. In fact, according with the severity of the clinical phenotype of patients, the MEFs exhibit an increased number of mitochondrial dysfunctions. We propose this cell model as a suitable tool to test drugs with potential therapeutic effect on mitochondrial diseases associated with OPA1 mutations. In a preliminary study we were able to to confirm the efficacy of few molecules previously identified in a yeast hight throuput screening as able to revert the pathological phenotype of a mutant Mgm1-OPA1 yeast chimera.