Deciphering OPA1 mutations pathogenicity by combined analysis of human, mouse and yeast cell models

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA “plus”. Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two “ad hoc” generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1−/− MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA “plus” phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions

Deciphering OPA1 mutations pathogenicity by combined analysis of human, mouse and yeast cell models

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA “plus”. Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two “ad hoc” generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1−/− MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA “plus” phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions

DNA polymerase [gamma] and disease: what we have learned from yeast

Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (PEO), Alpers' syndrome, and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissues with variable degrees of severity. In this review, we summarize the genetic and biochemical knowledge published on yeast mitochondrial DNA polymerase from 1989, when the MIP1 gene was first cloned, up until now. The role of yeast is particularly emphasized in (i) validating the pathological mutations found in human POLG and modeled in MIP1, (ii) determining the molecular defects caused by these mutations and (iii) finding the correlation between mutations/polymorphisms in POLGA and mtDNA toxicity induced by specific drugs. We also describe recent findings regarding the discovery of molecules able to rescue the phenotypic defects caused by pathological mutations in Mip1, and the construction of a model system in which the human Pol γ holoenzyme is expressed in yeast and complements the loss of Mip1

Validation of a MGM1/OPA1 chimeric gene for functional analysis in yeast of mutations associated with dominant optic atrophy

Mutations in OPA1 are associated with DOA or DOA plus. Novel mutations in OPA1 are periodically identified, but often the causative effect of the mutation is not demonstrated. A chimeric protein containing the N-terminal region of Mgm1, the yeast orthologue of OPA1, and the C-terminal region of OPA1 was constructed. This chimeric construct can be exploited to evaluate the pathogenicity of most of the missense mutations in OPA1 as well as to determine whether the dominance of the mutation is due to haploinsufficiency or to gain of function

Screening of native plants from central Argentina against the leaf-cutting ant Acromyrmex lundi (Guérin) and its symbiotic fungus

Leaf-cutting ants are major agricultural and forestry pests in the New World. Attempts to control themhave most frequently involved the use of chemical insecticides, with mixed results. Among alternativemethods, botanical pesticides may provide a sustainable and efficient control of leaf-cutting ants. In thepresent study, we screened the activity of plant extracts derived from 89 species native to Argentina,against the leaf-cutting ant Acromyrmex lundi (Guérin) and its mutualistic fungus, Leucoagaricus gongy-lophorus, through a pick-up assay and bioautography, respectively. The pick-up assay revealed moderateto strong anti-foraging activity for just over 13.5% of the assayed species, including complete ant foraginginhibition for Aristolochia argentina, Flourensia oolepis, Gaillardia megapotamica, Lantana grisebachii andLithrea molleoides. Most plant extracts were well tolerated by fungi, with only 12.3% of the species testedshowing some degree of fungus growth inhibition. Among these, A. argentina, F. oolepis and Pterocaulonalopecuroides were the strongest inhibitors, whereas Baccharis flabellata, Dalea elegans and Zanthoxylumcoco revealed a more moderate activity. Only A. argentina and F. oolepis extracts showed strong antifor-aging effects and affected fungus growth at the same time. Values of IC50and MIC indicated that extractsinhibiting ant foraging at lower concentrations did not necessarily also inhibit fungus growth at lowerdoses. The active principle of A. argentina, on both ant foraging and fungal growth, was identified asargentilactone.Fil: Diaz Napal, Georgina Nata. Area de Cs. Agrarias, Ingeniería, Cs. Biológicas y de la Salud de la Universidad Catolica de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Buffa, Liliana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Nolli, Laura C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Defagó, María Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Valladares, Graciela Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Carpinella, Maria Cecilia. Area de Cs. Agrarias, Ingeniería, Cs. Biológicas y de la Salud de la Universidad Catollica de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ruiz, Gustavo Miguel. Area de Cs. Agrarias, Ingeniería, Cs. Biológicas y de la Salud de la Universidad Catolica de Córdoba; ArgentinaFil: Palacios, Sara Maria. Area de Cs. Agrarias, Ingeniería, Cs. Biológicas y de la Salud de la Universidad Catolica de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Deciphering OPA1 mutations pathogenicity by combined analysis of human, mouse and yeast cell models

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA “plus”. Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two “ad hoc” generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1−/− MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA “plus” phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions