Autosomal dominant and recessive forms of progressive external ophthalmoplegia (adPEO and arPEO) are mitochondrial disorders characterized by the presence of multiple deletions of mitochondrial DNA in affected tissues. Four adPEO-associated missense mutations have been identified in the ANT1 gene. In order to investigate their functional consequences on cellular physiology, we introduced three of them at equivalent positions in AAC2, the yeast orthologue of human ANT1. We demonstrate here that expression of the equivalent mutations in aac2-defective haploid strains of Saccharomyces cerevisiae results in (a) a marked growth defect on non-fermentable carbon sources, and (b) a concurrent reduction of the amount of mitochondrial cytochromes, cytochrome c oxidase activity and cellular respiration. The efficiency of ATP and ADP transport was variably affected by the different AAC2 mutations. However, irrespective of the absolute level of activity, the AAC2 pathogenic mutants showed a significant defect in ADP versus ATP transport compared with wild-type AAC2. In order to study whether a dominant phenotype, as in humans, could be observed, the aac2 mutant alleles were also inserted in combination with the endogenous wild-type AAC2 gene. The heteroallelic strains behaved as recessive for oxidative growth and petite-negative phenotype. In contrast, reduction in cytochrome content and increased mtDNA instability appeared to behave as dominant traits in heteroallelic strains. Our results indicate that S. cerevisiae is a suitable in vivo model to study the pathogenicity of the human ANT1 mutations and the pathophysiology leading to impairment of oxidative phosphorylation and damage of mtDNA integrity, as found in adPEO.

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Introduction: We present a family comprising a clinically normal mother and two daughters, each with severe encephalopathy with onset in late childhood. A third daughter had died previously of an earlier onset but neuropathologically similar disease. Methods: Sequence analysis of the entire mtDNA was carried out in muscle, fibroblasts, and lymphocytes of the affected daughters and unaffected mother. Biochemical analysis of individual respiratory chain enzymes was performed on the same tissues, and on several transmitochondrial cybrid clones containing the nucleus of a 143B.206 osteosarcoma cell line and the mutant mtDNA. Results: Genetic analyses revealed in both daughters and mother the presence of a novel mutation in the tRNA Ile gene of mtDNA, which was homoplasmic in fibroblasts, lymphocytes, and skeletal muscle of the two patients. It was also homoplasmic in fibroblast and skeletal muscle samples of the mother, and approximately 97% heteroplasmic in her lymphocytes. Combined defects of complexes I and IV of the mitochondrial respiratory chain were found not only in fibroblasts of the two probands, but surprisingly also in those of their clinically unaffected mother. The respiratory chain defect segregated in transmitochondrial cybrids containing the nucleus of a 143B.206 osteosarcoma cell line and the mutant mtDNA, indicating that the latter was responsible for the biochemical phenotype. Discussion: Our results support the concept that homoplasmic mutations in tRNA genes can be responsible for mitochondrial disorders characterised by extremely variable penetrance. Albeit still unexplained, this phenomenon has important consequences in the nosological characterisation, clinical management, and genetic counselling of mitochondrial disorders.

Background/AimWe compared the quality of human donor corneas stored in a cold storage medium containing 2.5 μg/ml of amphotericin B (Kerasave, AL.CHI.MI.A. S.R.L., Ponte San Nicolò, Italy) and Optisol-GS (Bausch & Lomb Inc., Bridgewater, NJ, USA) for 14 days.MethodsSixteen pairs of human donor corneas were collected in Eusol-C (AL.CHI.MI.A. S.R.L., Ponte San Nicolò, Italy). Next, all tissues underwent the first evaluation that included the assessments of central corneal thickness (CCT), endothelial cell density (ECD) measured using both trypan blue staining and specular microscopy, endothelial cell (EC) mortality and morphology, and corneal transparency within 24 hours from recovery (Day 1). Afterwards, one cornea of each pair was transferred into Kerasave or Optisol-GS. ECD and CCT were also assessed at Day 7, and all the metrics were evaluated again at the end of the storage period (Day 14).ResultsAt all tested time points, no differences were found in the qualitative (corneal transparency, EC morphology) and quantitative metrics (ECD, CCT, EC mortality) between the Kerasave and the Optisol-GS storage groups. At Day 14, the corneas stored in Kerasave and Optisol-GS showed ECD of 2312±98 and 2335±128 cells/mm2 (p=0.886), CCT of 717±17 and 697±19 μm (p=0.454) and central EC mortality of 0.54%±0.40% and 0.14%±0.14% (p=0.719), respectively.ConclusionsThe new amphotericin B−containing medium Kerasave was comparable to Optisol-GS in terms of preservation of corneal characteristics at 2–8°C for 14 days.

Corneal endothelial (CE) dysfunction is the main indication for corneal transplantation, an invasive procedure with several limitations. Developing novel strategies to reactivate CE regenerative capacity is, therefore, of fundamental importance. This goal has proved to be challenging as corneal endothelial cells (CEnC) are blocked in the G0/G1 phase of the cell cycle in vivo and, albeit retaining proliferative capacity in vitro, this is further hindered by endothelial-to-mesenchymal transition. Herein we investigated the mechanisms regulating CEnC proliferation in vitro. Comparing the proteome of non-proliferating (in vivo-G0/G1) and proliferating (in vitro-G2/M) rabbit CEnC (rCEnC), 77 proteins, out of 3,328 identified, were differentially expressed in the two groups (p < 0.005). Literature and Gene Ontology analysis revealed β-catenin and transforming growth factor (TGF-β) pathways to be correlated with the identified proteins. Treatment of rCEnC with a β-catenin activator and inhibitor showed that β-catenin activation was necessary during rCEnC proliferation, but not sufficient for its induction. Furthermore, both pro-proliferative activity of basic fibroblast growth factor and anti-proliferative effects of TGF-β were regulated through β-catenin. Overall, these results provide novel insights into the molecular basis underlying the proliferation process that CEnC reactivate in vitro, consolidating the role of β-catenin and TGF-β. The corneal endothelium (CE) is a monolayer of cells localised in the innermost segment of the cornea, regulating solutes transport from and to the aqueous humor (pump-leak hypothesis) 1. Corneal endothelial cells (CEnC) are generally considered non-dividing in vivo 2 and arrested in the G0/G1 phase of the cell cycle 3,4. The presence of anti-proliferative factors in the aqueous humor, the stress-induced premature senescence, and the contact inhibition between cells are the main events triggering the expression of cell cycle regulators that induce CEnC mitotic block 2. For this reason, as CEnC density decreases by 0.6% each year 5,6 , cell loss is compensated through migration and cell enlargement 2,7. When, as a consequence of pathologies or surgical treatments, endothelial cell density falls below 500 cells/mm 2 , cell enlargement is no longer able to compensate for the passive leaking 7. In this case, the impaired CE results in corneal swelling and, if untreated, in the subsequent severe loss of corneal transparency, ultimately leading to blindness. CEnC dysfunction is the underlying cause of about 40% of all corneal transplants performed worldwide 7. Corneal grafts, despite decisive advancements in the last decades, are still complex and invasive procedures, with some severe limitations, including immune graft rejection, graft failure, and a general scarcity of donor corneas 7. Alternative clinical procedures have been recently proposed in order to increase the number of patients that can be treated and to improve their quality of life. Currently, descemetorhexis is the only therap...