Searching for Frataxin Function: Exploring the Analogy with Nqo15, the Frataxin-like Protein of Respiratory Complex I from Thermus thermophilus.

Nqo15 is a subunit of respiratory complex I of the bacterium Thermus thermophilus, with strong structural similarity to human frataxin (FXN), a protein involved in the mitochondrial disease Friedreich's ataxia (FRDA). Recently, we showed that the expression of recombinant Nqo15 can ameliorate the respiratory phenotype of FRDA patients' cells, and this prompted us to further characterize both the Nqo15 solution's behavior and its potential functional overlap with FXN, using a combination of in silico and in vitro techniques. We studied the analogy of Nqo15 and FXN by performing extensive database searches based on sequence and structure. Nqo15's folding and flexibility were investigated by combining nuclear magnetic resonance (NMR), circular dichroism, and coarse-grained molecular dynamics simulations. Nqo15's iron-binding properties were studied using NMR, fluorescence, and specific assays and its desulfurase activation by biochemical assays. We found that the recombinant Nqo15 isolated from complex I is monomeric, stable, folded in solution, and highly dynamic. Nqo15 does not share the iron-binding properties of FXN or its desulfurase activation function.

Human frataxin, the Friedreich ataxia deficient protein, interacts with mitochondrial respiratory chain.

Friedreich ataxia (FRDA) is a rare, inherited neurodegenerative disease caused by an expanded GAA repeat in the first intron of the FXN gene, leading to transcriptional silencing and reduced expression of frataxin. Frataxin participates in the mitochondrial assembly of FeS clusters, redox cofactors of the respiratory complexes I, II and III. To date it is still unclear how frataxin deficiency culminates in the decrease of bioenergetics efficiency in FRDA patients' cells. We previously demonstrated that in healthy cells frataxin is closely attached to the mitochondrial cristae, which contain both the FeS cluster assembly machinery and the respiratory chain complexes, whereas in FRDA patients' cells with impaired respiration the residual frataxin is largely displaced in the matrix. To gain novel insights into the function of frataxin in the mitochondrial pathophysiology, and in the upstream metabolic defects leading to FRDA disease onset and progression, here we explored the potential interaction of frataxin with the FeS cluster-containing respiratory complexes I, II and III. Using healthy cells and different FRDA cellular models we found that frataxin interacts with these three respiratory complexes. Furthermore, by EPR spectroscopy, we observed that in mitochondria from FRDA patients' cells the decreased level of frataxin specifically affects the FeS cluster content of complex I. Remarkably, we also found that the frataxin-like protein Nqo15 from T. thermophilus complex I ameliorates the mitochondrial respiratory phenotype when expressed in FRDA patient's cells. Our data point to a structural and functional interaction of frataxin with complex I and open a perspective to explore therapeutic rationales for FRDA targeted to this respiratory complex.

Ecotoxicological Impact of the Marine Toxin Palytoxin on the Micro-Crustacean Artemia franciscana.

Palytoxin (PLTX) is a highly toxic polyether identified in various marine organisms, such as Palythoa soft corals, Ostreopsis dinoflagellates, and Trichodesmium cyanobacteria. In addition to adverse effects in humans, negative impacts on different marine organisms have been often described during Ostreopsis blooms and the concomitant presence of PLTX and its analogues. Considering the increasing frequency of Ostreopsis blooms due to global warming, PLTX was investigated for its effects on Artemia franciscana, a crustacean commonly used as a model organism for ecotoxicological studies. At concentrations comparable to those detected in culture media of O. cf. ovata (1.0-10.0 nM), PLTX significantly reduced cysts hatching and induced significant mortality of the organisms, both at larval and adult stages. Adults appeared to be the most sensitive developmental stage to PLTX: significant mortality was recorded after only 12 h of exposure to PLTX concentrations > 1.0 nM, with a 50% lethal concentration (LC50) of 2.3 nM (95% confidence interval = 1.2-4.7 nM). The toxic effects of PLTX toward A. franciscana adults seem to involve oxidative stress induction. Indeed, the toxin significantly increased ROS levels and altered the activity of the major antioxidant enzymes, in particular catalase and peroxidase, and marginally glutathione-S-transferase and superoxide dismutase. On the whole, these results indicate that environmentally relevant concentrations of PLTX could have a negative effect on Artemia franciscana population, suggesting its potential ecotoxicological impact at the marine level.

Biomarkers and Precision Therapy for Primary Immunodeficiencies: An In Vitro Study Based on Induced Pluripotent Stem Cells From Patients.

Ataxia telangiectasia (AT) and Aicardi-Goutières syndrome (AGS) are inherited disorders of immunity with prevalent neurological phenotype. Available treatments are only partially effective, and the prognosis is poor. Induced pluripotent stem cells (iPSCs) are obtained by reprogramming patient somatic cells, preserving the donor individual genetic heritage and creating patient-specific disease models, useful to investigate pathogenesis and drug effects and to develop precision therapies. The aim is to investigate the cytotoxicity of a panel of immunomodulators using iPSCs of patients with AT or different forms of AGS (AGS1, AGS2, and AGS7). iPSCs were obtained by reprogramming AT and AGS patients' cells and, as a control, the BJ normal human fibroblast line, using Sendai virus. Cytotoxic effects of two drugs proposed to treat respectively AT and AGS (dexamethasone and mepacrine) were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 72 hours' exposure. Data were obtained also for other immunomodulatory drugs (thioguanine, mercaptopurine, thalidomide, and lenalidomide). Relative expression of genes involved in the tested drug pathways was analyzed. AGS7-derived iPSCs displayed altered viability when treated with a low dose of mepacrine and higher expression of cyclic guanosine monophosphate-adenosine monophosphate synthase, which is the main target for mepacrine action. AGS7-derived iPSCs were also more sensitive to thioguanine, while AGS2 and AT iPSCs were less sensitive to this medication than the BJ-iPSC. All iPSCs were equally sensitive to mercaptopurine and resistant to dexamethasone, thalidomide, and lenalidomide. This work establishes an innovative in vitro model that is useful to investigate the mechanisms of drugs potentially effective in AT and AGS.

Acute Oral Toxicity of Pinnatoxin G in Mice.

Pinnatoxin G (PnTx-G) is a marine cyclic imine toxin produced by the dinoflagellate Vulcanodiniumrugosum, frequently detected in edible shellfish from Ingril Lagoon (France). As other pinnatoxins, to date, no human poisonings ascribed to consumption of PnTx-G contaminated seafood have been reported, despite its potent antagonism at nicotinic acetylcholine receptors and its high and fast-acting toxicity after intraperitoneal or oral administration in mice. The hazard characterization of PnTx-G by oral exposure is limited to a single acute toxicity study recording lethality and clinical signs in non-fasted mice treated by gavage or through voluntary food ingestion, which showed differences in PnTx-G toxic potency. Thus, an acute toxicity study was carried out using 3 h-fasted CD-1 female mice, administered by gavage with PnTx-G (8-450 µg kg-1). At the dose of 220 µg kg-1 and above, the toxin induced a rapid onset of clinical signs (piloerection, prostration, hypothermia, abdominal breathing, paralysis of the hind limbs, and cyanosis), leading to the death of mice within 30 min. Except for moderate mucosal degeneration in the small intestine recorded at doses of 300 µg kg-1, the toxin did not induce significant morphological changes in the other main organs and tissues, or alterations in blood chemistry parameters. This acute oral toxicity study allowed to calculate an oral LD50 for PnTx-G equal to 208 g kg-1 (95% confidence limits: 155-281 µg kg-1) and to estimate a provisional NOEL of 120 µg kg-1.

Induced pluripotent stem cells for therapy personalization in pediatric patients: Focus on drug-induced adverse events.

Adverse drug reactions (ADRs) are major clinical problems, particularly in special populations such as pediatric patients. Indeed, ADRs may be caused by a plethora of different drugs leading, in some cases, to hospitalization, disability or even death. In addition, pediatric patients may respond differently to drugs with respect to adults and may be prone to developing different kinds of ADRs, leading, in some cases, to more severe consequences. To improve the comprehension, and thus the prevention, of ADRs, the set-up of sensitive and personalized assays is urgently needed. Important progress is represented by the possibility of setting up groundbreaking patient-specific assays. This goal has been powerfully achieved using induced pluripotent stem cells (iPSCs). Due to their genetic and physiological species-specific differences and their ability to be differentiated ideally into all tissues of the human body, this model may be accurate in predicting drug toxicity, especially when this toxicity is related to individual genetic differences. This review is an up-to-date summary of the employment of iPSCs as a model to study ADRs, with particular attention to drugs used in the pediatric field. We especially focused on the intestinal, hepatic, pancreatic, renal, cardiac, and neuronal levels, also discussing progress in organoids creation. The latter are three-dimensional in vitro culture systems derived from pluripotent or adult stem cells simulating the architecture and functionality of native organs such as the intestine, liver, pancreas, kidney, heart, and brain. Based on the existing knowledge, these models are powerful and promising tools in multiple clinical applications including toxicity screening, disease modeling, personalized and regenerative medicine.