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Mutations in the X-linked methyl-CpG-binding 2 (MECP2) gene lead to Rett Syndrome (RTT; OMIM 312750), a devasting neurodevelopmental disorder. RTT clinical manifestations are complex and with different degrees of severity, going from autistic-like behavior to loss of acquired speech, motor skills and cardiac problems. Furthermore, the correlation between the type of MECP2 mutation and the clinical phenotype is still not fully understood. Contextually, different genotypes can differently affect the patient's phenotype and omics methodologies such as proteomics could be an important tool for a molecular characterization of genotype/phenotype correlation. The aim of our study was focused on evaluating RTT oxidative stress (OS) responses related to specific MECP2 gene mutations by using proteomics and bioinformatics approaches. Primary fibroblasts isolated from patients affected by R133C and R255× mutations were compared to healthy controls (HC). After clustering primary dermal fibroblasts based on their specific MECP2 mutations, fibroblast-derived protein samples were qualitative and quantitative analyzed, using a label free quantification (LFQ) analysis by mass spectrometry (MS), achieving a preliminary correlation for RTT genotype/phenotype. Among the identified proteins involved in redox regulation pathways, NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) was found to be absent in R255× cells, while it was present in R133C and in HC fibroblasts. Moreover, NQO1 aberrant gene regulation was also confirmed when cells were challenged with 100 μM hydrogen peroxide (H 2 O 2). In conclusion, by employing a multidisciplinary approach encompassing proteomics and bioinformatics analyses, as well as molecular biology assays, the study uncovered phenotypic responses linked to specific MECP2 gene mutations. These findings contribute to a better understanding of the complexity of RTT molecular pathways, confirming the high heterogeneity among the patients. [Display omitted] • Rett syndrome is a neurodevelopmental disorder mainly caused by MECP2 gene mutations. • Correlations among MECP2 mutations and severity's degrees consequences are unclear. • Proteomics paves the way for biomarkers discoveries and for personalizing therapies. • Molecular mechanisms underlying oxidative stress are still unclear in Rett syndrome. [ABSTRACT FROM AUTHOR]

Exposure of human lung epithelial cells (A549 cell line) to the oxidant pollutant ozone (O 3) alters cell membrane currents inducing its decrease, when the cell undergoes to a voltage-clamp protocol ranging from −90 to +70mV. The membrane potential of these cells is mainly maintained by the interplay of potassium and chloride currents. Our previous studies indicated the ability of O 3 to activate ORCC (Outward Rectifier Chloride Channel) and consequently increases the chloride current. In this paper our aim was to understand the response of potassium current to oxidative stress challenge and to identify the kind potassium channel involved in O 3 induced current changes. After measuring the total membrane current using an intracellular solution with or without potassium ions, we obtained the contribution of potassium to the overall membrane current in control condition by a mathematical approach. Repeating these experiments after O 3 treatment we observed a significant decrease of I potassium. Treatment of the cells with Iberiotoxin (IbTx), a specific inhibitor of BK channel, we were able to verify the presence and the functionality of BK channels. In addition, the administration of 4-Aminopyridine (an inhibitor of voltage dependent K channels but not BK channels) and Tetraethylammonium (TEA) before and after O 3 treatment we observed the formation of BK oxidative post-translation modifications. Our data suggest that O 3 is able to inhibit potassium current by targeting BK channel. Further studies are needed to better clarify the role of this BK channel and its interplay with the other membrane channels under oxidative stress conditions. These findings can contribute to identify the biomolecular pathway induced by O 3 allowing a possible pharmacological intervention against oxidative stress damage in lung tissue. [Display omitted] • Subjecting A549 cells to ozone at 0.1 ppm decreased the total membrane current with respect to control. • AS ozone increases Chloride current, Potassium channels were analyzed. • Immunocytochemical test and patch clamp experiments with Iberiotoxin, indicated the presence and the activity of BK channels. • Western blot analyses and patch clamp experiments with 4-Aminopyridine and ozone, showed that BK is a target of ozone. [ABSTRACT FROM AUTHOR]