Redox imbalance and morphological changes in skin fibroblasts in typical Rett syndrome - PubMed (original) (raw)

doi: 10.1155/2014/195935. Epub 2014 May 29.

Silvia Leoncini 2, Claudio De Felice [ 3](#full-view-affiliation-3 "Neonatal Intensive Care Unit, University Hospital AOUS, Policlinico "S. M. alle Scotte," 53100 Siena, Italy."), Alessandra Pecorelli 2, Ilaria Meloni 4, Francesca Ariani 4, Francesca Mari 4, Sonia Amabile 4, Eugenio Paccagnini 5, Mariangela Gentile 5, Giuseppe Belmonte 6, Gloria Zollo 2, Giuseppe Valacchi 7, Thierry Durand 8, Jean-Marie Galano 8, Lucia Ciccoli 1, Alessandra Renieri 9, Joussef Hayek 10

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Redox imbalance and morphological changes in skin fibroblasts in typical Rett syndrome

Cinzia Signorini et al. Oxid Med Cell Longev. 2014.

Abstract

Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (-43.6%) and GSH/GSSG ratio (-3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.

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Figures

Figure 1

Figure 1

Increased levels of total (i.e., sum of free and esterified form) F2-IsoPs, total F4-NeuroPs, 4-HNE PAs, and NPBI in RTT skin fibroblast as compared to the control cells. *P < 0.0001, **P = 0.0013. Data are expressed as means ± standard deviation. Legend: F2-IsoPs, F2-isoprostanes; F4-NeuroPs, F4-neuroprostanes; 4-HNE PAs, 4-hydroxy-2-nonenal protein adducts; NPBI, nonprotein bound iron.

Figure 2

Figure 2

Significant reduction in cellular GSH and significant increase of GSSG in RTT skin fibroblast as compared to control cells. *P < 0.0001, **P = 0.0033. Data are expressed as means ± standard deviation. Legend: GSH reduced glutathione; GSSG, oxidized glutathione.

Figure 3

Figure 3

Transmission electron microscopy of control (a) and RTT (b) fibroblasts cultures. Skin fibroblasts, either from control subjects or RTT patients, show a flattened morphology with extensive tapering cytoplasmic processes. An euchromatic and oval-shaped nucleus was present in central position of the cells, with clumps of heterochromatin next to the nuclear envelope. The cytoplasm contains many vesicles with variable electron density, a prominent Golgi complex, and mitochondria. Rough endoplasmic reticulum (RER) cisternae in RTT fibroblasts appear more dilated than in control. Some large multilamellar bodies (MLB) are frequently detectable in the cytoplasm of the RTT fibroblast cells. (G) Golgi complex, (M) mitochondrion, and (V) vesicle. Bar = 1 _μ_m.

Figure 4

Figure 4

Double immunofluorescence staining shows the localization of type I collagen (central column, red color) and type III collagen (left column, green color). Images are merged in the right panel and the yellow color indicates overlap of the staining. The colocalization of types I and III collagen is reduced in RTT skin fibroblasts. Legend: Col I, type I collagen; Col III, type III collagen.

Figure 5

Figure 5

Relative intensity of fluorescence for types I and type III collagen in RTT and control skin fibroblasts. Software LEICA AF6000 (Leica Microsystems-Germany). Data are expressed as median ± semiinterquartile range *P = 0.0062; N.S.: no significant difference (P = 0.4361). Legend: Col I, type I collagen; Col III, type III collagen.

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