Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization

et al. (2014) Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization. PLoS ONE 9(3): e93181. doi:10.1371/journal.pone.0093181 Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization Alessio Cortelazzo 0 Claudio De Felice 0 Alessandra Pecorelli 0 Giuseppe Belmonte 0 Cinzia Signorini 0 Silvia Leoncini 0 Gloria Zollo 0 Antonietta Capone 0 Cinzia Della Giovampaola 0 Claudia Sticozzi 0 Giuseppe Valacchi 0 Lucia Ciccoli 0 Roberto Guerranti 0 Joussef Hayek 0 Maurizio D'Esposito, Institute of Genetics and Biophysics, Italy 0 1 Department of Medical Biotechnologies, University of Siena , Siena , Italy , 2 Child Neuropsychiatry Unit, University Hospital, Azienda Ospedaliera Universitaria Senese (AOUS) , Siena , Italy , 3 Neonatal Intensive Care Unit, University Hospital , AOUS, Siena , Italy , 4 Department of Molecular and Developmental Medicine, University of Siena , Siena , Italy , 5 Department of Medical Sciences Surgical and Neuroscience, University Hospital , AOUS, Siena , Italy , 6 Department of Life Science, University of Siena , Siena , Italy , 7 Department of Sciences of Life and Biotechnologies, University of Ferrara , Ferrara , Italy , 8 Department of Food and Nutrition, Kyung Hee University , Seoul , South Korea Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenance of cell shape to transcription regulation, was evaluated in the erythrocyte membranes from patients with typical Rett syndrome (RTT) and methyl CpG binding protein 2 (MECP2) gene mutations. RTT, affecting almost exclusively females with an average frequency of 1:10,000 female live births, is considered the second commonest cause of severe cognitive impairment in the female gender. Evaluation of beta-actin was carried out in a comparative cohort study on red blood cells (RBCs), drawn from healthy control subjects and RTT patients using mass spectrometry-based quantitative analysis. We observed a decreased expression of the beta-actin isoforms (relative fold changes for spots 1, 2 and 3: 21.8260.15, 22.1560.06, and 22.5960.48, respectively) in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, betaactin from RTT patients also showed a dramatic increase in oxidative posttranslational modifications (PTMs) as the result of its binding with the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE). Our findings demonstrate, for the first time, a beta-actin down-regulation and oxidative PTMs for RBCs of RTT patients, thus indicating an altered cytoskeletal organization. - Funding: The work was supported by Tuscany Region [Bando Salute 2009, Antioxidants (v-3 Polyunsaturated Fatty Acids, lipoic acid) supplementation in Rett syndrome: A novel approach to therapy], Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Actins are highly conserved proteins ubiquitously expressed in eukaryotic cells [1]. In vertebrates, three main groups of actin isoforms have been identified, i.e., alpha, beta and gamma. While the alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus, beta and gamma actins coexist in most cell types either as components of the cytoskeleton or mediators of internal cell motility [2]. In particular, beta-actin is a critical player in several cellular functions, ranging from cell motility and the maintenance of cell shape and polarity to transcription regulation. Interestingly, several syndromes associated with mental retardation are characterized by cortical dendritic abnormalities [3], and the neuronal cytoskeleton is essential for dendritic developmental processes. It includes microfilaments, neurofilaments and microtubules, each one being formed by a major protein, i.e., actin, neurofilament and tubulin, respectively [4,5]. Rett syndrome (RTT), the second commonest cause of severe mental retardation in the female gender, is represented by a devastating neurodevelopmental disorder with a wide phenotypical heterogeneity which is caused in the overwhelming majority of cases by loss-of-function de novo sporadic mutations in the X-linked gene encoding the methyl-CpG binding protein 2 (MeCP2) [6]. In its classical phenotype, RTT is characterized by an apparently normal development in the first few months followed by loss of neurodevelopmental milestones in a 4-stage progression [7,8]. Increasing evidence supports the concept that RTT is associated with impairment of dendritic arborization [9,10]. Several studies have described neuronal abnormalities in RTT human brains, and in male Mecp2-mutant mice [9,10,11,12,13]. Overall, this body of research suggests that MECP2 deficiency mutations are involved in cellular mechanisms regulating short term dynamics of dendritic spines early in development within the frame of maximum neural plasticity. However, the underlying cause for the dendritic arborization impairment in RTT is still largely unknown. While prior studies on Mecp2-deficient brains show striking changes in neuronal maturation [14], recent evidence indicates that MeCP2 deficiency affects microtubule dynamics in RTT astrocytes and impairs microtubule stability in RTT primary fibroblast cultures [15,16]. These data suggest that MeCP2 has a stabilizing role on microtubule dynamics and that its deficiency could lead to impaired microtubule stability which may at least partly underlie the dendritic abnormalities detected in RTT brains. We have previously demonstrated the existence of an abnormal erythrocyte shape in typical RTT patients showing a striking prevalence of circulating leptocytes with enhanced membrane oxidative stress (OS). Therefore, in the present study, we tested the hypothesis that a beta-actin deficiency with an increased oxidative posttranslational modification (PTMs) could underlie the red blood cells (RBCs) shape abnormalities in patients affected by the typical form of the disease [17]. Materials and Methods Study Population and Ethical Statement The study included 20 female patients with clinical diagnosis of typical RTT (median age: 5.0 years inter-quartile range 36, values range 310 years) with demonstrated MECP2 gene mutations. RTT diagnosis and inclusion/exclusion criteria were based on RTT nomenclature consensus [18]. All the patients were admitted to the Siena Rett Syndrome National Reference Centre. Gender-matched healthy control subjects of comparable age (N = 20, median age: 5.0 years inter-quartile range 35.5, values range 310 years) with a typical development were selected as a control population. Blood samplings in (...truncated)