Abnormal Expression of Cerebrospinal Fluid Cation Chloride Cotransporters in Patients with Rett Syndrome Sofia Temudo Duarte 1,4,5 *, Judith Armstrong 1,3 , Ana Roche 1 , Carlos Ortez 1,3 , Ana Pe ´ rez 1 , Maria del Mar O’Callaghan 1 , Antonina Pereira 4 , Francesc Sanmartı´ 1 , Aida Ormaza ´ bal 2,3 , Rafael Artuch 2,3 , Mercedes Pineda 1,3 , Angels Garcı ´a-Cazorla 1,3 1 Department of Neurology, Hospital Sant Joan de De ´u (HSJD), Barcelona, Spain, 2 Department of Biochemistry, Hospital Sant Joan de De ´u (HSJD), Barcelona, Spain, 3 CIBER-ER (Biomedical Network Research Centre on Rare Diseases, Instituto de Salud Carlos III), Madrid, Spain, 4 Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal, 5 Instituto Gulbenkian de Cie ˆ ncia, Oeiras, Portugal Abstract Objective: Rett Syndrome is a progressive neurodevelopmental disorder caused mainly by mutations in the gene encoding methyl-CpG-binding protein 2. The relevance of MeCP2 for GABAergic function was previously documented in animal models. In these models, animals show deficits in brain-derived neurotrophic factor, which is thought to contribute to the pathogenesis of this disease. Neuronal Cation Chloride Cotransporters (CCCs) play a key role in GABAergic neuronal maturation, and brain-derived neurotrophic factor is implicated in the regulation of CCCs expression during development. Our aim was to analyse the expression of two relevant CCCs, NKCC1 and KCC2, in the cerebrospinal fluid of Rett syndrome patients and compare it with a normal control group. Methods: The presence of bumetanide sensitive NKCC1 and KCC2 was analysed in cerebrospinal fluid samples from a control pediatric population (1 day to 14 years of life) and from Rett syndrome patients (2 to 19 years of life), by immunoblot analysis. Results: Both proteins were detected in the cerebrospinal fluid and their levels are higher in the early postnatal period. However, Rett syndrome patients showed significantly reduced levels of KCC2 and KCC2/NKCC1 ratio when compared to the control group. Conclusions: Reduced KCC2/NKCC1 ratio in the cerebrospinal fluid of Rett Syndrome patients suggests a disturbed process of GABAergic neuronal maturation and open up a new therapeutic perspective. Citation: Duarte ST, Armstrong J, Roche A, Ortez C, Pe ´rez A, et al. (2013) Abnormal Expression of Cerebrospinal Fluid Cation Chloride Cotransporters in Patients with Rett Syndrome. PLoS ONE 8(7): e68851. doi:10.1371/journal.pone.0068851 Editor: Maurizio D’Esposito, Institute of Genetics and Biophysics, Italy Received December 12, 2012; Accepted June 5, 2013; Published July 19, 2013 Copyright: ß 2013 Duarte et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by ‘‘Real Patronato’’ (Spanish Ministry of Health and Social Policy), Tecnifar Epilepsy Research grant and FIS PS09/01132. Dr. Sofia T. Duarte has received a research grant from Portuguese League Against Epilepsy and from Tecnifar S.A. (Epilepsy research grant). Currently, Dr. Sofia Duarte integrates the Portuguese Programme for Advanced Medical Education, sponsored by Calouste Gulbenkian Foundation and Portuguese Foundation for Science and Technology. Dr. Carlos Ortez was supported by a grant from Caja Navarra. Dr. Mercedes Pineda is funded by a grant from ‘‘Real Patronato’’, Spanish Ministry of Social Politics. Dr. Angels Garcia-Cazorla is funded by the grant FIS PS09/01132. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Dr. Sofia T. Duarte received a research grant from Portuguese League Against Epilepsy and from Tecnifar S.A. (Epilepsy research grant). There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: sofia.duarte22@hotmail.com Introduction Rett syndrome (RTT) is an X-linked neurodevelopmental disorder with an incidence of 1:10000 live female births and is one of the leading causes of mental retardation and autistic behavior in females [1]. Loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2) cause most cases of RTT. Individuals affected with RTT experience normal development up to the age of 6–18 months, at which time they fail to acquire new skills and enter a period of motor regression [2]. Autistic features are a hallmark of this disorder and epilepsy is frequent [3]. RTT patient brain does not show obvious signs of neurodegeneration, atrophy, gliosis, demyelination, or neuronal migration defects [4,5], suggesting that neurological symptoms may primarily stem from subtle defects of subcellular compart- ments such as dendrites, axons, or synaptic structures [6]. MeCP2 is a transcriptional regulatory protein, and in its absence, a large number of genes exhibit abnormal expression with implications in the balance between synaptic excitation and inhibition [7,8]. MeCP2 might be particularly important to GABAergic function and there is evidence that the expression of MeCP2 is approximatelly 50% higher in GABAergic neurons when com- pared to non GABAergic neurons. Mice with conditional deletion of Mecp2 in GABAergic neurons initially show normal behavior but in the course of development start displaying forepaw stereotyped movements, compulsive grooming, impaired motor coordination, learning/memory deficits, abnormal EEG hyperex- PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e68851