1. Pediatric Neurology Department, Hospital D. Estefânia-CHLC 2. Instituto de Medicina Molecular, Universidade de Lisboa 3.Department of Neurology, CIBER-ER Biomedical Network Research Centre on Rare Diseases, Instituto de Salud Carlos III, Barcelona, Spain
- 11th European Paediatric Neurology Society (EPNS) Congress. 27-30 Maio de 2015, Vienna, Austria (poster)
- 9th FENS Forum of Neuroscience, 2014, Milão, Itália (poster)
- Published Abstract: European Journal of Paediatric Neurology 2015; 19:S149
Objective: Induced pluripotent stem cells (iPs cells) are a promising tool for the study of neurological diseases. Disruption of GABAergic activity in Rett Syndrome (RTT) can be related, at least in part, with a failure to complete the GABA functional switch that occurs during normal development, from excitation in immature neurons to inhibition in mature neurons. This switch is mainly mediated by a developmental increase in KCC2 expression, a co-transporter that drives Cl- efflux outside neurons, with the consequent decrease in intracellular Cl- concentration and GABA-mediated hyperpolarization in mature neurons. We documented reduced KCC2 levels in the cerebrospinal fluid of RTT patients and aimed to test GABAergic function in neurons derived from RTT patients’ iPs cells.
Methods: Human skin biopsies were collected in accordance with European and National ethical regulation and IPS cells were generated from fibroblast cells upon infection with a retroviral vector expressing four transcription factors (Oct4, Sox2, Klf4, and Myc). IPs cell lines were obtained from patients carrying MECP2 mutations (R306C and R255X) and controls. Neuronal cortical populations were derived in a monolayer culture system using the dual-SMAD inhibition protocol. Perforated patch recordings were performed in these neurons and GABA-evoked postsynaptic currents were measured to evaluate GABA A receptor equilibrium potencial. Adittionally, we performed molecular studies in a RTT mouse model (Mecp2tm1.1Bird/J).
Results: Preliminary data indicate that recordings from RTT cells exhibit a GABA A receptor equilibrium potential that is more positive than in recordings from control cells, indicating changes in chloride gradient, characteristic of an immature state. The level of KCC2 protein expression is lower in Mecp2-KO mice when compared to control littermates, as addressed by western blot analysis of 6 week old hippocampi.
Conclusion: Results form the animal model, iPs cell-based models and human cerebrospinal fluid studies suggest a GABAergic dysfunction in RTT that includes alterations in KCC2 expression and/or activity.