Anales RANF

P.45 ARE micro-RNAs THERAPEUTIC TARGETS FOR SPINAL CORD INJURY EXCITOTOXICITY? microRNA-135a-5p REDUCES P2X 7 - DEPENDENT RISE IN INTRACELLULAR CALCIUM AND PROTECTS AGAINST EXCITOTOXICITY. D. Reigada 1 , A.A. Calderón-García 1,2 , M. Soto-Catalán 1 , M. Nieto-Díaz 1 , T. Muñoz- Galdeano 1 , A. del Águila 1,3 , A. Soto-Neira 1 , M.A. Barreda-Manso 1 , R.M. Maza 1 . 1 Hospital Nacional de Parapléjicos. Servicio de Salud de Castilla La Mancha (SESCAM), Toledo. Spain. 2 Institute of Neurosciences of Castilla y León (INCYL). Institute of Biomedical Research of Salamanca (IBSAL).University of Salamanca. Salamanca, Spain. 3 Division of Developmental Biology, Cincinnati Children's Hospital Medical Center.Cincinnati, OH, USA. Release of large amounts of neurotransmitters after traumatic spinal cord injury (SCI) causes excitotoxicity, contributing to extend neural damage and functional deficits. The P2X 7 receptor participates in the excitotoxic processes that follow SCI, leading to a massive influx of calcium and the activation of the apoptotic death. miRNAs are a family of small non-coding RNAs capable of post-transcriptionally silencing the expression of protein-coding genes to regulate major cell processes. We hypothesize that the downregulation of miRNAs targeting P2X 7 contributes to receptor overexpression after SCI and that restoring their levels to pre-injury conditions will reduce P2X 7 -mediated excitotoxicity. We combined data from gene expression analyses, predictive algorithms, computational tools and luciferase reporter gene assays to identify microRNA-135a-5p (miR-135a) as a post-transcriptional modulator of P2X 7 expression. RT-qPCR confirmed that miR-135a expression decreases after SCI, in inverse correlation with P2X 7 overexpression. Transfection of Neuro2a neuroblastoma cell line with an inhibitory sequence (antagomiR-135a), simulating the miR-135a downregulation that occurs after SCI, led to an increase in mRNA and protein levels of P2X 7 . Functionally, Fura-2AM and flow cytometry assays indicated that miR-135a inhibition results in an increase of P2X 7 -dependent intracellular calcium concentration and excitotoxic cell death induced by ATP (300 µM) and Bz-ATP (100 µM). On the contrary, transfection with a miR-135a mimic reduced P2X 7 expression and calcium- dependent excitotoxic cell death. Therefore, we conclude that restoration of miR-135a expression after SCI may reduce the deleterious effects of ATP-dependent excitotoxicity. In this direction, we are developing a therapeutic strategy for the administration of miR-135a in the spinal cord of rat models of SCI using polymeric vehicles to reduce the extension of the secondary phase of neural cell death processes following SCI, that are induced, in part, by massive ATP release and P2X 7 -dependent excitotoxicity.