Viral vectors designed for NMDA receptor subunits silencing

CECILIA A VÁZQUEZ; MAGALÍ C CERCATO; ALEJANDRA I AGUIRRE; ANNA SALVETTI; ALBERTO L. EPSTEIN; DIANA A. JERUSALINSKY; MA. VERÓNICA BÁEZ

Mar del Plata

Congreso; XXX Annual Meeting and SAN-ISN Small Conference and Course; 2015

Sociedad Argentina de Investigación en Neurociencias

N-methyl-D-aspartate receptors (NMDAR) are found in the post-synapses of neurons in the central nervous system (CNS). These receptors are formed by two constitutive subunits (GluN1) and two variable ones: GluN 2 A-D or GluN3 A-B, which determine the receptor´s subtype and characteristics. In the hippocampus, GluN2A and GluN2B are NMDAR most abundant regulatory subunits.It was suggested that memory acquisition is related to either an increase of NMDAR, a variation of its subtype?s relative abundance in the hippocampus, or both. In our laboratory, we showed that GluN1 and GluN2A expression increases temporarily 70 minutes after plasticity induction.To further study NMDAR?s role in learning, our group generated adeno-associated viral vectors (AAV) carrying greenfluorescent protein gene (GFP) and short-hairpin RNA (shRNA) against GluN2A (shN2A), to knock-down this NMDAR subunit. Another vector, carrying a similar, scrambled sequence (shSc) was built as a control.AAV?s expression was assayed in neuroblastoma cells (Neuro2a) with the shSc AAV, allowing to determine an optimal multiplicity of infection (MOI) of 105. After essays in primary cultured neurons, optimal MOI was established in 104. No differences in GFP expression were observed after 6 or 7 days post infection (DPI). On day 8, GFP quantification was restricted by a significant decrease in cell viability. Currently, experiments are being made to assess the effect of shN2A on its target messenger RNA and its expression.