Abstract 134: Agonaute 2 Regulates Neurogenesis In Ischemic Neural Progenitor Cells

Abstract

Background: Molecular mechanisms underlying stroke-induced neurogenesis have not been fully investigated. Argonaute 2 (Ago2) is the central component of RNA-induced silencing complex (RISC) that regulates miRNA activities. We investigated the biological functions of Ago2 in neural progenitor cells (NPCs) under normal and ischemic conditions. Methods and Results: Adult rats were subjected to permanent right middle cerebral artery occlusion (MCAo). Double immunofluorescent data revealed that stroke significantly (p<0.05) increased the number of Ago2 + neuroblasts (doublecortin, DCX + , 31±4 vs 13±4 in non-MCAo control, n=6/group) in the subventricular zone (SVZ). To examine the role of stroke-upregulated Ago2, we selectively attenuated endogenous Ago2 in NPCs isolated from the ischemic SVZ with siRNA. Attenuation of endogenous Ago2 suppressed stroke-induced NPC proliferation (bromodeoxyuridine + , BrdU + cells, 45±3 vs 61±5% in scrambled siRNA group, n=6/group, p<0.05), reduced the size of neurospheres (169±44 vs 224±40 μm, n=7/group, p<0.05), and increased the percentage of TUNEL + cells (37±5 vs 15±5%, n=6/group, p<0.05). Ablation of Ago2 also significantly reduced the percentage of stroke-increased neuroblasts (Tuj1 + cells, 4±1 vs 9±1%, n=6/group, p<0.05). Using high-throughput deep-sequencing of RNAs isolated by Ago2 crosslinking immunoprecipitation (Ago2 HITS-CLIP) analysis, we found that stroke substantially increased the copy numbers of miRNAs including miR-21 and miR-214, which were expressed at relatively low levels in non-ischemic NPCs. Attenuation of endogenous Ago2 by siRNA reduced Ago2-enriched miRNAs induced by stroke, whereas overexpression of Ago2 increased these miRNA expression. Conclusion: Our data suggest that Ago2 regulates miRNA biogenesis in NPCs after stroke, which provides new insight into molecular mechanisms of stroke-induced neurogenesis.