Abstract TP264: Actin Reorganization is a Rapid and Reversible Adaptive Response by Neurons Exposed to Ischemic and Excitotoxic Injury in vitro and in vivo

Abstract

In initial stages of ischemic injury, glutamate receptor hyperactivation mediates extensive neuronal cell death. Most of this initial death is due to cytotoxic edema, whereby sodium and chloride entry cause cell swelling. Early subcellular responses to neuronal edema are poorly understood. We observed that actin filaments (F-actin) undergo a dramatic reorganization within the somatodendritic domain of neurons exposed to ischemia and/or sublethal excitotoxicity in vivo and in vitro. Fluorescence imaging was performed on embryonic rat hippocampal neurons cultured for 21-days in vitro. “Ministrokes” were induced in vivo by photothrombosis of a single penetrating vessel in mouse sensorimotor cortex. Animals were fixed 4 or 6 hours after vessel occlusion. Fluorescence images were analyzed in infarct, peri-infarct, and contralateral regions. All experiments were repeated 3-4 times, and quantified in a blinded fashion In control tissue F-actin is concentrated within dendritic spines, with little F-actin in the dendrite shaft. Within 5 min of applying 50μM NMDA, F-actin is lost from spines (inducing spine shrinkage) and polymerizes into long filament bundles within the dendrite shaft and soma. A similar redistribution of actin occurs after oxygen/glucose deprivation in vitro and after stroke in vivo. In vitro these actin changes are reversible within 1-3 hours of transient NMDA exposure. Increased Ca2+ is necessary but not sufficient for iniduction. Conditions that reduce cytotoxic edema prevent actinification; conditions that promote cytotoxic edema induce actinfication. The actin nucleator inverted formin-2 (INF2) was found to mediate dendrite actinification. Pharmacological inhibition of formins but not Arp2/3, completely prevented actinification. Moreover, silencing of endogenous INF2 using shRNA prevented NMDA-induced actinification; overexpression of wildtype INF2 sensitized neurons to actinification. Silencing of INF2 rendered neurons more vulnerable to NMDA-induced cell death. Results indicate that ischemia-induced actin filament accumulation within the somatodendritic compartment is a pro-survival response that protects neurons from death induced by cell swelling.