Abstract P800: Liposomal Delivery of PICK1 Inhibitor, FSC231, Prevents Ischemic/Reperfusion-Induced Degradation of GluA2 AMPA Receptor Subunit

Stroke remains to be a leading cause of disability within the United States. Despite an enormous amount of research effort within the scientific community, very few therapeutics are available for stroke patients. Cytotoxic accumulation of intracellular calcium is a well-studied phenomenon that occurs following ischemic stroke. This intracellular calcium overload results from excessive release of the excitatory neurotransmitter glutamate, a process known as excitotoxicity, eventually leading to delayed neuronal death. The hippocampus is particularly susceptible to AMPA receptor-mediated delayed neuronal death. AMPA receptors (AMPARs) are homo- or hetero-tetramers composed of GluA1-4 subunits. The majority of AMPARs are impermeable to calcium due to a post-transcriptional modification in the channel pore of the GluA2 AMPAR subunit. Calcium-permeable AMPARs lacking the GluA2 subunit, contribute to calcium cytotoxicity and subsequent neuronal death. The internalization and subsequent degradation of GluA2 AMPAR subunits following oxygen-glucose deprivation/reperfusion (OGD/R) is, at least in part, mediated by protein-interacting with C Kinase-1 (PICK1). We hypothesize that disrupting the PICK1—GluA2 interaction will prevent the degradation of GluA2, thereby protecting neurons within the hippocampus from AMPAR-mediated delayed neuronal death. Pretreatment with liposome-encapsulated FSC231, an inhibitor of PICK1, in acute rodent hippocampal slices prevents the OGD/R-induced association of PICK1—GluA2. FSC231 treatment during OGD/R rescues total GluA2 AMPAR subunit protein levels. This work is the first to utilize a liposomal drug delivery system for the delivery of a small molecule in ex vivo acute rodent hippocampal slices exposed to ischemia/reperfusion injury. These results suggest that the interaction between GluA2 and PICK1 serves as an important step in the ischemic/reperfusion-induced reduction in total GluA2 levels and is a potential therapeutic target for the treatment of ischemic stroke.

Oxidative Stress Underlies the Ischemia/Reperfusion-Induced Internalization and Degradation of AMPA Receptors

Stroke is the fifth leading cause of death annually in the United States. Ischemic stroke occurs when a blood vessel supplying the brain is occluded. The hippocampus is particularly susceptible to AMPA receptor-mediated delayed neuronal death as a result of ischemic/reperfusion injury. AMPA receptors composed of a GluA2 subunit are impermeable to calcium due to a post-transcriptional modification in the channel pore of the GluA2 subunit. GluA2 undergoes internalization and is subsequently degraded following ischemia/reperfusion. The subsequent increase in the expression of GluA2-lacking, Ca2+-permeable AMPARs results in excitotoxicity and eventually delayed neuronal death. Following ischemia/reperfusion, there is increased production of superoxide radicals. This study describes how the internalization and degradation of GluA1 and GluA2 AMPAR subunits following ischemia/reperfusion is mediated through an oxidative stress signaling cascade. U251-MG cells were transiently transfected with fluorescently tagged GluA1 and GluA2, and different Rab proteins to observe AMPAR endocytic trafficking following oxygen glucose-deprivation/reperfusion (OGD/R), an in vitro model for ischemia/reperfusion. Pretreatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a superoxide dismutase mimetic, ameliorated the OGD/R-induced, but not agonist-induced, internalization and degradation of GluA1 and GluA2 AMPAR subunits. Specifically, MnTMPyP prevented the increased colocalization of GluA1 and GluA2 with Rab5, an early endosomal marker, and with Rab7, a late endosomal marker, but did not affect the colocalization of GluA1 with Rab11, a marker for recycling endosomes. These data indicate that oxidative stress may play a vital role in AMPAR-mediated cell death following ischemic/reperfusion injury.