The objective of this proposal is to examine the relationship of the transcription factor nuclear factor-kappaB (NF-kappaB), and its activation pathway, to neuronal damage induced by intracerebral hemorrhage. The experiments proposed are largely based upon preliminary data linking cellular death to NF-kappaB activation after cerebral infarction and hemorrhage, and our recent finding of involvement of a novel enzyme in the NF-kappaB regulatory pathway, inhibitor kappaB-kinase (IKK). Our overall hypothesis is that the acute phase response following cerebral hemorrhage is in part a dynamic inflammatory response coordinated at the gene transcription level. We hypothesize that NF-kappaB activation leads to neuronal death that is controlled up-stream by the phosphorylation of inhibitor kappaB (IkappaB) by IKK. We propose two specific aims. First using carefully selected pharmacologic (15d-PGJ2, IKK inhibiting peptide and proteasome inhibitor) and molecular (knockout and transgenic mice) probes, affecting important components of the NF-kappaB transduction pathway at different up-stream levels of its activation, we will determine the causal relationship of the NF-kappaB pathway to brain damage (neuronal loss and behavioral dysfunction) after intracerebral hemorrhage. Next using relevant biochemical, microscopic and molecular techniques and brains from animals that benefited from inhibition of up-stream components of NF-kappaB activation, we will describe the temporal and spatial characteristics of this inhibition and pin down the exact components of the pathway down-stream from the inhibition site, including IKK activity, IkappaB-phosphorylation, NF-kappaB DNA binding and NF-kappaB gene transactivation that were affected by these inhibitions. Subsequently, using a single cell level analysis we will identify what cell type(s) represent the primary target of anti-NF-kappaB therapy in treatment of ICH. These laboratory studies in animal models of hemorrhage and infarction will provide the framework for developing a possible new approach to therapy for stroke in humans.
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