Multi-pronged Approach to Recalibrating the Inflammatory Cascade in Ischemic Stroke with BET blockade

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Abstract Neuroinflammation after stroke significantly contributes to neuronal cell death. Bromodomain and Extra Terminal Domain (BET) proteins are essential to inflammatory gene transcription. There are four BET proteins: BRD2, BRD3, BRD4, and BRDT. BRD2 and BRD4 are abundant and ubiquitously expressed. BRD3 expression is very low in most tissues including the brain, and BRDT is testis specific. BET proteins contain two conserved bromodomains that associate with acetylated lysines, and an extraterminal domain. BET proteins have varied effects including chromatin remodeling, histone acetyltransferase activity, and as scaffolds to recruit transcription factors; they couple chromatin remodeling with transcription. We hypothesize that BET blockade will provide a multipronged approach to reducing cell death after stroke. BRD2 normally represses peroxisome proliferator activator γ (PPARγ) activity, which has an anti-inflammatory effect, so we expect that inhibiting BRD2 will increase anti-inflammatory gene transcription. BRD2 knockdown also decreases nuclear factor-B (NF-B) activation, which is a major regulator of pro-inflammatory gene transcription in stroke. BRD4 acts as an NF-B co-activator, therefore we predict that BRD2 and BRD4 inhibition will decrease pro-inflammatory gene transcription in the ischemic brain. Furthermore, because BRD2 and BRD4 constitutively inhibit nuclear factor (erythroid-derived 2)-related factor (Nrf2) which is essential to antioxidant gene transcription, we expect BRD2/4 inhibition to increase expression of antioxidant genes, reducing oxidative stress. Little is known regarding the role of BET proteins in stroke, but our preliminary data shows that BET inhibition reduces infarct in a rodent model of stroke. Our long-term goal is to reduce the spread of stroke damage by limiting the effects of secondary inflammation. Our hypothesis is that BET inhibition is neuroprotective in ischemic stroke by limiting the deleterious effects of secondary inflammation. Our main objective is to determine the mechanism by which BET inhibition is protective in ischemic stroke. In Aim 1, we will determine the neuroprotective effect of BET blockade after ischemia using JQ1 (BET inhibitor) and dBET1 (a proteolysis-targeting chimera that degrades BET proteins). We will utilize male and female aged mice and rats subjected to ischemic stroke and will investigate the effects of BET blockade on infarct size and long-term behavioral outcomes. In Aim 2, we will determine the effects of BET blockade on stroke-induced neuroinflammation. In Aim 3, we will dissect the cell-specific role of BRD4 in the neuroinflammatory process after stroke by using BRD4 floxed mice crossed with lines producing Cre recombinase in specific cell types. We will study the contribution of BRD4 from myeloid-lineage cells (BRD4floxed/floxed x LysMCre/Cre) as well as from brain-specific endothelial cells (BRD4floxed/floxed x Slco1c1-CreERT2) to stroke injury. This project will provide mechanistic insights into how BET proteins contribute to secondary injury after ischemic stroke. These data will yield a positive impact as it will provide a strong foundation for future development of novel therapeutic strategies targeting BET proteins to reduce stroke damage.