Astrocyte-specific TLR4 signaling and Blood Brain Barrier permeability following acute focal cerebral ischemia

项目总结/文摘

PROJECT SUMMARY/ABSTRACT Currently approved stroke therapies include underutilized, time-limited, systemic thrombolysis and mechanical recanalization options. Therefore, there is a need for more affordable and flexible approaches to treatment that can be disseminated widely. One approach includes targeting robustly expressed innate pathways such as the Toll-like Receptor (TLR) signaling pathways activated after focal cerebral ischemia. There is a lack of knowledge regarding the timing and cellular specificity of downstream pathways activated by pathways such as TLR4 in astrocytes and other components of the neurovascular unit. In this proposal, we will determine the key downstream targets in astrocyte TLR4 signaling and the effect of astrocyte-specific TLR4 signaling on blood brain barrier permeability (BBB) and neurobehavioral outcomes following acute focal cerebral ischemia. We will use a clinically relevant, endogenous danger associated molecular pattern (DAMP), HMGB1, a known TLR4 ligand to determine the key downstream targets in astrocyte TLR4 signaling. We will also use in vitro models of ischemia, such as Oxygen Glucose Deprivation (OGD), to determine the TLR4-dependent downstream pathways activated by other DAMPs in astrocytes. Characterization of the downstream effectors of astrocyte TLR4 signaling has important implications for decreasing BBB permeability and secondary brain damage and improving outcomes after stroke. The overall hypothesis of this proposal is that stroke-induced, astrocyte-specific TLR4 signaling induces BBB disruption in the acute phase of stroke, and that inhibiting ischemia-relevant DAMP-TLR4 signaling in astrocytes will decrease BBB permeability following acute focal cerebral ischemia and improve behavioral outcomes. This central hypothesis will be tested in the following aims: Aim 1: We will determine the signaling pathways active in TLR4-reactive and TLR4 non-reactive penumbral astrocytes using a model of middle cerebral artery occlusion (MCAO) and transcriptomics following acute cerebral ischemia Aim 2) Using HMGB1 stimulation of cultured astrocytes and an in vitro model of cerebral ischemia, OGD, we will identify downstream targets of TLR4 signaling in astrocytes via Western blot and phosphoproteomics. Aim 3: Using mice with inducible, astrocyte-specific deletion of TLR4, we will determine the effect of astrocyte-specific TLR4 deletion on BBB permeability and neurobehavioral outcomes following MCAO. At the end of these studies, we will have a better understanding of the molecular mechanisms that underlie TLR4 signaling in astrocytes. Results from these studies will lay the foundation for the development of novel therapeutics that can decrease brain damage after stroke.