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.