Astrocytic TGFB Signaling Pathways and Neuroinflammation Following Stroke

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DESCRIPTION (provided by applicant): Stroke is one of the leading causes of death and disability in the world. The only FDA approved therapy for stroke is tissue plasminogen activator and it is only effective within approximately 4.5 hours following stroke. One promising strategy with a longer treatment window is limiting excessive neuroinflammation following stroke. Excessive inflammation worsens infarct sizes and perpetuates the inflammatory response. Promoting resolution of neuroinflammation after allowing its beneficial effects to occur may improve outcome in stroke. Transforming growth factor betas (TGF?'s) are master regulatory cytokines that are secreted in an inactive form and sequestered in the extracelluar matrix. They are primarily anti-inflammatory and their signaling is elevated days after stroke. Astrocytes are one of the cell types responding to increased levels of TGF? during this subacute period after stroke. Because astrocytes are glial cells that interact with neurons, blood vessels, and immune cells, they are well situated to coordinate neuroinflammatory responses. However TGF?'s regulation and function in astrocytes is not completely understood. Our preliminary data suggests that thrombospondin-1 production by astrocytes in the stroke border activates TGF? in the extracellular matrix, and the increase upregulates TGF? and thrombospondin-1 in astrocytes. Additionally, inhibiting astrocytic TGF? signaling approximately doubles the inflammatory response, decreases neuroprotective Akt signaling in surviving neurons adjacent to the infarct, and worsens outcomes after stroke. Our central hypothesis is therefore that astrocytes utilize TGF? in the subacute time period after stroke to trigger a positive feedback loop that leads to higher levels of active TGF?, which then reduces neuroinflammation and improves outcomes. We aim to determine whether astrocytic TGF? signaling is necessary and sufficient to trigger a positive feedback loop that increases active TGF? subacutely after stroke. We also aim to determine whether increasing global or astrocytic TGF? signaling over baseline is sufficient to decrease neuroinflammation and improve functional outcomes after stroke. To determine whether astrocytic TGF? signaling is necessary for this positive feedback loop we will use a double transgenic model with reduced TGF? specifically in astrocytes and supplement this deficit by activating global TGF?. We will also determine whether this astrocytic TGF? signaling is sufficient for this positive feedback loop with a different double transgenic model, upregulating astrocytic TGF? signaling. We will use ELISA, Western blot, immunohistochemistry, and qPCR to measure global levels of TGF? and thrombospondin-1, and signaling pathways of TGF? in each of these models. In addition, we will use these models to determine which conditions of TGF? signaling provide reduced neuroinflammation and improved functional outcomes following stroke. Regardless of our hypothesis being correct, the proposed experiments will provide a better understanding of the mechanisms that astrocytes use to influence neuroinflammation following stroke, which be applicable to other types of central nervous system inflammation as well.