Project Summary Wnt signaling in stroke Intracerebral hemorrhage (ICH) is the most lethal but under-researched type of stroke. Therapeutic targets to minimize pathologic processes focus on mitigating inflammation and promoting hematoma clearance. Wnt signaling regulates diverse cellular activities and modulates an inflammatory response in microglia in certain brain diseases. Although much work has investigated Wnt-dependent cell genesis, a critical gap exists with respect to our understanding of how Wnt signaling regulates inflammatory responses after ICH. The secreted frizzled-related protein 3 (sFRP3, a synonym of Frzb) regulates both canonical and noncanonical Wnt pathways, and dysregulation of sFRP3 is associated with certain pathologic conditions, including osteoarthritis, astrocytoma, and heart failure. However, very few studies have investigated sFRP3 in brain, and little is currently known about the role of sFRP3/Wnt signaling in injury from stroke or ICH. The scientific objective of this R01 is to investigate whether astrocyte sFRP3 inhibits proinflammatory response, thereby reducing acute ICH injury and improving functional outcomes. By using multiple cellular, molecular, genetic, pharmacologic, histologic, and flow cytometric methods, we have obtained strong evidence to suggest a key role for sFRP3 in protection against early ICH injury: 1) sFRP3 expression level is upregulated in astrocytes in the perihematomal region early after ICH; 2) Loss/gain of sFRP3 function exacerbates/mitigates ICH injury; and 3) sFRP3 deletion increases expression of Toll-like receptor (TLR)4 and Wnt downstream molecule RhoA, as well as MMP-9 activity in the ICH brain. These novel observations support the scientific premise that astrocyte sFRP3 protects against early ICH injury and led us to hypothesize that astrocyte sFRP3 promotes microglial M2 polarization through inhibition of TLR4 signaling and that astrocyte sFRP3 attenuates brain damage through TLR4/Wnt signaling after ICH. In three specific aims, we will determine whether loss/gain of sFRP3 function exacerbates/mitigates ICH injury (Aim 1), whether astrocyte sFRP3 promotes microglial M2 polarization through inhibition of TLR4 signaling (Aim 2), and whether astrocyte sFRP3 attenuates brain injury through the TLR4/Wnt signaling pathway (Aim 3). Using multidisciplinary approaches, this study will elucidate the role of astrocyte sFRP3 after ICH and the cellular and molecular mechanisms by which astrocyte sFRP3-mediated microglial phenotypic switching mitigates gray and white matter injury, and improves functional recovery. This novel proof-of-concept work has translational potential and will stimulate more studies of sFRP3 in various brain diseases. Investigating innate immunity after stroke is a critical priority identified by the recent NINDS-SPRG.