Abstract P750: 3K3A-APC Restores Oligodendrocyte Pools in Models of White Matter Stroke via PAR1 Signaling

Abstract

A recombinant variant of activated protein C, 3K3A-APC, was recently tested in a successful phase II clinical trial for ischemic stroke (RHAPSODY), which demonstrated that the treatment may reduce hemorrhages and is safe in patients that were treated with tPA and/or thrombectomy. Animal models of large ischemic stroke (middle cerebral artery occlusion) and neurodegeneration have shown that the effects of 3K3A-APC are mediated by preserved APC cell signaling activities (anti-apoptotic neuronal protection, protection of blood-brain barrier (BBB) integrity and anti-inflammatory activities). Because recent studies have suggested a role of protease activated receptor 1 (PAR1) in oligodendrocyte recovery after white matter damage, we wanted to test whether biased PAR1 signaling mediated by 3K3A-APC would protect oligodendrocytes in in vivo and ex vivo models of ischemic white matter stroke. First, we used a mouse model of white matter stroke induced by injecting a vasoconstrictor N 5 -(1-Iminoethyl)-L-ornithine (L-NIO) into the corpus callosum. Our results show that treatment with recombinant murine 3K3A-APC (0.2 mg/kg, starting 4h after stroke) protected oligodendrocytes (OLS, Olig2+CNPase+) and oligodendrocyte precursor cells (OPCs, Olig2+PDGFRα+) from cell death at 1 and 7 days after stroke. In vitro , 3K3A-APC protected primary mouse oligodendrocytes from apoptosis when exposed to OGD conditions at nanomolar concentrations (EC50 concentration was 5.2 nM). Improved oligodendrocyte viability in vivo was associated by about ~67% reduction in overall cell death (cresyl violet staining, T2w and diffusion tensor MRI) and reduced BBB leakage (fibrin staining and DCE-MRI) as well as improved functional recovery (adhesive removal and grid walking tests). Protective effects of 3K3A-APC were abolished by silencing of PAR1 with small interfering RNA (siRNA) in vivo and in vitro . In conclusion, PAR1 mediated signaling initiated by 3K3A-APC protects oligodendrocyte pools in models of ischemic white matter damage with preservation of neurovascular unit integrity.