Abstract 64: Scar-Forming Glial Cells Engulf Synapse and Worsen Stroke Outcome Through Megf10 and Mertk

Abstract

Objective: Previous studies showed that microglia and astrocytes actively engulf synapse through MEGF10 and MERTK in the developing brain. But whether microglia and astrocytes in ischemic stroke (IS) or hemorrhagic stroke (HS) mice are still phagocytotic and how they affect the outcome of stroke remain unclear. Methods: Microglia or astrocyte-specific MEGF10 or MERTK knockout mice were generated, and subjected to transient middle cerebral artery occlusion (tMCAO), or collagenase-induced intracerebral hemorrhage. Immunostaining, Transmission electron microscope and Western blot were used to evaluate synapse engulfment, and dendritic spines were quantified using Golgi staining. Cresyl violet staining was used to evaluate atrophy volume and lateral ventricle enlargement of stroke mice. Neurobehavioral test including mNSS , rotarod, grid-walking and smart cage were performed to assess neurofunctional recovery. Single-cell RNA sequencing was further conducted to compare gene expression between IS and HS mice. Results: We showed that 14 days after IS, 25% of microglia and 50% of astrocytes were phagocytotic and engulfed synapses in the glial scar. However, in HS, 25% of microglia and 5% of astrocytes were phagocytotic, and only microglia were found to engulf synapses. We further demonstrated that MEGF10 and MERTK were upregulated in both astrocytes and microglia of mice subjected to IS and HS, and knockout of MEGF10 or MERTK in astrocytes or microglia inhibited synapse engulfment, which further increased synaptic density, reduced brain atrophy volume and improved neurobehavioral recovery after IS. However, knockout of MEGF10 or MERTK in microglia but not astrocytes in HS mice resulted in increased synaptic density and neurobehavioral recovery improvement. Single-cell RNA sequencing revealed that phagocytosis-related processes were downregulated in a subtype of astrocytes in HS brain than that in IS brain. Conclusion: Our study revealed a previously unknown role for astrocytes and microglia in engulfing synapse in the glial scar in stroke brain, and showed phagocytic astrocytes and microglia contribute differently to brain function recovery of mice that subjected to IS and HS, opening a new avenue to develop effective strategy for stroke treatment.