Upregulation Of Microglial PGC-1α Ameliorates Brain Damage After Acute Ischemic Stroke

Objective:Immune and inflammatory responses play crucial roles in neurological diseases.Brain-resident immune cells aggravate inflammatory reactions via interacting with peripheral infiltrating immune cells.Microglia,as brain-resident immune cells,greatly contribute to the initiation and propagation of neuroinflammation after acute ischemic stroke（AIS）.A better understanding of the specific role of microglia may lead to more effective therapies for AIS.Here,we report that PPARγcoactivator 1a（PGC-1α）expression is altered in microglia,both in brains of humans and mice of a stroke model.Nevertheless,it remains unclear whether and how microglia PGC-1αis involved in the pathogenesis after AIS.Methods:Post-mortem formalin-fixed brain sections were obtained from the Department of Pathology of Ohio State University（Columbus,OH）and the Brain and Body Donation program of Banner Sun Health Research Institute（Sun City,AZ）.PGC-1αin microglia of the ischemic penumbra region was detected by immunostaining.Meanwhile,the levels of PGC-1αin microglia from mice with ischemic stroke were evaluated.We conditionally overexpressed PGC-1αin microglia through the Cre-loxP system.PGC-1α^f/f mice carried a loxP-flanked allele of PGC-1αand an enhanced GFP（eGFP）gene.To generate mice with microglia-specific overexpression of PGC-1α,PGC-1α^f/f mice were crossbred with B6.129P2（Cg）-Cx3cr1^{tm2.1（cre/ERT2）Litt}/WganJ mice.Mice were performed to induce focal cerebral ischemia using monofilament under transient middle cerebral artery occlusion（tMCAO）procedure.Microglia in cortex were scanned step by step using confocal microscope,and images were reconstructed in 3D using IMARIS software.Primary microglia from adult mice were isolated using the MojoSort^TM mouse P2RY12 selection kit.To investigate the underlying mechanism of PGC-1α,we performed microarray analysis and ChIP-Seq analysis using sorted microglia.To determine whether PGC-1αregulates the inflammatory profiles of microglia after ischemic stroke,we employed mouse inflammatory cytokine array,and conducted FACS analyses to confirm the results.BV2 cells were cultured and transfected with lentivirus containing PGC-1αfragment to confirm results from in vivo experiments.Results:Microglia PGC-1αis increased at 1 day after stroke onset compared to non-neurological control,and decreased in stained sections from patients who died3-10 days after stroke onset.We also verified that microglia-specific expression of PGC-1αchanges in a time-dependent manner in our tMCAO model.We successfully induced PGC-1αspecifically overexpressed in microglia via the Cre-loxP system.PGC-1αin microglia protects against ischemia-induced brain damage in mice.PGC-1αenhanced microglia ramification and altered gene expression profiles of microglia.ChIP-Seq analysis was used to identify the targets of PGC-1αin microglia.KEGG pathway analysis of these genes identified the mitophagy signaling pathway as one of the most highly enriched pathways.These mitophagy-related genes were divided into two categories,one related to hypoxia（KRAS,ULK1,CSNK2A1,and CITED2）and the other related to non-hypoxia（TFEB,OPTN,EIF2AK3,BCL2L13,and UBB）.PGC-1αinduces mitophagy by regulating ULK1 expression in an ERRα-dependent manner.PGC-1α-associated attenuation of neurological deficits was blocked by mitophagy inhibitor treatment.PGC-1αeffectively inhibits ROS production and protects neurons against oxidative stress.PGC-1αattenuates neuroinflammation by suppressing NLRP3 inflammasome hyperactivation.Conclusion:In this study,we discovered a previously unknown role for microglia-specific PGC-1αin the pathophysiology of AIS.Our data show that microglia PGC-1αattenuates ischemia-induced neuroinflammation and neural injury.Moreover,these effects are mainly mediated by PGC-1α-induced mitophagy through enhanced ULK1 expression in an ERRα-dependent manner.This,in turn,leads to decreased ROS generation and inhibition of NLRP3-ASC-caspase-1pathway-mediated inflammatory responses.Thus,our findings highlight an important neuroprotective role of PGC-1αin microglia after ischemic brain injury,and promotion of microglial PGC-1αmay function as a promising target for AIS therapy through modifying microglial function and limiting neuroinflammation.