Project description:To test the functional role of ApoA-I on microglia in the context of stress, we treated primary microglia with LPS stress with or without ApoA-I protein, and used bulk RNA-seq to evaluate if ApoA-I exhibits an anti-inflammatory role.
Project description:We found ApoA-I from blood can be taken up by microglia in the brain. To determine if ApoA-I from circulation is sufficient to restore microglial transcriptomic state to a more wild-type-like state, we performed a rescue experiment by intravenously injecting ApoA-I protein into adult Apoa1 KO mice and sorted microglia with or without ApoA-I uptake for bulk transcriptomic analysis.
Project description:Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes, many of which have been implicated in several diseases and disorders of the central nervous system including alcohol use disorders and disorders. By utilizing a method optimized for sensitive and rapid quantitative proteomic analysis of microglia involving suspension trapping (S-Trap), we were able to produce efficient and reproducible protein extraction from low cell yielding primary mouse brains. Using a ~2 h gradient on a 75 cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole-Orbitrap mass spectrometer (QE Plus), 5,062 total proteins were identified where 4,928 of those proteins were quantifiable by label-free quantitation (with 5 biological replicates). This analysis resulted in the most comprehensive proteomic dataset for ethanol- and LPS-treated primary mouse microglia to date and even expanded upon the well-characterized macrophage/microglia response to LPS treatment. This study also highlights the subtle, yet significant changes ethanol exposure can induce when compared to control. Interestingly, these changes are not consistent with the robust classical activation induced by LPS treatment, but instead align with the emerging theory that ethanol-treated microglia yield an alternative activation response. The contrast to LPS-treated microglia leads us to conclude that ethanol does not elicit a strong inflammatory response but rather might have a general inhibitory effect on multiple pathways such as phagocytosis and cell migration.
Project description:Our study demonstrated that the expression of Igf2bp1 in activated microglia was significantly up-regulated, implying a role of Igf2bp1 in LPS-induced m6A modifications in microglia. To understand the roles of Igf2bp1 on LPS-induced m6A modification in microglia, we performed Igf2bp1 loss-of-function (LOF) approach. Microglia stimulated by LPS were transfected with either scrambled siRNA control or Igf2bp1 siRNA for 48 hours. To m6A modification profiles in control and Igf2bp1 LOF microglia were determined by MeRIP-seq analysis.
Project description:Primary microglia were derived from neonatal mice and were activated via LPS+ATP treatment. test mice were treated with the drug Ladostigil and transcriptome was compared to untreated controls
Project description:The experiment is to demonstrate the global m6A methylation signatures and their correlation with mRNA expression patterns in microglia stimulated by LPS. The results also identified m6A modifiers with significantly altered expression, which reveals the key one that regulate the m6A modifications in microglia.
Project description:We investigated the expression patterns of transcripts in mouse primary microglia that are stimulated by LPS and transfected with miR-106b mimics. Three experiment groups were utilized in the RNA-seq, that are PBS+mimics control group, LPS+mimics group, and LPS+miR-106b mimics group. Samples were collected 2 days after LPS treatment and mimics transfection.
Project description:Ischemic stroke is a common acute CNS disorder leading to nearly half a million deaths per year in Europe. The high mortality is primarily owed to the limited treatment options of restoring blood flow in a narrow time window of several hours. Furthermore, inflammatory processes in the days and weeks after ischemic stroke contribute to tissue loss and neurological deficits. The key cells that influence and control this inflammatory cascade are microglia, the innate immune cells of the CNS. Microglia can be influenced and activated by e.g. lipopolysaccharide (LPS),a bacterial cell membrane component. It has been previously shown, that repetitive LPS stimuli prior to infarction (termed immunological preconditioning) lead to reduced infarct volumina in mouse models of ischemic stroke. Furthermore, our laboratory has shown, that phosphoinositide-3 kinase gamma mediates microglial functions after LPS-preconditioning. Hence, the aim of this work was to characterize proteomic alterations in microglia with (I) ischemic stroke in general in the tMCAO (transient middle cerebral artery occlusion) mouse model of ischemic stroke, (II) the influence of LPS-preconditioning on microglial proteomic alterations after tMCAO and (III) the role of PI3Ky in the microglial proteomic changes after tMCAO and preconditioning. This was done by a single LPS injection 3 days before tMCAO in wildtype mice and mice with PI3Ky knockout or knockin of the kinase dead form of PI3Ky.