Project description:In this study, we derived iPS-derived microglia-like cells (iMGLs) from a healthy donor (Sample: Control) CRISPR engineered to carry a dead CAS9 fused to the transcriptional repressor KRAB (dCAS9-KRAB) and treated these cultures with the pro-inflammatory stimulant LPS (Sample: LPS).
Project description:In this study, we derived iPS-derived microglia-like cells (iMGLs) from a healthy donor (Sample: Control) CRISPR engineered to carry a dead CAS9 fused to the transcriptional repressor KRAB (dCAS9-KRAB) and treated these cultures with pre-formed amyloid beta 1-42 fibrils. We then performed gene expression profiling analysis using data obtained from RNA-seq of treated and untreated cells at 4 different time points.
Project description:Proteomic approaches revealed that primary cilia, small hair-like structures found on cells, played a role in the regulation of microglial secretory function. Notably, primary cilia were transiently observed in less than 10% of microglia, and their presence was significantly reduced in microglia from Alzheimer's disease (AD) mice. We observed significant changes in the expression and distribution of secretomes after inhibiting the primary cilia gene intraflagellar transport particle 88 (Ift88) in microglia. Intriguingly, inhibiting primary cilia in the SEM of AD mice resulted in the expansion of extracellular amyloid plaques and damage to adjacent neurites. These results indicate that DAM-like microglia are present in the septumLS, a critical target region for hippocampal nerve bundles, and that the primary ciliary signaling system regulates microglial secretion, affecting extracellular proteostasis. Age-related primary ciliopathy probably contributes to the selective sensitivity of microglia, thereby exacerbating AD. To elucidate the dynamic changes in microglial proteomics resulting from silenced Ift88 and Aβ treatment, we performed an analysis of BV2 cell lysates and extracellular vesicles (EVs) by downregulating Ift88 expression. To investigate the specific proteomic alterations in EVs associated with CD63 and CD81, we conducted a proteomic characterization of CD63- or CD81-bound EVs in the lysate and EVs secreted from BV2 cells transfected with siIft88 and treated with Aβ, utilizing co-immunoprecipitation (Co-IP) with anti-CD63 or anti-CD81 antibodies.
Project description:Microglia-mediated neuroinflammation has been implicated in the pathogenesis of Alzheimer's disease (AD). Although microglia in aging and neurodegenerative disease model mice show a loss of homeostatic phenotype and activation of disease-associated microglia (DAM), a correlation between those phenotypes and the degree of neuronal cell loss has not been clarified. In this study, we performed RNA sequencing of microglia isolated from three representative neurodegenerative mouse models, AppNL-G-F/NL-G-F with amyloid pathology, rTg4510 with tauopathy, and SOD1G93A with motor neuron disease by magnetic activated cell sorting. In parallel, gene expression patterns of the human precuneus with early Alzheimer's change (n=11) and control brain (n=14) were also analyzed by RNA sequencing. We found that a substantial reduction of homeostatic microglial genes in rTg4510 and SOD1G93A microglia, whereas DAM genes were uniformly upregulated in all mouse models. The reduction of homeostatic microglial genes was correlated with the degree of neuronal cell loss. In human precuneus with early AD pathology, reduced expression of genes related to microglia- and oligodendrocyte-specific markers was observed, although the expression of DAM genes was not upregulated. Our results implicate a loss of homeostatic microglial function in the progression of AD and other neurodegenerative diseases. Moreover, analyses of human precuneus also suggest loss of microglia and oligodendrocyte functions induced by early amyloid pathology in human.
Project description:We studied the membrane protein compositions of Streptococcus pneumoniae WT and scRNA mutant strains, with or without the CSP1 induction into competence state.