The role of mTOR-dependent translation in aged microglia phenotype

Abstract

Aged microglia are known to become primed, acquiring the ability to respond more strongly to immune stimuli. By RNAseq, we discovered an upregulation of pathways controlling translation depending on mTORC1 signaling. Indeed, mTOR-dependent phosphorylation of 4EBP1 and S6 were increased in aged microglia, as assessed by phospho flow cytometry, and this correlated with an increase in inflammatory cytokines upon in vivo LPS challenge only at protein level. Attenuation of mTOR signaling through deletion of Rheb1, specifically in microglia and immune cells in Rheb1^fl/fl:Csf1r^Cre, showed two opposing effects on microglia priming genes: an NF-kappaB–dependent upregulation at mRNA level but a downregulation at protein level. Indeed, Rheb1^fl/fl:Csf1r^Cre mice showed milder symptoms of inflammation upon in vivo LPS injection, including milder sickness behavior. Mechanistically, the decrease in protein levels of inflammatory mediators in aged microglia were due to a reduction of the mTOR-eIF4E-dependent protein synthesis. In particular, Rheb1 loss caused diminished phosphorylation of 4EBP1, which resulted in decreased binding of eIF4E to eIF4G, a step required for the formation of the initiation complex eIF4F. Similar changes were also present in aged human microglia and in damage-associated microglia (DAM), a subset of microglia identified in neurodegeneration, indicating that the upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration. Whether translation regulatory elements play a role in regulating microglia immune gene expression in aging remains unclear. To address this point, we conducted a comprehensive multi-omics approach by analyzing the transcriptome, translatome and proteome of microglia upon <em>in vivo</em> LPS challenge from Rpl22^HA:Rheb1^fl/fll:CX₃CR1^CreER/+ animals. We confirmed our previous evidences that aging microglia upregulated components of protein synthesis machinery to boost their immune response to LPS. Further analysis will be carried out by integrating the datasets in order to discover cis-regulatory elements that might be underlying to translation regulation of immune genes. These findings will offer potential avenues for therapeutic interventions by targeting mTOR-dependent translation to alleviate age-related neuro-inflammatory diseases in the elderly.