mTOR-dependent translation amplifies microglia priming in aging mice
Lily Keane, … , Michael T. Heneka, Melania Capasso
Lily Keane, … , Michael T. Heneka, Melania Capasso
Published October 27, 2020
Citation Information: J Clin Invest. 2021;131(1):e132727. https://doi.org/10.1172/JCI132727.
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Research Article Aging Inflammation

mTOR-dependent translation amplifies microglia priming in aging mice

Abstract

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB–dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration.

Authors

Lily Keane, Ignazio Antignano, Sean-Patrick Riechers, Raphael Zollinger, Anaelle A. Dumas, Nina Offermann, Maria E. Bernis, Jenny Russ, Frederike Graelmann, Patrick Neil McCormick, Julia Esser, Dario Tejera, Ai Nagano, Jun Wang, Claude Chelala, Yvonne Biederbick, Annett Halle, Paolo Salomoni, Michael T. Heneka, Melania Capasso

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Figure 7

In Rheb-KO cells, diminished binding of eIF4E to its partner eIF4G results in diminished translation of cytokines.

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In Rheb-KO cells, diminished binding of eIF4E to its partner eIF4G resul...
(A) Immunofluorescence staining of p-4EBP1 Thr 37/46 (green) and F4/80 (macrophage marker, red) in WT, Rheb-KO, or WT BMDMs pretreated for 90 minutes with 100 nM rapamycin and stimulated with 100 ng/mL LPS for 1 hour (not shown) and 6 hours. Scale bar: 10 μm. Bottom panel: p-4EBP1 Thr37/46 quantification (n = 3). (B) Proximity ligation assay (PLA) of eIF4E and eIF4G1 in neonatal primary microglia cultures from WT and Rheb-KO brains. The schematic represents mTOR modulation of translation and the principle of PLA. Bottom panel: Quantification of eIF4E/eIF4G binding (PLA events, red dots). Scale bar: 25 μm (objective 40×). n = 3 biological replicates, each with 4 technical replicates. (C) Western blot and densitometry analysis of eIF4E and eIF4G1 from WT and Rheb-KO microglia (n = 6 for eIF4G1, n = 4 for eIF4E) and from WT and Rheb-KO BMDMs stimulated with 100 ng/mL LPS for the indicated time points (n = 5 for eIF4G1, n = 3 eIF4E). (D) TNF measured by ELISA of culture supernatant from WT and Rheb-KO primary microglia. Cells were pretreated for 90 minutes with 25 μM of the eIF4E/eIF4G inhibitor 4EGI-1 and then stimulated with 200 ng/mL LPS for 3 hours (n = 3). (E) TNF measured by ELISA of culture supernatant from acute brain slices of WT mice, pretreated for 2 hours with either 100 μM ribavirin, 40 μM cycloheximide, or vehicle and then stimulated with 2 μg/mL LPS for 6 hours. Data are represented as the fold change normalized to LPS samples; n = 6 from 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; 2-way ANOVA (A, C [BMDMs], and D; Student’s t test (B, C [microglia], and E). For A, P = 0.0041 for WT versus Rheb-KO and NS (P = 0.1465) for WT versus rapamycin; 2-way ANOVA.