Integrated stress response associated with dark microglia contributes to neurodegeneration [TRAP-seq]

Microglia, the brain's primary resident immune cells, can assume various phenotypes with diverse functional outcomes on brain homeostasis. In Alzheimer's disease (AD), where microglia are a leading causal cell type, the identity of microglia subsets that drive neurodegeneration remains unresolved. Here, we identify a microglia phenotype characterized by a conserved stress signaling pathway, the integrated stress response (ISR). Using mouse models to activate or inhibit ISR in microglia, we show that ISR underlies the ultrastructurally distinct “dark” microglia subset linked to pathological synapse loss. Inducing microglial ISR in murine AD models exacerbates neurodegenerative pathologies, such as Tau pathology and synaptic terminal loss. Conversely, inhibiting microglial ISR in AD models ameliorates these pathologies. Mechanistically, we present evidence that ISR promotes the secretion of toxic long- chain lipids that impair neuron and oligodendrocyte homeostasis in vitro. Accordingly, inhibition of lipid synthesis in AD models ameliorates synaptic terminal loss. Our results demonstrate that activation of ISR within microglia represents a pathway contributing to neurodegeneration and suggest that this may be sustained, at least in part, by the secretion of long-chain lipids from ISR-activated microglia. Overall design: To investigate the impact of microglial ISR on microglia molecular phenotypes, we performed ribosome profiling by translating ribosome affinity purification (TRAP) sequencing on microglia from TRAP (n=5, Cx3cr1CreErt2/+;Eef1a1LSL-EGFP-L10a/+), ASV-injected 5xFAD-TRAP (n=4, Cx3cr1CreErt2/+;5xFAD;Eef1a1LSL-EGFP-L10a/+), vehicle-injected iPKR (PEG:DMSO, n=4, Cx3cr1CreErt2/+;Eef1a1LSL.NS3.eGFP-L10a.iPKR/+), ASV-injected iPKR (n=5, Cx3cr1CreErt2/+;Eef1a1LSL.NS3.eGFP-L10a.iPKR/+), Eif2A (n=4, Cx3cr1CreErt2/+; Eif2s1A/A;Tg(flEifs1)), ASV-injected 5xFADiPKR (n=4, Cx3cr1CreErt2/+;5xFAD;Eef1a1LSL.NS3.eGFP-L10a.iPKR/+), and 5xFADEif2A (n=3, Cx3cr1CreErt2/+;5xFAD;Eif2s1A/A;Tg(flEifs1)) mice. Mice were euthanized with CO2 and brain regions of interest were dissected and flash-frozen for TRAP. The cortices were micro-dissected and GFP-tagged ribosomes from the cell type of interest were immunoprecipitated with a GFP antibody. RNA sequencing was performed on the GFP antibody-bound fractions.