Mitochondrial respiration in microglia is essential for response to demyelinating injury but not proliferation

Microglia are necessary for CNS function during development and play roles in aging, Alzheimer’s Disease (AD) and the response to demyelinating injury. Mitochondrial respiratory chain (RC) is necessary for conventional T cell proliferation6 and macrophage-dependent immune responses. However, whether mitochondrial RC is essential for microglia proliferation or function is not known. We conditionally deleted the mitochondrial complex III subunit Rieske Iron-Sulfur Protein (RISP) in the microglia of adult mice to assess the requirement of microglial RC for survival, proliferation, and adult CNS function in vivo. Surprisingly, mitochondrial RC function was not required for survival or proliferation of microglia in vivo. RNA-seq analysis showed that loss of RC function in microglia caused changes in gene expression distinct from aged or disease-associated microglia (DAM). Microglia-specific loss of mitochondrial RC function is not sufficient to induce cognitive decline. Amyloid-β plaque coverage decreased and microglial interaction with Amyloid-β plaques increased in the hippocampus of 5xFAD mice with mitochondrial RC-deficient microglia. Microglia-specific loss of mitochondrial RC function did impair remyelination following an acute, reversible demyelinating event. Thus, mitochondrial respiration in microglia is dispensable for proliferation but is essential to maintain a proper response to CNS demyelinating injury. For FACS of microglia for RNAseq and microglial enumeration, single cell suspension from whole brain were initially incubated in 1:50 Anti-mouse CD16/32 (biolegend 101302, clone 93) and, subsequently, stained with the following antibodies: 1:100 Anti-Mouse CD45.2-PacBlue (ebioscience 48-0454-82, clone 104) and 1:200 Anti-mouse CD11b(Mac-1)-APC (ebioscience, 50-0112-80, clone M1/70) in 200µL final volume of FACS Buffer. Viability was assessed with addition of DAPI 3 minutes before collection on flow cytometer (Fisher, D1306 0.1µg/mL final concentration). RNA quality and quantity were assessed using TapeStation 4200 High Sensitivity RNA tapes (Agilent), and RNA-seq libraries were prepared from 5-10ng of total RNA using the SMARTer Stranded Total RNA-seq Kit v2 (Takara Bio). For analysis of allele status, libraries were prepared using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB E7370L). Library QC was then performed using TapeStation 4200 High Sensitivity DNA tapes (Agilent). Dual-indexed libraries were pooled and sequenced on a NextSeq 500 instrument (Illumina) for untreated RISP-/- and wild-type, age-matched animals, or a NextSeq2000 instrument for all other samples (Illumina) 75 or 100 cycles, respectively, single-end, to an average sequencing depth of 27.5 million reads per sample.