Comprehensive analysis of cellular senescence in the central nervous system revealed microglial senescence and its involvement in demyelinating diseases

Cellular senescence is a state of irreversible cell-cycle arrest caused by various cellular stressors. Accumulating evidence indicates that cellular senescence is one of the key factors causing age-related tissue dysfunction in various organs. However, the features of cells that undergo cellular senescence and their significance in neural impairment are still unclear in the central nervous system. We showed that microglia, particularly in the white matter, undergo cellular senescence in the brain and spinal cord during ageing and in disease models involving demyelination through comprehensive investigations utilizing single-cell transcriptome analysis and various mouse models. Microglial senescence is predominantly detected in disease-associated microglia (DAM), which emerge with ageing and in neurodegenerative diseases. Knockouts of the p16INK4a gene, a key inducer of cellular senescence, ameliorated the neuroinflammatory phenotype in damaged spinal cords in mice. Finally, we showed that commensal bacteria promote the accumulation of senescent microglia and DAM associated with ageing. These results advanced our understanding of cellular senescence's role in the central nervous system and opened new strategies for treating age-related neural disorders. Overall design: Young and old GF and SPF mice (young GF, 2 months, N = 2; young SPF, 2 months, N = 2; old GF, 22 months, N = 2; old SPF, 22 months) were anaesthetized by intraperitoneal injection of three anaesthetic agents and heparin and transcardially perfused with ice-cold HBSS containing heparin. The brains were cut in sagittal midlines and the cerebral hemispheres were roughly minced by scissors, followed by digestion according to the standard protocol of Neural tissue dissociation kits (P). The brain homogenate was filtered through a 70 µm cell strainer, and cell debris was removed by centrifugation according to the standard protocol of Debris Removal Solution. The cells in HBSS with 1% BSA were then treated on ice with CD16/32 FcR-blocking reagent for 10 minutes, followed by 15 minutes of staining with fluorescence-conjugated antibodies. The following antibodies were used (all antibodies were purchased from BioLegend): Fluorescein isothiocyanate (FITC)-conjugated anti-Cd45 (cat#: 103108) and allophycocyanin (APC)-conjugated anti-Cd11b (cat#: 101212). Before sorting, cells were filtered through a 40 µm cell strainer and stained with propidium iodide (PI). PI-negative, Cd45-low, and Cd11b-positive cells were sorted into 1.5 ml Protein LoBind Tubes containing 1% BSA in HBSS and then centrifuged. After removal of the supernatant, the remaining pellet was lysed and total RNA was isolated according to the standard protocol of the RNeasy Micro Kit. Full-length cDNA was prepared using a SMART-Seq HT Kit (Takara Bio, Japan) according to the manufacturer's instructions. According to the SMARTer kit instructions, an Illumina library was then prepared using a NexteraXT DNA Library Preparation Kit (Illumina). An Illumina NovaSeq 6000 sequencer (Illumina) were used for DNA Sequencing. Generated reads were mapped to the mouse (mm10) reference genome using TopHat v2.1.1. Fragments per kilobase of exon per million mapped fragments (FPKMs) were calculated using Cuffdiff 2.2.1.