Spatial and single-cell transcriptomics reveal the reorganization of cerebellar microglia with aging

Aging induces region-specific functional decline across the brain. The cerebellum, critical for motor coordination and cognitive function, undergoes significant structural and functional changes with age. The molecular mechanisms driving cerebellar aging—particularly the role of cerebellar glia, including microglia—remain poorly understood. Here, we used single-nuclei RNA sequencing (snRNA-seq), microglial bulk RNA-seq, and multiplexed error-robust fluorescence in situ hybridization (MERFISH) to characterize transcriptional changes associated with cellular aging in the mouse cerebellum. We discovered that microglia exhibited the most pronounced age-related changes of all cell types and that their transcriptional signatures pointed to enhanced neuroprotective immune activation and reduced lipid-droplet accumulation compared to hippocampal microglia. Furthermore, cerebellar microglia in aged mice, compared to young mice, were found in closer proximity to granule cells. This relationship was characterized using the newly defined neuron-associated microglia score, which captures proximity-dependent transcriptional changes and suggests a novel microglial responsiveness. These findings underscore the unique adaptations of the cerebellum during aging and its potential resilience to Alzheimer's disease (AD) related pathology, providing crucial insight into region-specific mechanisms that may shape disease susceptibility. Overall design: Nuclei from the cerebellum of two female and two male mice, aged 3, 12, 18, and 24 months, were isolated using Fluorescence-Activated Cell Sorting (FACS) based on NeuN expression status and subsequently analyzed via single-nucleus RNA sequencing (snRNA-seq).