Carver, Chase

Abstract Brain white matter tracts undergo structural and functional changes linked to late-life cognitive decline, but the cellular and molecular contributions to their selective vulnerability are not well defined. In naturally aged mice, we demonstrate that senescent and disease-associated microglia (DAM) phenotypes converge in hippocampus-adjacent white matter. Through gold-standard gene expression and immunolabeling combined with high-dimensional spatial mapping, we identified microglial cell fates in aged white matter characterized by aberrant morphology, microenvironment reorganization, and expression of senescence and DAM markers, including galectin 3 (GAL3/Lgals3), B-cell lymphoma 2 (Bcl2), and cyclin dependent kinase inhibitors, including Cdkn2a/p16ink4a. Pharmacogenetic or pharmacological targeting of p16ink4a or BCL2 reduced white matter GAL3+ DAM abundance and rejuvenated microglial fimbria organization. Our results demonstrate dynamic changes in microglial identity in aged white matter that can be reverted by senotherapeutic intervention to promote homeostatic maintenance in the aged brain.

Abstract Cellular senescence is a conserved mechanism of aging characterized by cell cycle arrest and secretion of a senescence-associated secretory phenotype comprised of proinflammatory cytokines, chemokines, proteases, and growth factors that disrupt cellular homeostasis by promoting sterile inflammation and aberrant tissue remodeling. Tissue microenvironment, induction mechanism, and duration since senescence onset contribute to senescent cell heterogeneity, making identification challenging. Senescent cell contribution to age-related cognitive decline suggests that the hippocampus may be a key brain region for uncovering functional identities of senescence. Therefore, our goal is to contextually and spatially map senescent cells in comparison of cell-type specific morphological, transcriptional, and functional features that distinguish them among highly-ordered brain microenvironments. GeoMx Digital Spatial profiling was used to investigate cell identity and senescence markers in the aging brain to develop transcriptomic profiles from 22-24-month old mice. We also adapted Imaging Mass Cytometry with a curated panel of antibody-labeled proteins in fixed brain sections. We observed that senescent-like phenotypes of cells from myeloid and non-myeloid origin accumulate in the hippocampus and cortex, whereas CD45+ immune cells primarily accumulate in the white matter regions, in close proximity to microglia and GAL3+ cells. We profiled differences in ramified and ameboid microglia, in comparison of young and old mice, with emphasis on the chemotactic microenvironment involving activation of inflammatory signals. These advanced, multi-plex imaging modalities enable in-depth characterization of senescent cell features in the aging brain. Beyond morphological profiling, we demonstrate enhanced resolution and detection of individualized senescence in the aging brain.

Abstract Surgical parabiosis is widely used to study the mechanistic influence of the circulating milieu on aging and regeneration. This powerful model presents diverse complications based on age, strain, sex, and other experimental parameters. In young (Y) and old (O) male and female C57BL6 mice, we optimized heterochronic (n=12) and isochronic (n=10 Y-Y, n=7 O-O) parabiosis. Throughout protocol development, we identified several complications including variable responses to anesthesia, external and internal dehiscence, dehydration, and weight loss. We identified and implemented solutions during surgical and post-surgical periods, including titrated anesthesia, reinforced internal sutures, topical agents to promote wound healing, and increased supplementation. By consistently adopting protocol changes we were able to significantly increase survival. Separately, we confirmed the time course of chimerism in heterochronic pairs of C57BL6 and Tg(act-EGFP)Y01Osb (eGFP) mice. Baseline and longitudinal blood samples were collected via tail vein. Flow cytometry was used to visualize GFP-positive cells from the parabiont blood sample. Through blood analysis we found that chimerism occurs as early as 2 days post-operatively. Exploitation of our optimized protocol may enable others to efficiently adopt the surgical parabiosis model to dynamically study mechanisms of aging and regeneration.

Abstract New strategies to preserve cognitive function could broadly benefit the aging population. Cellular senescence is a hypothesized mediator of inflammation-related tissue dysfunction in aging. Using single-cell RNA-sequencing, we discovered an age-related brain myeloid population exhibiting overlapping senescent and disease-associated activation signatures, including upregulation of chemoattractant factors. We confirmed senescent brain myeloid cells promote peripheral immune cell chemotaxis in vitro. Through mass cytometry, we demonstrate age- and sex-dependent increases in activated resident and infiltrating brain immune cells are reduced through systemic targeting of p16-positive senescent cells, which is associated with improvements in executive function and spatial learning tasks. Our high-dimensional results reveal dynamic remodeling of the age-dependent brain immune cell landscape and implicate senescent cell targeting as a strategy to counter inflammatory changes and cognitive decline.