Temporal tracking of microglia activation in neurodegeneration at single-cell resolution. Mus musculus

In this study, we used single-cell RNA-sequencing to gain unprecedented insight into the phenotypic heterogeneity and the transcriptional dynamics of microglia cells during the progression of neurodegeneration. Briefly, by using a severe neurodegeneration mouse model with Alzheimer’s-like pathology and phenotypes (CK-p25 model), we surveyed microglia activation by RNA sequencing longitudinally at fine temporal- and single-cell resolution. In summary, our work identified previously unobserved heterogeneity in the response of microglia to neurodegeneration, discovered novel microglia cell states, revealed the trajectory of cellular reprogramming of microglia in response to neurodegeneration, and uncovered the underlying transcriptional programs. These insights into the molecular programs underlying microglia activation provided by our study may pave the way for designing new rational and efficient strategies to treat Alzheimer’s and other neurodegenerative diseases. Overall design: To determine the phenotypic heterogeneity and the transcriptional dynamics of microglia cells during the progression of neurodegeneration, we used the CK-p25 mouse model of severe neurodegeneration which develops AD-like pathology in an inducible and temporally predictable manner. We isolated cells expressing the microglia markers CD11b and CD45 from the hippocampus of three to four CK-p25 animals and three CK control littermates at each of the following four time points during the progression of neurodegeneration: before p25 induction (0 weeks), 1 week, 2 weeks, and 6 weeks after p25 induction. Single microglial cells (expressing the cell surface markers CD11b and CD45) were sorted directly into RNA lysis buffer in 96-well plates using Fluorescence-Activated Cell Sorting (FACS). We prepared a total of 2183 single-cell RNA-sequencing libraries using a modified version of the Smart-Seq2 protocol and sequenced the libraries to a depth of 251353 (median) mapped reads per cell.