Rapid and robust isolation of microglia and vascular cells from brain subregions for integrative single-cell analyses.

Cell isolation protocols from brain tissue include prolonged ex vivo processing durations, rendering them suboptimal for transcriptomic studies. Particularly for microglia and vascular cells, current isolation methods produce lower yields, necessitating addition of an enrichment step, and use of large tissue volumes - in most cases whole brain tissue - to obtain sufficient yields. Here, we developed a simple, rapid, and reproducible cell isolation method for generating single-cell suspensions from micro-dissected brain regions, enriched for microglia and vascular cells, without an enrichment step. Cells isolated using this method are suitable for molecular profiling studies using 10 × Genomics Chromium single-cell RNA sequencing with high reproducibility. Our method is valuable for longitudinal unbiased molecular profiling of microglia and vascular cells within different brain regions, spanning multiple time points across physiological development or disease progression.

EDA2R reflects the acute brain response to cranial irradiation in liquid biopsies.

BACKGROUND: Cranial radiotherapy is standard of care for high-grade brain tumors and metastases; however, it induces debilitating neurocognitive impairments in cancer survivors, especially children. As the numbers of pediatric brain cancer survivors continue improving, the numbers of individuals developing life-long neurocognitive sequalae are consequently expected to rise. Yet, there are no established biomarkers estimating the degree of the irradiation-induced brain injury at completion of radiotherapy to predict the severity of the expected neurocognitive complications. We aimed to identify sensitive biomarkers associated with brain response to irradiation that can be measured in easily accessible clinical materials, such as liquid biopsies. METHODS: Juvenile mice were subjected to cranial irradiation with 0.5, 1, 2, 4, and 8 Gy. Cerebrospinal fluid (CSF), plasma, and brains were collected at acute, subacute, and subchronic phases after irradiation, and processed for proteomic screens, and molecular and histological analyses. RESULTS: We found that the levels of ectodysplasin A2 receptor (EDA2R), member of tumor necrosis factor receptor superfamily, increased significantly in the CSF after cranial irradiation, even at lower irradiation doses. The levels of EDA2R were increased globally in the brain acutely after irradiation and decreased over time. EDA2R was predominantly expressed by neurons, and the temporal dynamics of EDA2R in the brain was reflected in the plasma samples. CONCLUSIONS: We propose EDA2R as a promising potential biomarker reflecting irradiation-induced brain injury in liquid biopsies. The levels of EDA2R upon completion of radiotherapy may aid in predicting the severity of IR-induced neurocognitive sequalae at a very early stage after treatment.

ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain.

Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1+ microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1+ microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1- microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.