Journal: Nature Communications

Article Title: Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice

doi: 10.1038/s41467-022-33932-3

Figure Lengend Snippet: a Schemes of the in vitro astrocytic engulfment assay using pHrodo-labeled microglial debris and starvation-induced autophagy. Astrocytic LAPosomes in microglial debris engulfment ( b ) and autophagosomes in starvation-induced autophagy ( c ) in vitro. d Quantification of LAPosome and autophagosome diameters in vitro. N = 81 phagosomes from 4 to 5 mice of each group. Two-tailed independent t test. e Electron microscopy of microglial debris-containing LAPosomes in astrocytes cultured with debris. f Quantification of LAPosomes in astrocytes in vitro by electron microscopy. N = 4 (without debris) and 5 (with debris) mice of each group. Two-tailed independent t test. g Validation of RUBICON knockout and knockdown in primary cultured astrocytes by Western blotting. N = 3 independent biological replicates of each group. Two-tailed independent t test. h Rubcn knockout or knockdown prevents the formation of microglial debris-containing LAPosomes in vitro. The puncta of LC3 + autophagosomes are significantly increased in these knockout or knockdown cells. Each experiment is independently repeated using 4 mice with similar results. i , j RUBICON knockout or knockdown reduces the degradative product (neutral lipids) in astrocytes in vitro. N = 3 biological replicates of each group. Two-tailed independent t test. k Schemes of studying the in vivo consequences of RUBICON conditional knockout in astrocytes on the degradation of engulfed microglial debris in astrocytes. l , m RUBICON conditional knockout in astrocytes in vivo dampens the degradation of engulfed microglial debris and thus results in the accumulation of nondegraded debris in astrocytes. N = 5 and 6 ALDH1L1-CreER::Ai14 (WT) mice and ALDH1L1-CreER::Ai14::RUBICON fl/fl (RUBICON-cKO) mice, respectively. Two-tailed independent t test. WT wild-type, SCR scramble; KO RUBICON knockout, KD RUBICON knockdown, PLX5622 PLX5622-formulated AIN-76A diet, CD control AIN-76A diet, MFI mean fluorescence intensity. Data are presented as mean ± SD (bar plot) or median ± quartile (violin plot). The source data are provided as a Source Data file.

Article Snippet: siRNA oligonucleotides targeting mouse Rubcn (CTCAGAGTAACAGGACCTT) were synthesized by OBiO Technology (Shanghai).

Techniques: In Vitro, Labeling, Two Tailed Test, Electron Microscopy, Cell Culture, Biomarker Discovery, Knock-Out, Knockdown, Western Blot, In Vivo, Control, Fluorescence