doi: 10.1016/j.gendis.2023.101178.
eCollection 2024 Nov.
Loss of homeostatic functions in microglia from a murine model of Friedreich's ataxia
Affiliations
- PMID: 39100202
- PMCID: PMC11295442
- DOI: 10.1016/j.gendis.2023.101178
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Loss of homeostatic functions in microglia from a murine model of Friedreich's ataxia
Genes Dis.
.
No abstract available
Conflict of interest statement
The authors declare no competing financial interests.
Figures
Loss of homeostatic functions in microglia from a murine model of Friedreich's ataxia. (A) Representative images of microglia from WT and KIKO mice stained with CD11b (red), phalloidin (green), and DAPI (blue). The transformation index is calculated as (perimeter)/4π × area. Scale bar = 20 μm. The data represent mean ± standard error of the mean (SEM); n = 3 independent experiments. Statistical significance was calculated by t-test; ∗P < 0.05, ∗∗P < 0.01. (B) In the migration assay, the number of cells migrated into the gap was counted after 24 and 48 h and reported as a percentage of the number of WT cells. (C) Representative fluorescence images showing WT and KIKO microglia stained with phalloidin (green) after incubation with fluorescent latex beads (red, merged signals in yellow). The percentage of phagocytic cells/total cells and the number of beads/cells were calculated. Scale bar = 20 μm. In (B,C), the data represent mean ± SEM; n = 3 independent experiments, n = 10 fields/experiment. Statistical significance was calculated by t-test; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (D) The volcano plot of genes showing the magnitude (log2 (fold change/FC), x-axis) and significance (−log10 (P value), y-axis) for KIKO compared with WT microglia (n = 4/group). Differentially expressed (DE) genes are marked in red (up-regulated genes) or green (down-regulated genes). Gene names of the top 10 DE genes are shown. (E) Hierarchical clustering heatmap of DE genes with P value < 0.01 and FC > 1.3 of KIKO microglia compared with WT microglia. (F) Gene Ontology analysis of DE genes between KIKO and WT mice microglia. DE genes with P value < 0.01 and FC > 1.3 were analyzed by the Enrichr analysis tool. The top 12 enriched terms for Gene Ontology analysis are displayed based on decreasing −log10 (P value). BP, biological process. (G) Real time PCR for Il1B, Cd68, and Cybb in WT and KIKO microglia. The data were normalized to Actb and expressed as mean ± SEM of 3 (n) experiments performed in triplicate. (H) Representative western blots and quantification of CD68 and gp91phox in WT and KIKO microglia. GAPDH was used as a loading control. (I) Representative images of dihydroethidium (DHE)-stained WT and KIKO microglia and relative DHE positive cell quantification. Scale bar = 20 μm. Representative western blots and quantification of P2Y12, arginase 1 (ARG1), ferritin (J), and C-X-C chemokine receptor type 3 (CXCR3) (K) in WT and KIKO microglia. GAPDH was used as a loading control. In (H–K), the values were expressed as mean ± SEM; n = 3 independent experiments. Statistical significance was calculated by t-test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001. (L) Quantification of the flow cytometry analysis of mitochondrial mass, using the Mito Tracker Green (MTG) fluorescent dye. (M) Measurement of the rate of oxygen consumption rate (OCR) in WT and KIKO microglia. Individual parameters for maximal respiration and spare respiratory capacity are indicated. (N) Glycolysis, glycolytic capacity, and non-glycolytic acidification were analyzed in WT and KIKO microglia. In (L–N), the data were presented as mean ± SEM; n = 4 independent experiments. Statistical significance was calculated by t-test; ∗P < 0.05. (O) Representative images of neurons grown in microglial conditioned medium derived from WT microglia (WT_MCM) and from KIKO microglia (KIKO_MCM), after labeling neurons with anti-β-tubulin III (green). Cell survival and length and number of neurites per cell were analyzed using the ImageJ software NeuronJ plug-in. The data represent mean ± SEM of 3 (n) independent experiments performed in quadruplicate. Statistical significance was calculated by t-test; ∗∗∗P < 0.001, ∗P < 0.05. (P) Real time PCR for P2ry12, Trem2, and Cx3cr1 mRNA in the cerebellum of WT and KIKO mice at postnatal day 15. The data were normalized to Actb and expressed as mean ± SEM of 3 (n) independent experiments performed in triplicate. (Q) Representative confocal images of cerebellar sections from WT and KIKO mice at postnatal day 15 immunostained for P2Y12 (green) and calbindin (red). DAPI was used for nuclei staining. Scale bar = 100 μm. GL, granular layer; ML, molecular layer; PCL, Purkinje cell layer. P2Y12 signal quantification was performed by ImageJ software. (R) Example of original, binary, and skeletonized microglia. Analysis of the microglial branches, average branch length, and triple points of microglia (junctions with exactly three branches). In (Q,R), the values were expressed as mean ± SEM; n = 3 mice/group; at least 3 sections for mice. Statistical significance was calculated by t-test; ∗P < 0.05.
References
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- Khan W., Corben L.A., Bilal H., et al. Neuroinflammation in the cerebellum and brainstem in Friedreich ataxia: An [18F]-FEMPA PET study. Mov Disord. 2022;37(1):218–224. - PubMed
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