Autophagy Inhibition Reduces Irradiation-Induced Subcortical White Matter Injury Not by Reducing Inflammation, but by Increasing Mitochondrial Fusion and Inhibiting Mitochondrial Fission

Abstract

Radiotherapy is an effective tool in the treatment of malignant brain tumors, but irradiation-induced late-onset toxicity remains a major problem. The purpose of this study was to investigate if genetic inhibition of autophagy has an impact on subcortical white matter development in the juvenile mouse brain after irradiation. Ten-day-old selective neural Atg7 knockout (KO) mice and wild-type (WT) littermates were subjected to a single 6-Gy dose of whole-brain irradiation and evaluated at 5 days after irradiation. Neural Atg7 deficiency partially prevented myelin disruption compared to the WT mice after irradiation, as indicated by myelin basic protein staining. Irradiation induced oligodendrocyte progenitor cell loss in the subcortical white matter, and Atg7 deficiency partly prevented this. There was no significant change between the KO and WT mice in the number of microglia and astrocytes in the subcortical white matter after irradiation. Transcriptome analysis showed that the GO mitochondrial gene expression pathway was significantly enriched in the differentially expressed genes between the KO and WT group after irradiation. Compared with WT mice, expression of the mitochondrial fusion protein OPA1 and phosphorylation of the mitochondrial fission protein DRP1 (P-DRP1) were dramatically decreased in KO mice under physiological conditions. The protein levels of OPA1and P-DRP1 showed no differences in WT mice between the non-irradiated group and the irradiated group but had remarkably increased levels in the KO mice after irradiation. These results indicate that inhibition of autophagy reduces irradiation-induced subcortical white matter injury not by reducing inflammation, but by increasing mitochondrial fusion and inhibiting mitochondrial fission.

Keywords: Astrocyte; Autophagy; Fusion and fission; Microglia; Mitochondria; White matter injury.

Fig. 1

Cerebral irradiation interrupts subcortical white matter development in young mice a Representative sagittal hemisphere MBP staining in WT-Sham and Atg7 KO-Sham and irradiated mouse pups. The red line represents the subcortical white matter. b The subcortical white matter volume assessed as the volume of MBP-positive staining. Atg7 KO protected against the irradiation-induced reduction in subcortical white matter. c Representative MBP staining in subcortical white matter and myelinated fibers. The red line represents myelinated fibers within the cortex. d The mean lengths of MBP-positive myelinated fibers between the end of myelinated axons and the cortical plate at fixed levels. e Representative MBP immunostaining in myelinated fibers within the cortex. f The MBP-positive immunodensity in myelinated fibers presented as the percentage of controls. n = 7/group for the immunostaining. *p < 0.05, **p < 0.01.

Fig. 2

Atg7 KO reduces irradiation-induced OPC loss in the subcortical white matter a Representative images of OPCs in the subcortical white matter after irradiation that were immunostained for PDGFRα in WT-Sham and Atg7 KO-Sham and irradiated mouse pups. b Quantitative analysis of the PDGFRα-labeled cells in the subcortical white matter. c Bar graphs showing the mRNA expression of Olig2, Cldn11, CNP, and MBP in the cortical tissue, including the subcortical white matter, at 5 days after irradiation. n = 7/group for the immunostaining, and n = 5/group for qRT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

Astrocyte and microglia changes in the subcortical white matter after irradiation a Representative images of GFAP-labeled cells in the subcortical white matter. b Quantification of GFAP-labeled cells in the subcortical white matter. c Representative Iba-1 immunostaining in the subcortical white matter. d Quantification of Iba-1–labeled cells in the subcortical white matter. e The protein levels of IL-1β, IL-2, IL-4, IL-6, IL-10, and KC in the cortical tissue at 5 days after irradiation as detected by Luminex assay in the Atg7 KO and WT pups. n = 7/group for the immunostaining and Luminex assay. *p < 0.05, ***p < 0.001.

Fig. 4

Irradiation induces transcriptome alterations in WT and Atg7 KO mice a Volcano plot showing DEGs between WT irradiated mice and WT non-irradiated mice in the subcortical white matter. b Volcano plot showing DEGs between KO irradiated mice and KO non-irradiated mice in the subcortical white matter. c Venn diagram showing the intersection of DEGs between Atg7 KO and WT mice after irradiation. d The graph shows the top eight classified GO terms in three ontologies. GO classification was performed based on the 1931 DEGs. The x-axis represents the number of DEGs, and the y-axis represents the GO terms. MF, molecular biological function; CC, cellular component; BP, biological process. n = 6/group for RNA sequencing.

Fig. 5

Irradiation induces mitochondria-related gene alterations a Heatmap showing the overall distribution of 1109 mitochondria-related genes. b GSEA analysis showed DEGs between WT-irradiated mice and KO-irradiated mice in the GO Mitochondrial gene expression pathway. The top part of each plot shows the enrichment score that represents running-sum statistics calculated by “walking down” the ranked list of genes. The middle part shows the position of a member of a gene set in the ranked list of genes. The bottom part depicts the ranking metric that measures a gene’s correlation with a biological function. c Correlation heatmap for mitochondria fusion and fission-related genes and oligodendrocyte and myelin-related genes between WT-irradiated mice and KO-irradiated mice. Some genes were negatively related and others were positively related as represented in different colors, with red representing positive correlation and blue representing negative correlation. The number in the correlation heatmap is the correlation coefficient.

Fig. 6

Effect of Atg7 deficiency on mitochondrial fission and fusion in the subcortical white matter after irradiation a Bar graphs showing mRNA expression of Opa1, Drp1, and Fis1 at 5 days after irradiation. b Representative immunoblots of mitochondrial fusion protein (OPA1), fission proteins (P-DRP1/DRP1 and Fis1), and mitophagy-related protein PINK1 in WT and KO mice under physiological conditions and after irradiation. c–g Quantification of OPA1, P-DRP1, DRP1, FIS1, and PINK1. n = 5/group for qRT-PCR, n = 6/group for immunoblotting. *p < 0.05, **p < 0.01.