Selective Neural Deletion of the Atg7 Gene Reduces Irradiation-Induced Cerebellar White Matter Injury in the Juvenile Mouse Brain by Ameliorating Oligodendrocyte Progenitor Cell Loss

Abstract

Radiotherapy is an effective tool for treating brain tumors, but irradiation-induced toxicity to the normal brain tissue remains a major problem. Here, we investigated if selective neural autophagy related gene 7 ( Atg7 ) deletion has a persistent effect on irradiation-induced juvenile mouse brain injury. Ten-day-old Atg7 knockout under a nestin promoter (KO) mice and wild-type (WT) littermates were subjected to a single dose of 6 Gy whole-brain irradiation. Cerebellar volume, cell proliferation, microglia activation, inflammation, and myelination were evaluated in the cerebellum at 5 days after irradiation. We found that 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 (OPC) loss in the white matter of the cerebellum, and Atg7 deficiency partly prevented this. The mRNA expression of oligodendrocyte and myelination-related genes ( Olig2 , Cldn11 , CNP , and MBP ) was higher in the cerebellum in Atg7 KO mice compared with WT littermates. The total cerebellar volume was significantly reduced after irradiation in both Atg7 KO and WT mice. Atg7 -deficient cerebellums were in a regenerative state before irradiation, as judged by the increased OPC-related and neurogenesis-related transcripts and the increased numbers of microglia; however, except for the OPC parameters these were the same in both genotypes after irradiation. Finally, there was no significant change in the number of astrocytes in the cerebellum after irradiation. These results suggest that selective neural Atg7 deficiency reduces irradiation-induced cerebellar white matter injury in the juvenile mouse brain, secondary to prevention of OPC loss.

Autophagy inhibition reduces irradiation-induced subcortical white matter injury not by reducing inflammation, but by increasing mitochondrial fusion and inhibiting mitochondrial fission

“…In our previous studies, we demonstrated that selective Atg7 deletion prevents irradiation-induced caspase-3 activation, microglia activation, and in ammation and reduces irradiation-induced neural stem and progenitor cell death during the acute injury phase [8]. We also showed that selective inhibition of autophagy in neural cells reduces irradiation-induced subacute cerebellar white matter injury by decreasing OPC loss [7]. However, the mechanism through which autophagy de ciency prevents white matter injury after irradiation in the subacute injury phase is still unclear, and further research is needed.…”

“…Floxed Atg7 mice were characterized as previously described [6,7] and were crossed with a nestin-Credriven line to produce Atg7 ox/ ox; Nes−Cre knockout (Atg7 KO) and Atg7 ox/+; Nes−Cre mice (WT). All of the mice were housed in a temperature-controlled and pathogen-free environment with a 12:12-hour light-dark cycle.…”

“…Iba-1, PDGFRα, and GFAP-positive cells were counted in the subcortical white matter under a stereo microscope (MicroBrightField, Magdeburg, Germany). The counting area in the subcortical white matter was traced, and the number of cells was expressed as cells/mm 2 [7,8]. The counting was performed by a person who did not have prior knowledge of the groups.…”

“…The immunodensity of MBP-positive staining in the myelinated axons was determined using the ImageJ software and manually setting the threshold to include the MBP-stained area, followed by measuring the proportion of the eld that was positive for MBP staining in the subcortical white matter. The MBP immunodensity was determined by measuring the integrated density and normalizing it to the WT non-irradiation group [7,9]. RNA isolation, cDNA synthesis, and RNA sequencing Samples of subcortical white matter from WT and KO mice after irradiation at 5 days and from the nonirradiated group were prepared for RNA isolation and RNA sequencing.…”

“…As a marker of OPCs, we used PDGFRα staining to determine the extent of irradiation-induced white matter injury [7]. PDGFRα-positive cells were located mainly in the subcortical white matter (Fig.…”