Asparagine endopeptidase deficiency mitigates radiation-induced brain injury by suppressing microglia-mediated neuronal senescence

Summary Mounting evidence supports the role of neuroinflammation in radiation-induced brain injury (RIBI), a chronic disease characterized by delayed and progressive neurological impairment. Asparagine endopeptidase (AEP), also known as legumain (LGMN), participates in multiple malignancies and neurodegenerative diseases and may potentially be involved in RIBI. Here, we found AEP expression was substantially elevated in the cortex and hippocampus of wild-type (Lgmn+/+) mice following whole-brain irradiation. Lgmn knockout (Lgmn−/−) alleviated neurological impairment caused by whole-brain irradiation by suppressing neuronal senescence. Bulk RNA and metabolomic sequencing revealed AEP’s involvement in the antigen processing and presentation pathway and neuroinflammation. This was further confirmed by co-culturing Lgmn+/+ primary neurons with the conditioned media derived from irradiated Lgmn+/+ or Lgmn−/− primary microglia. Furthermore, esomeprazole inhibited the enzymatic activity of AEP and RIBI. These findings identified AEP as a critical factor of neuroinflammation in RIBI, highlighting the prospect of targeting AEP as a therapeutic approach.

(E and F) T1-weight MRI scan with contrast of the brain of Lgmn +/+ -Sham, Lgmn +/+ -IR and Lgmn - /--IR mice (E) and the quantification of MRI signal intensity of the cortex (F) at 8 weeks following whole-brain irradiation (scale bar = 1 mm, n = 5 per group; F value of genetic type = 18.15;F value of irradiation = 64.00).
LGMN = legumain.Inability to walk on a 3-cm-wide beam 1 Inability to walk on a 2-cm-wide beam 1 Inability to walk on a 1-cm-wide beam 1 Inability to balance on a 0.7-cm-wide beam 1 Inability to balance on a 0.5-cm-wide round stick 1 Failure to exit a 30-cm-diameter circle in 2 minutes 1 Inability to walk straight 1

Loss of startle behavior 1
Loss of seeking behavior 1 The maximum total score 10

Figure S2 .
Figure S2.AEP expression was not elevated in astrocytes in the cortex following whole-brain irradiation, related to Figure 1 (A and B) Whole-brain section images (A, scale bar = 1 mm) and localized cortical images (B, scale bar = 20 μm) of immunofluorescence analysis of AEP and Sox9 following whole-brain irradiation.AEP = asparagine endopeptidase; LGMN = legumain; IR = irradiation.

Figure S5 .
Figure S5.The hippocampal neurological impairment following whole-brain irradiation was partially reduced by Lgmn knockout, related to Figure 3 (A) Open field tests of Lgmn +/+ -Sham, Lgmn +/+ -IR, Lgmn -/--Sham and Lgmn -/--IR mice (n =6 per group).The left panel is the total distance moved in the whole field, the middle panel is the distance moved in the center, and the right panel is the time spent in the center (F value of genetic type = 2.38, 16.36 and 9.81 in the left, middle and right panel respectively; F value of irradiation = 15.01,21.89 and 14.09 in the left, middle and right panel respectively).(B) Novel object recognition tests of Lgmn +/+ -Sham, Lgmn +/+ -IR, Lgmn -/--Sham and Lgmn -/--IR mice (n = 6 per group).The left panel is the percentage of the time spent exploring the novel object divided by the time spent exploring both objects, and the right panel is the percentage of the time

Figure S6 .
Figure S6.Heatmap of the bulk RNA sequencing data, related to Figure 5

Figure S7 .
Figure S7.Enzymatic activity assay of AEP, related to Figure 7