Loss of C/EBPδ Exacerbates Radiation-Induced Cognitive Decline in Aged Mice due to Impaired Oxidative Stress Response

Abstract

Aging is characterized by increased inflammation and deterioration of the cellular stress responses such as the oxidant/antioxidant equilibrium, DNA damage repair fidelity, and telomeric attrition. All these factors contribute to the increased radiation sensitivity in the elderly as shown by epidemiological studies of the Japanese atomic bomb survivors. There is a global increase in the aging population, who may be at increased risk of exposure to ionizing radiation (IR) as part of cancer therapy or accidental exposure. Therefore, it is critical to delineate the factors that exacerbate age-related radiation sensitivity and neurocognitive decline. The transcription factor CCAAT enhancer binding protein delta (C/EBPδ) is implicated with regulatory roles in neuroinflammation, learning, and memory, however its role in IR-induced neurocognitive decline and aging is not known. The purpose of this study was to delineate the role of C/EBPδ in IR-induced neurocognitive decline in aged mice. We report that aged Cebpd^-/- mice exposed to acute IR exposure display impairment in short-term memory and spatial memory that correlated with significant alterations in the morphology of neurons in the dentate gyrus (DG) and CA1 apical and basal regions. There were no significant changes in the expression of inflammatory markers. However, the expression of superoxide dismutase 2 (SOD2) and catalase (CAT) were altered post-IR in the hippocampus of aged Cebpd^-/- mice. These results suggest that Cebpd may protect from IR-induced neurocognitive dysfunction by suppressing oxidative stress in aged mice.

Keywords: C/EBPδ; Cebpd; behavior; hippocampus; ionizing radiation; novel object recognition; oxidative stress; reactive oxygen species; short-term memory; spatial learning.

Figure 1

Short-term memory analyzed by Y-maze test in sham and irradiated aged Cebpd^+/+ and Cebpd^−/− mice. (A–C) Cebpd^+/+-sham, Cebpd^+/+-IR, and Cebpd^−/−-sham mice were able to successfully distinguish the novel arm, by spending significantly more time exploring it. (D) Cebpd^−/−-IR mice were not able to distinguish between the three Y-maze arms, and spent an approximately equal time exploring all arms failing to recognize the novel environment when exposed to it 4 h later. N = 5/7 mice/treatment, Average ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

NOR of sham-irradiated and irradiated aged Cebpd^+/+ and Cebpd^−/− mice. (A) Cebpd^+/+-sham, (B) Cebpd^+/+-IR, and (C) Cebpd^−/−-sham irradiated mice showed novel object recognition and spent more time exploring the novel than the familiar object. However, Cebpd^−/−-IR (D) mice did not show any preference for the novel object. N = 5/7 mice/treatment. Average ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Sholl analyses of neurons in the dentate gyrus. (A) Dendritic length, measured by Sholl analysis, radiation greatly decreased length at 90–100 µm from the soma when Cebpd^−/−-sham were compared to Cebpd^−/−-IR. (B) Treatment decreased length at 100–170 µm from the soma when Cebpd^+/+-IR were compared to Cebpd^−/−-IR. There were no significant differences observed when (C) Cebpd^+/+-sham were compared to Cebpd^−/−-sham or (D) Cebpd^+/+-sham were compared to Cebpd^+/+-IR. Average ± SEM (n = 5); * p < 0.05, ^† p < 0.01. ^‡ p < 0.001.

Figure 4

Sholl analyses of neurons in the CA1 apical. (A) Dendritic length, measured by Sholl analysis radiation decreased length at 80–160 µm from the soma when Cebpd^−/−-sham were compared to Cebpd^−/−-IR. (B) Treatment greatly decreased length at 80–160 µm from the soma when Cebpd^+/+-IR were compared to Cebpd^−/− -IR. There were no significant differences observed when (C) Cebpd^+/+-sham were compared to Cebpd^−/−-sham or (D) Cebpd^+/+-sham were compared to Cebpd^+/+-IR. Average ± SEM (n = 5) * p < 0.05, ^† p < 0.01. ^‡ p < 0.001.

Figure 5

Sholl analyses of neurons in the CA1 basal. (A) Dendritic length, measured by Sholl analysis radiation decreased length at 90–140 µm from the soma when Cebpd^+/+-sham were compared to Cebpd^+/+-IR. (B) There was a decrease in length at 90–140 µm from the soma when Cebpd^+/+-sham were compared to Cebpd^−/− -sham. (C) Radiation decreased length at 50–110 µm from the soma when Cebpd^+/+-IR were compared to Cebpd^−/−-IR. (D) Treatment greatly decreased length at 50–110 µm from the soma when Cebpd^−/−-sham were compared to Cebpd^−/−-IR. Average ± SEM (n = 5).

Figure 6

Expression of markers of inflammation and activated microglia in sham and irradiated Cebpd^+/+ and Cebpd^−/− hippocampal extracts of aged mice. Immunoblotting of TLR4 and CD68 normalized to β-actin used as a loading control, n = 4 mice per genotype per treatment.

Figure 7

Expression of antioxidant response proteins in hippocampal extracts of sham and irradiated Cebpd^+/+ and Cebpd^−/− mice. Immunoblotting of hippocampal extracts probed for NRF2, SOD2, CAT, γ-GCSm and β-actin, n = 4 mice per genotype per treatment. * p < 0.05; *** p < 0.001.