UV irradiation to mouse skin decreases hippocampal neurogenesis and synaptic protein expression via HPA axis activation

Abstract

The skin senses external environment, including ultraviolet light (UV). Hippocampus is a brain region that is responsible for memory and emotion. However, changes in hippocampus by UV irradiation to the skin have not been studied. In this study, after 2 weeks of UV irradiation to the mouse skin, we examined molecular changes related to cognitive functions in the hippocampus and activation of the hypothalamic-pituitary-adrenal (HPA) axis. UV exposure to the skin decreased doublecortin-positive immature neurons and synaptic proteins, including N-methyl-D-aspartate receptor 2 A and postsynaptic density protein-95, in the hippocampus. Moreover, we observed that UV irradiation to the skin down-regulated brain-derived neurotrophic factor expression and ERK signaling in the hippocampus, which are known to modulate neurogenesis and synaptic plasticity. The cutaneous and central HPA axes were activated by UV, which resulted in significant increases in serum levels of corticosterone. Subsequently, UV irradiation to the skin activated the glucocorticoid-signaling pathway in the hippocampal dentate gyrus. Interestingly, after 6 weeks of UV irradiation, mice showed depression-like behavior in the tail suspension test. Taken together, our data suggest that repeated UV exposure through the skin may negatively affect hippocampal neurogenesis and synaptic plasticity along with HPA axis activation.

Figure 1

UV irradiation of mouse skin decreased adult hippocampal neurogenesis. Representative immunohistochemical images of (a) DCX-positive and (b) Ki-67-positive cells in the DG. DCX- and Ki-67-positive cells were counted in 6 hippocampal sections from each mouse and in 8 mice from each group, and mean values were calculated. (c) The protein expression levels of DCX, Bcl-2, and cleaved caspase-3 were assessed in hippocampal lysates using Western blot. Relative band density was analyzed using Image J software. Tubulin was used as the endogenous control. The blots are cropped raw images are provided in Supplementary Figure S2. Scale bars: 100 µm. Graphs show means ± SEM (n = 8 mice/group). *P < 0.05 and ***P < 0.001 indicate significant differences when compared to the control group. Bcl-2, B-cell lymphoma 2; C.caspase-3, cleaved caspase-3; DCX, doublecortin; DG, dentate gyrus.

Figure 2

The expression levels of synaptic proteins NMDAR2A and PSD-95 were reduced by UV irradiation. (a) Representative western blot analysis results, and (b) density graphs of NMDAR2A, NMDAR2B, GluA1, GluA2, PSD-95, and SYP in the DG. Relative band density was analyzed using Image J software and normalized by tubulin. The blots are cropped raw images are provided in Supplementary Figure S3. Graphs show means ± SEM (n = 8 mice/group). *P < 0.05 and ***P < 0.001 indicate significant differences when compared to the control group. GluA1, glutamate receptor 1; GluA2, glutamate receptor 2, NMDAR2A, N-methyl-D-aspartate receptor 2 A; NMDAR2B, N-methyl-D-aspartate receptor 2B; PSD-95, post-synaptic density protein 95; SYP, synaptophysin.

Figure 3

UV irradiation suppressed the expression of BDNF and phosphorylated ERK in the hippocampus. (a) Neurotropic factors (BDNF, NGF) and growth factors (VEGF, IGF-1, and FGF-2) mRNA expression levels in the hippocampus were analyzed using RT-qPCR. GAPDH was used to normalize the mRNA expression levels. Representative Western blot images and density graphs of (b) BDNF, p-ERK, and t-ERK in the hippocampus. (c) Relative band density was analyzed using image J software. Tubulin and t-ERK were used as endogenous controls, respectively. The blots are cropped raw images are provided in Supplementary Figure S4. Graphs show means ± SEM (n = 8mice/group). *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences when compared to control group. BDNF, brain-derived neurotrophic factor; FGF-2, fibroblast growth factor 2; IGF-1, insulin-like growth factor 1; NGF, nerve growth factor; p-ERK, phosphorylated extracellular signal-related kinase; t-ERK, total extracellular signal-related kinase VEGF, vascular endothelial growth factor.

Figure 4

UV irradiation stimulated both central and cutaneous HPA axes. Serum levels of (a) CORT and (c) ACTH were measured using ELISA assay. (b) MC2R, STAR, and CYP11B1 mRNA levels in the adrenal glands, and (d) CRH mRNA expression levels in the hypothalamus were measured using RT-qPCR. (e) UCN and POMC expression levels in mouse skin with or without ultraviolet radiation exposure were examined using immunohistochemistry. Scale bars: 50 µm under 400x magnification. (f) The mRNA expression levels of UCN, POMC, MC2R, and CYP11B1 in the skin were analyzed using RT-qPCR. All relative mRNA levels were normalized to those of the endogenous control GAPDH. Graphs show means ± SEM (n = 8mice/group). *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences between groups. ACTH, adrenocorticotropic hormone; CORT, corticosterone; CRH, corticotropin-releasing hormone; CYP11B1, cytochrome P450 family 11 subfamily B member 1; MC2R, melanocortin 2 receptor; POMC, pro-opiomelanocortin; STAR, steroidogenic acute regulatory protein; UCN, urocortin.

Figure 5

UV irradiation of mouse skin induced nuclear translocation of GR and modulated the expression levels of its target genes. (a) Representative immunohistochemical images of GR-positive cells under 200x (upper panel) and high magnification (lower panel) of insert area in the hippocampus. Scale bars: 100 µm and 50 µm, respectively. The graphs show (b) GR-positive cell numbers counted in 8 brain sections from each mouse. (c) Relative GR, ARRB2, and TTP mRNA expression levels in the hippocampus were analyzed using RT-qPCR and normalized to GAPDH expression levels. Graphs show means ± SEM (n = 8mice/group). *P < 0.05 and ***P < 0.001 indicate significant differences between groups. ARRB2, arrestin beta 2; GR, glucocorticoid receptor; TTP, tristetraproline.

Figure 6

UV irradiation of mouse skin results in depression-like behavior. To assess a depression-like phenotype (immobility) in UV-irradiated mice, we performed the tail suspension test. UV light was applied to the dorsal skin under anesthesia 3 days a week for 2 or 6 weeks, and the immobility time was measured during the 6 min test session and compared with that of sham-irradiated mice. Graphs show means ± SEM (n = 8 mice/group). *P < 0.05, indicate significant differences compared with control group values. Cont., Sham-UV irradiated group; 2w, 2 weeks UV-irradiated group; 6w, 6 weeks UV-irradiated group.