Cranial irradiation affects cognitive function and the maturation of dendritic spine

(A) A scheme illustrating the schedule of ionizing radiation (IR) and overall behavioral tests. (B) The results of spontaneous alteration (left) and spatial memory test (right) after IR using Y-maze. The parameters of spontaneous alteration and spatial memory are described in methods. (C) The result of novel object recognition test after IR. (D) Left: representative fluorescence image of filamentous actin (F-actin, green) after IR in cortical neuron. Right: The density and distribution of dendritic spine in IR group compared to control group. Scale bars, 10 μm. (E) The alterations of F/G-actin ratio after IR in cortical neuron, frontal cortex, and hippocampus. Statistical analysis was performed with Student’s t test for (B, left of C, and E) and one-way ANOVA plus a Tukey’s multiple comparisons test for (right of C and D). ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

IR decreases phosphorylation of LIMK1 and Cofilin via PAK3 downregulation

(A) The heatmap for the expression of genes in the term ‘‘synapse organization’’ and “dendritic spine development” in the irradiated brain. (B) The changes of PAK3 mRNA and protein levels after IR in cortical neuron. Each western blot bands are quantified by ImageJ. (C) Representative fluorescence image of F-actin (green) and PAK3 (red) in cortical neuron. Scale bars, 10 μm. (D) Schematic illustration of PAK3-LIMK1-cofilin signaling. (E) Left: the protein levels of phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after IR in cortical neuron. Right: each western blot bands are quantified by ImageJ. (F-G) Representative fluorescence image of F-actin (green) and phosphorylated LIMK1 (F, red) or phosphorylated cofilin (G, red) in cortical neuron. Scale bars, 10 μm. (H) Left: the protein levels of phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after IR in frontal cortex and hippocampus. Right: each western blot bands are quantified by ImageJ. Statistical analysis was performed with Student’s t test. ns, non-significant; **p < 0.01; ***p < 0.001; ****p < 0.0001.

PAK3 regulates F/G-actin and dendritic spine in neuron

(A) The changes of PAK3 mRNA levels after PAK3 downregulation (shPAK3) in cortical neuron. (B) The F/G-actin ratio after expression of PAK3 downregulation (shPAK3) in cortical neuron. Each F-actin and G-actin are quantified by ImageJ. (C) The protein levels of phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after PAK3 downregulation (shPAK3) in cortical neuron. (D) Upper: representative fluorescence image of filamentous actin (F-actin, green) after PAK3 downregulation (shPAK3) in cortical neuron. Lower: the density and distribution of dendritic spine in shPAK3 group compared to control group. Scale bars, 10 μm. (E) The protein levels of PAK3, phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after PAK3 overexpression in cortical neuron. (F) The F/G-actin ratio after IR or IR with PAK3 overexpression in cortical neuron. Each F-actin and G-actin are quantified by ImageJ. (G) Upper: representative fluorescence image of filamentous actin (F-actin, green) after IR and/or PAK3 overexpression in cortical neuron. Lower: the density and distribution of dendritic spine in control, IR and/or PAK3 overexpression group. Scale bars, 10 μm. Statistical analysis was performed with Student’s t test for (B and D) and one-way ANOVA plus a Tukey’s multiple comparisons test for (A, D, F, and G). ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

IR decreases PAK3-LIMK1-Cofilin signaling in differentiated human neuron

(A) The F/G-actin ratio after IR in differentiated neuron. Each F-actin and G-actin are quantified by ImageJ. (B) The mRNA level of PAK3 after IR in differentiated neuron. (C) The protein levels of PAK3, phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after IR in differentiated neuron. (D) Upper: representative fluorescence image of filamentous actin (F-actin, green) after IR in differentiated neuron. Lower: the density and distribution of dendritic spine in IR group compared to control group. Scale bars, 10 μm. (E) Upper: representative fluorescence image of filamentous actin (F-actin, green) after PAK3 downregulation (shPAK3) in differentiated neuron. Lower: the density and distribution of dendritic spine in shPAK3 group compared to control group. Scale bars, 10 μm. Statistical analysis was performed with Student’s t test for (A, B, D, and E) and one-way ANOVA plus a Tukey’s multiple comparisons test for (D and E). ns, non-significant; **p < 0.01; ***p < 0.001; ****p < 0.0001.

IR-induced miR-206-3p affects PAK3-LIMK1-cofilin signaling

(A) The relative luciferase activity of PAK3 promoter after IR in cortical neuron. (B) Venn diagram of predictable PAK3-targeting miRNAs from databases (miRDB, TargetScan, and TarBase). (C) The relative miRNA expression of 3 overlapped miRNAs after IR in cortical neuron and differentiated neuron. (D) The relative miRNA expression of miR-206-3p after IR and/or antagomiR-206-3p treatment in cortical neuron and differentiated neuron. (E) The mRNA level of PAK3 after IR and/or treatment of antagomiR-206-3p in cortical neuron and differentiated neuron. (F) The protein levels of PAK3, phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after treatment of miR-206-3p mimic in differentiated neuron. (G) The protein levels of PAK3, phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after IR and/or treatment of antagomiR-206-3p in differentiated neuron. (H) Upper: representative fluorescence image of filamentous actin (F-actin, green) after IR and/or treatment of antagomiR-206-3p in cortical neuron. Lower: the density and distribution of dendritic spine in control, IR and/or treatment of antagomiR-206-3p group. Scale bars, 10 μm. Statistical analysis was performed with Student’s t test for A and one-way ANOVA plus a Tukey’s multiple comparisons test for the others. ns, non-significant; ***p < 0.001; ****p < 0.0001.

Intranasal administration of antagomiR-206-3p recovers PAK3 signaling and cognitive impairment after IR

(A) A scheme illustrating the schedule of IR and antagomiR-206-3p treatment and overall behavioral tests. (B) Schematic illustration of antagomir-206-3p delivery via intranasal administration. (C) The fluorescence images and relative units of cy5-antagomiR-206-3p in the brain of mice 24 h after intranasal administration. (D) The relative miRNA expression of miR-206-3p after IR and/or antagomiR-206-3p treatment in frontal cortex and hippocampus. (E) The results of spontaneous alteration (left) and spatial memory test (right) after IR and/or antagomiR-206-3p using Y-maze. The parameters of spontaneous alteration are described in methods. (F) The result of novel object recognition test after IR and/or antagomiR-206-3p. (G) The protein levels of PAK3, phosphorylated LIMK1, LIMK1, phosphorylated cofilin, and cofilin after IR and/or treatment of antagomiR-206-3p in frontal cortex and hippocampus. (H) The alterations of F/G-actin ratio after IR and/or treatment of antagomiR-206-3p in frontal cortex and hippocampus. Statistical analysis was performed with one-way ANOVA plus a Tukey’s multiple comparisons test. ns, non-significant; **p < 0.01; ***p < 0.001; ****p < 0.0001.