Moesin KO neurons decreased cell viability and the expression of neuronal cell markers
To examine the involvement of moesin in neuronal cell viability and morphology, primary cortical neurons from moesin KO mouse pups were harvested, cell viability was checked, and stained with each antibody. Moesin was stained in the cell body and neurites in the neuronal cells of WT mice but not in the KO mice (Fig. 1B). Next, we examined the expression of Sox2 and DCX as neural stem cell markers and MAP2 as differentiation markers by immunohistochemistry. The intensity of MAP2 (WT, 109 ± 4.9; KO, 43 ± 12.7, p < 0.01), Sox2 (WT, 103.5 ± 9.2; KO, 54.9 ± 6.07, p < 0.01), and GFAP (WT, 103.9 ± 5.9; KO, 41.1 ± 4.7, p < 0.01) was significantly reduced in the cortical neurons of moesin KO mice compared to that of WT mice (Fig. 1B and D). In moesin-deleted neurons, cell viability decreased by 40% (WT, 95.8 ± 11.76; KO, 48.01 ± 8.92, p < 0.001). Moesin KO neurons significantly decreased Sox2 (WT, 100.33 ± 16.77; KO, 31.67 ± 4.04, p < 0.01), DCX (WT, 100.33 ± 4.93; KO, 88.01 ± 4.36, p < 0.05), and MAP2 (WT, 100.34 ± 24.70; KO, 19.67 ± 3.51, p < 0.01) expression on DIV7 neurons, and the expression of GFAP (WT, 97.67 ± 37.29; KO, 0.33 ± 0.01, p < 0.01) as a glial cell marker was also decreased in the moesin KO neurons (Fig. 1E). Moreover, moesin KO significantly decreased SNAP25 (WT, 99.67 ± 2.89; KO, 39.33 ± 8.50, p < 0.001) as a presynaptic protein and PSD95 (WT, 99.68 ± 12.09; KO, 67.33 ± 4.01, < 0.01) as a postsynaptic protein (Fig. 1E).
At 14 days after seeding the cortical neurons, moesin KO neurons significantly decreased Sox2 (WT, 100.01 ± 24.43; KO, 39.01 ± 19.47, p < 0.05), DCX (WT, 100.14 ± 8.19; KO, 57.67 ± 7.77, p < 0.01), PSD95 (WT, 99.67 ± 25.51; KO, 54.12 ± 14.01, p < 0.05) and GFAP (WT, 100.01 ± 10.44; KO, 13.67 ± 8.50, p < 0.001) but not MAP2 (WT, 103.01 ± 6.08; KO, 56.04 ± 44.27) and SNAP25 (WT, 100.06 ± 1.07; KO, 89.67 ± 10.97) (Fig. 1F).
Moesin Ko Mice Show Abnormal Behaviors
To observe whether there are developmental disorders in moesin-deficient mice, surface righting, negative geotaxis, and cliff avoidance were performed on 7-day-old mice. Male moesin KO pups showed a significant decrease in scores in the surface righting test (WT, 1.8 ± 0.2; KO, 0.8 ± 0.37, p < 0.05) and in the cliff avoidance test on PND 7 (WT, 2 ± 0; KO, 1.4 ± 0.24, p < 0.05) but not in the negative geotaxis test (WT, 2 ± 0; KO, 2 ± 0) (Fig. 2A).
Next, Y-maze and passive avoidance tests were performed on mice between 3 and 5 months of age to observe whether there was memory impairment in moesin KO mice. In the Y maze test, moesin KO exhibited a lower spontaneous alteration rate compared to that of WT mice (Fig. 2B, WT, 69.60 ± 1.468; KO, 59.83 ± 2.426, p < 0.05) and lower total entry number (Fig. 1B, WT, 34.8 ± 3.07; KO, 26 ± 0.96, p < 0.01). In the passive avoidance test, there was no significant difference between WT and KO mice until day 2, but on days 3 and 4, the latency time for WT to enter the dark room was significantly longer than that of KO (Fig. 2C, WT, 269.2 ± 24.81; KO, 37.9 ± 21.13, p < 0.001, WT, 209.9 ± 52.76; KO, 37.7 ± 13.15, p < 0.05). An open-field test was performed to measure anxiety status. KO mice showed a significant decrease in the total distance (WT, 31.841 ± 3.00; KO, 22.937 ± 2.33, p < 0.05), center distance (WT, 2.587 ± 0.32; KO, 1.246 ± .191, p < 0.01), the number of entries in the center (WT, 33.5 ± 4.41; KO, 15.8 ± 2.23, p < 0.01), and time in the center (WT, 82.6 ± 20.25; KO, 24.9 ± 5.15, p < 0.01) compared to WT mice (Fig. 2D, E), suggesting increased anxiety-like behavior.
Moesin KO decreased the expression of synaptic proteins and phosphorylation of signaling proteins in the adult brain.
To examine the downstream targets of moesin, the expression or activation levels of synaptic vesicle proteins were confirmed by immunoblotting in the cortex and hippocampus. First, the amount of radixin, an ERM family member, was observed, and there was no difference in the expression in cells that could not express moesin (Fig. 3A). Previously, we reported that clozapine-induced chloride channel 4 and moesin activation modulates CDK5 expression [16]. Thus, we examined CDK5 expression in the hippocampus and cortex of moesin-KO mice. The expression level of CDK5 was meaningfully suppressed by moesin deletion in both neuronal tissues (cortex, p < 0.01; hippocampus, p < 0.05; Fig. 3A). Moreover, synaptic proteins, such as syntaxin 1A, synaptophysin, MUNC18, and synapsin I, were examined in the hippocampus and cortex of moesin KO mice. Syntaxin 1A expression was reduced in the brain (cortex, p < 0.01; hippocampus, p < 0.05), but synaptophysin, MUNC18, and synapsin I were not (Fig. 3A & B). However, the phosphorylation levels of synapsin I (S605, S549, and S9) and mammalian uncoordinated (MUNC) 18 were significantly decreased in the moesin KO brain (Fig. 3C). PSD95 expression was decreased in the cortex of adult mice, but not in the hippocampus; however, phosphorylation was reduced in the hippocampus but not in the cortex (Fig. 3E). The activation levels of signaling molecules, such as ERK and CREB, were checked using phospho-specific antibodies. The expression levels of ERK and CREB were not changed in the moesin KO brain, Still, phosphorylation was significantly decreased in the brain of moesin KO mice compared to WT mice (Fig. 3E).
Risperidone Reversed Behavioral And Molecular Deficits In Moesin Ko Mice
Next, we tested whether the antipsychotic drug risperidone reversed cognitive impairment in moesin-KO mice. Risperidone was administered for 2 weeks according to a schedule and validated by behavioral experiments. Risperidone reversed the cognitive impairment of moesin KO mice in the passive avoidance test (WT versus KO p < 0.001; KO versus KO + Ris, p < 0.05, Fig. 4A), total entry number (WT versus KO, p < 0.001; KO versus KO + Ris, p < 0.001, Fig. 4B, left panel), and spontaneous alternation percentage (WT versus KO, p < 0.001; KO versus KO + Ris, p < 0.001, Fig. 4B, right panel).
Risperidone reversed the reduction in the expression of PSD95 (WT, 97.33 ± 3.70; KO, 75.0 ± 3.0; KO + R, 104.0 ± 6.0; p < 0.01). The phosphorylation of synapsin at Ser605 and Ser549 in the cortex was also restored in moesin KO mice by risperidone administration (WT, 100.33 ± 1.53; KO, 68.0 ± 10.0; KO + Ris, 110.0 ± 10.0; p < 0.05), and ERK phosphorylation was increased in risperidone-treated moesin KO mice compared to that of WT and untreated moesin KO mice (WT, 106.77 ± 10.02; KO, 75.0 ± 3.0; KO + Ris, 135.0 ± 10.0; p < 0.05; Fig. 4C and D).