CHI mouse serum pre-treatment led to Akt activation in iNSCs
After early CHI mouse serum treatment, we observed a dramatic increase in Crry expression in iNSCs [23, 24]. To explore the mechanism underlying this effect, we used RT-QPCR to determine the expression of the Erk, P38, Jnk and Akt genes in iNSCs among the PBS (iNSCs receiving PBS pre-treatment), HI-CHI (iNSCs receiving HI-CHI mouse serum pre-treatment), and CHI (iNSCs receiving CHI mouse serum pre-treatment) groups following treatment with CHI mouse serum (Fig. 1a-d). The levels of the Erk, P38 and Jnk genes in iNSCs were not significantly different among the three groups. However, Akt levels in iNSCs were substantially higher in the CHI group than in the other two groups (n=3/group, P<0.05).
Next, we performed a western blot analysis to detect the protein expression levels of p-Akt and Akt in iNSCs among the three groups after CHI mouse serum treatment (Fig. 1e-h). The levels of p-Akt, Akt and p-Akt/Akt in iNSCs were not significantly different between the PBS and HI-CHI groups. However, p-Akt, Akt and p-Akt/Akt levels in iNSCs of the CHI group were markedly higher than those in the other two groups (n=6/group, P<0.05). These data showed that the pre-treatment of iNSCs with CHI mouse serum led to Akt activation in iNSCs.
Akt inhibition reduced Crry expression in iNSCs pre-treated with CHI mouse serum
To examine the role of Akt signalling in mediating the immunoregulatory effect of iNSCs on complement activation, we utilized the Akt inhibitor LY294002 to pre-treat iNSCs (Fig. 2). Using western blot analysis, we observed that p-Akt, Akt and p-Akt/Akt levels in iNSCs of the CHI+LY294002 (iNSCs receiving CHI mouse serum and LY294002 pre-treatment) group were significantly higher than those in the PBS (iNSCs receiving PBS pre-treatment) group but substantially lower than those in the CHI (iNSCs receiving CHI mouse serum pre-treatment) group following treatment with CHI mouse serum (n=6/group, P<0.05). Furthermore, Crry levels in iNSCs of the CHI+LY294002 group were markedly higher than those in the PBS group but significantly lower than those in the CHI group after CHI mouse serum treatment (n=6/group, P<0.05). In contrast, the levels of C3d, C9 and active Caspase-3 in iNSCs of the CHI+LY294002 group were substantially lower than those in the PBS group but markedly higher than those in the CHI group following treatment with CHI mouse serum (n=6/group, P<0.05). These findings implied that the expression of Crry in iNSCs was positively associated with the level of Akt activation in iNSCs. Moreover, Akt inhibition clearly diminished the effect of the pre-treatment of iNSCs with CHI mouse serum on Crry expression in iNSCs. Additionally, the role of Crry in the reduction of complement-mediated injury to iNSCs was significantly attenuated by Akt inhibition.
Crry expression and Akt activation in astrocytes and neurons derived from iNSCs receiving CHI mouse serum pre-treatment
To evaluate the levels of Crry expression and Akt activation in iNSC-derived astrocytes, we used RT-QPCR and observed no significant differences in the levels of the Crry or Akt genes in astrocytes between the PBS (iNSCs receiving PBS pre-treatment) and HI-CHI (iNSCs receiving HI-CHI mouse serum pre-treatment) groups after CHI mouse serum treatment (Fig. 3a, b). In contrast, the levels of the Crry and Akt genes in astrocytes were substantially higher in the CHI (iNSCs receiving CHI mouse serum pre-treatment) group compared to the other two groups (n=3/group, P<0.05). Supporting these findings, a western blot analysis showed that the Crry, p-Akt, Akt and p-Akt/Akt levels in astrocytes between the PBS and HI-CHI groups were almost identical, whereas the levels of Crry, p-Akt, Akt and p-Akt/Akt in astrocytes of the CHI group were markedly higher than those in the other two groups (n=6/group, P<0.05) (Fig. 3c-g). These results indicated that the pre-treatment of iNSCs with CHI mouse serum enhanced Crry expression and Akt activation in iNSC-derived astrocytes.
Next, to detect Crry expression and Akt activation levels in iNSC-derived neurons, we utilized RT-QPCR and observed no significant differences in the levels of the Crry or Akt genes in neurons among the three groups after CHI mouse serum treatment (Fig. 3h, i). Furthermore, a western blot analysis also revealed that the Crry, p-Akt, Akt and p-Akt/Akt levels in neurons among the three groups were almost identical (Fig. 3j-n). These data suggested that the pre-treatment of iNSCs with CHI mouse serum did not affect the levels of Crry expression or Akt activation in iNSC-derived neurons.
Enhanced levels of Crry expression and Akt activation in iNSC-derived neurons treated with astrocyte culture supernatants
We previously reported that astrocytes derived from iNSCs pre-treated with CHI mouse serum could reduce the numbers of apoptotic neurons via Crry expression following CHI mouse serum treatment [24]. To further study the mechanism underlying the neuroprotection mediated by astrocytes, we performed a functional assay and observed no significant differences in the levels of the Crry or Akt genes in neurons among the i (CHI mouse serum diluted in DMEM/F12), iii (CHI mouse serum diluted in DMEM/F12 containing purified rat anti-mouse Crry antibody), and iv (CHI mouse serum diluted in the astrocyte culture supernatants containing purified rat anti-mouse Crry antibody) sub-groups (Fig. 4a, b). However, the levels of the Crry and Akt genes in neurons were substantially higher in the ii (CHI mouse serum diluted in the astrocyte culture supernatants) sub-group than in the other three sub-groups (n=3/group, P<0.05). Using western blot analysis, we observed that the Crry, p-Akt, Akt and p-Akt/Akt levels in neurons were not significantly different among the i, iii and iv sub-groups (Fig. 4c-g). In contrast, the levels of Crry, p-Akt, Akt and p-Akt/Akt in the neurons of the ii sub-group were markedly higher than those in the other three sub-groups (n=6/group, P<0.05). These findings revealed that the levels of soluble Crry in astrocyte culture supernatants were positively associated with the levels of Crry expression and Akt activation in iNSC-derived neurons.
Crry expression and Akt activation in iNSC-derived neurons treated with CR2-Crry in the presence of CHI mouse serum
To explore the effect of Crry treatment on iNSC-derived neurons, we utilized RT-QPCR and observed that the levels of the Crry and Akt genes in neurons were substantially higher in the CHI (neurons receiving CHI mouse serum treatment) group than in the PBS (neurons receiving PBS treatment) group (n=3/group, P<0.05) (Fig. 5a, b). Moreover, the levels of the Crry and Akt genes in neurons were significantly higher in the CHI+CR2-Crry (neurons receiving CHI mouse serum and CR2-Crry treatment) group than in the other two groups (n=3/group, P<0.05). Additionally, a western blot analysis revealed that the C3d, C9, Crry, active Caspase-3, p-Akt, Akt and p-Akt/Akt levels were low in the neurons of the PBS group (Fig. 5c-j). However, CHI mouse serum treatment induced obvious increases in the levels of C3d, C9, Crry, active Caspase-3, p-Akt, Akt and p-Akt/Akt in the neurons of the CHI group (n=6/group, P<0.05). Remarkably, the Crry, p-Akt, Akt and p-Akt/Akt levels in neurons were markedly higher in the CHI+CR2-Crry group than in the other two groups (n=6/group, P<0.05). Furthermore, the levels of C3d, C9 and active Caspase-3 in the neurons of the CHI+CR2-Crry group were substantially higher than those in the PBS group but significantly lower than those in the CHI group (n=6/group, P<0.05).
Subsequently, we performed immunofluorescence staining and observed that the numbers of p-Akt+/Crry+ and Akt+/Crry+ neurons were markedly higher in the CHI group than in the PBS group (n=6/group, P<0.05) (Fig. 6a-d). Moreover, the levels of p-Akt+/Crry+ and Akt+/Crry+ neurons were substantially higher in the CHI+CR2-Crry group than in the other two groups (n=6/group, P<0.05). Additionally, flow cytometry analysis demonstrated that the Crry, p-Akt and Akt levels in neurons were significantly higher in the CHI group than in the PBS group (n=3/group, P<0.05) (Fig. 6e-j). Furthermore, the levels of Crry, p-Akt and Akt in neurons were markedly higher in the CHI+CR2-Crry group than in the other two groups (n=3/group, P<0.05). Therefore, the treatment of iNSC-derived neurons with CR2-Crry clearly enhanced Crry expression and Akt activation in neurons following CHI mouse serum treatment. Moreover, the administration of CR2-Crry effectively reduced complement-mediated injury to neurons.
CR2-Crry pre-treatment enhanced Crry expression and Akt activation in iNSCs, iNSC-derived astrocytes and neurons
To evaluate the effect of CR2-Crry pre-treatment on iNSCs, we used RT-QPCR and observed that the levels of the Crry and Akt genes in iNSCs, iNSC-derived astrocytes and neurons were markedly higher in the CR2-Crry (iNSCs receiving CR2-Crry pre-treatment) group than in the PBS (iNSCs receiving PBS pre-treatment) group after CHI mouse serum treatment (n=3/group, P<0.05) (Supplementary Fig. S1a, b, S2a, b, and S3a, b). In support of these findings, western blot analysis indicated that the Crry, p-Akt, Akt and p-Akt/Akt levels in iNSCs, iNSC-derived astrocytes and neurons were substantially higher in the CR2-Crry group than in the PBS group (n=6/group, P<0.05) (Supplementary Fig. S1c-g, S2c-g and S3c-g). Furthermore, we performed the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay to measure the viability of iNSCs, iNSC-derived astrocytes and neurons from the PBS and CR2-Crry groups following CHI mouse serum treatment (Supplementary Fig. S1h, S2h and S3h). The cell viabilities between the two groups without CHI mouse serum treatment were almost identical. However, dramatic decreases in cellular viability within each group were observed after CHI mouse serum treatment (n=3/group, P<0.05). Moreover, the viabilities of iNSCs, iNSC-derived astrocytes and neurons were significantly lower in the PBS groups than in the CR2-Crry group (n=3/group, P<0.05). These data implied that, following treatment with CHI mouse serum, the pre-treatment of iNSCs with CR2-Crry clearly enhanced Crry expression and Akt activation in iNSCs, iNSC-derived astrocytes and neurons. Additionally, the pre-treatment of iNSCs with CR2-Crry markedly improved the survival of iNSCs, iNSC-derived astrocytes and neurons post-treatment with CHI mouse serum.
Increased levels of Crry expression and Akt activation in neurons in the brains of CHI mice receiving iNSCs pre-treated with CR2-Crry
To determine the effect of intracerebral-transplanted iNSCs receiving CR2-Crry pre-treatment on CHI mice, we performed double-labelling experiments and observed that Crry+/NeuN+, p-Akt+/NeuN+ and Akt+/NeuN+ neurons were present in the injured cortices of the iNSC (CR2-Crry; CHI mice receiving iNSCs pre-treated with CR2-Crry) group on day 14 post-trauma (Fig. 7a-c). In contrast, NeuN+/TUNEL+ neurons were evident in the injured cortices of the PBS (CHI mice receiving PBS) group at the same time point (Fig. 7d). Quantitatively, the levels of Crry+/NeuN+, p-Akt+/NeuN+ and Akt+/NeuN+ neurons were significantly higher in the iNSC (CHI mice receiving iNSCs pre-treated with PBS) group than in the PBS group (n=6/group, P<0.05) (Fig. 7e-g). Moreover, the numbers of Crry+/NeuN+, p-Akt+/NeuN+ and Akt+/NeuN+ neurons were markedly higher in the iNSC (CR2-Crry) group than in the other two groups (n=6/group, P<0.05). In addition, the levels of NeuN+/TUNEL+ neurons were substantially lower in the iNSC group than in the PBS group (n=6/group, P<0.05) (Fig. 7h). Furthermore, the numbers of NeuN+/TUNEL+ neurons were significantly lower in the iNSC (CR2-Crry) group than in the other two groups (n=6/group, P<0.05).
Subsequently, we utilized western blot analysis to evaluate the levels of C3d, C9, Crry, active Caspase-3, p-Akt, Akt and p-Akt/Akt in the brains of CHI mice among the three groups at 14 days after injury (Fig. 8). The levels of C3d, C9 and active Caspase-3 in the brains of the iNSC group were markedly lower than those in the PBS group (n=6/group, P<0.05). However, the levels of Crry, p-Akt, Akt and p-Akt/Akt in the brains of CHI mice were substantially higher in the iNSC group than in the PBS group (n=6/group, P<0.05). Remarkably, the levels of C3d, C9 and active Caspase-3 in the brains of CHI mice were significantly lower, whereas the levels of Crry, p-Akt, Akt and p-Akt/Akt were markedly higher in the iNSC (CR2-Crry) group than in the other two groups (n=6/group, P<0.05). In summary, intracerebral-transplanted iNSCs, pre-treated with CR2-Crry, could enhance Crry expression and Akt activation in neurons and reduce complement-mediated injury to neurons in the brains of CHI mice.