- Effect of treatment on PC12 cell viability
The differentiated PC12 cells were cultured for 3 days, and their cell viabilities were detected by the CCK8 assay. As showed in Fig.1A, compared with cell viability at 1 day, the cell viability of PC12 cells happened a small decease at 2 days (93.5±3.5%), followed by highly significant decreases at 3 days (71.4±4.4%). There was a significant difference in cell viability between the 2 days and 3 days (P<0.01). In addition, the cells showed cell damage and neuronal atrophy at 4 days of culture (results not shown), with cell viability reducing to nearly 20%, which are not suitable for the subsequent experiments.
To investigate the appropriate concentrations of FLU (Aladdin, China), SB216763 (SB, CRMP2 antagonist) and Wortmannin (WT, CRMP2 agonist; Selleck, USA), cells were divided into FLU group (0.01μM, 0.1μM, 1μM, 10μM) and SB group (1μM, 10μM, 100μM, 1000μM) and WT group (0.05μM, 0.50μM, 5μM, 50μM). As shown in Fig.1B, 1C and 1D, the cell viabilities of the FLU (1μM), SB (10µM) and WT (5µM) treatment were the highest (all P<0.05), which were used in subsequent experiments.
- FLU treatment has opposite effects on the regulation of cellular neuroplasticity and microtubule plasticity at 1 day than at 3 days
Cellular neuroplasticity in PC12 cells was determined by detection of tubulin, a spherical protein that is the basic structural unit of the cells, so that the IF results of tubulin directly reflects changes in cell morphology. FLU significantly improved cellular neuroplasticity at 1 day, but inhibited them at 3 days. The IF results found a more significant increase of cellular neuroplasticity, such as neurite outgrowth, neurite length and number of neuritis, in FLU group than NC group at 1 day (Fig. 2A shows). The cell morphology of NC group formed a network-like structure at 3 days, but FLU group did not exhibit the connected network structure observed in the normal control (NC) group (Fig. 2C shows).
We used western blotting and RT-PCR to further detect the expression of tubulin, which is microtubule plasticity marker. The results of western blotting (Fig.3 shows) showed the level of tubulin protein was increased with FLU treatment for 1 day (1.34±0.23, P=0.041), compared to that in NC group (0.86±0.17). But there was no significant difference in the tubulin content (0.73±0.29, P=0.123) between FLU group and NC group (1.40±0.33) at 2 days. The tubulin protein content with FLU treatment at 3 days (0.21±0.09, P=0.015) was lower than that in NC group (0.77±0.25). Similarly, the mRNA expression of tubulin was increased with FLU treatment for 1 day but decreased at 3 days. As shown in Figs. 4A-2, 4B-2, 4C-2, the mRNA expression of tubulin with FLU treatment for 1 day (1.35±0.05, P=0.017) and 2 days (1.63±0.05, P=0.001) were higher than that in NC group at 1 day (1.01±0.06) and 2 days (1.01±0.04). However, the tubulin mRNA level with FLU treatment at 3 days (0.64±0.07, P=0.026) was lower than that in NC group (1.05±0.07).
This study also found that treatment with FLU had effects on the expression of CRMP2,a protein closely related to cellular neuroplasticity. FLU treatment increased levels of CRMP2 protein at 1 day, but this level was decreased significantly at 3 days. Fig.3 shows CRMP2 in FLU group (0.84±0.26, P=0.004) was significantly higher than that in NC group (0.48±0.09) at 1 day,but there was no significant difference between FLU group (0.67±0.23, P=0.266) and NC group (0.86±0.28) at 2 days. CRMP2 protein content with FLU treatment (0.23±0.11, P=0.026) for 3 days was lower than that in NC group (0.56±0.31). Similarly, as showed in Fig. 4A-1, 4B-1 and 4C-1, the mRNA expression of CRMP2 with FLU treatment at 1 day (1.31±0.08,P=0.030) and 2 days (1.60±0.07, P=0.001) were significantly higher than that at 1 day (1.01±0.11) and 2 days (1.00±0.07) in NC group. However, CRMP2 mRNA level in FLU group (0.46±0.04, P=0.001) was lower than that in NC group (1.05±0.07) at 3 days.
- Effect of CRMP2 activity on cellular neuroplasticity and microtubule plasticity in PC12 cells
The above dates indicate that treatment with FLU has opposite effects on the regulation of cellular neuroplasticity and microtubule plasticity at 1 day than at 3 days, and it also affects the expression of CRMP2. What is more, the IF results showed there was colocalization between CRMP2 and tubulin (Fig.2 shows). To further investigate this result, we used Co-IP validates a direct interaction between CRMP2 and tubulin (Fig.3J shows), which is consistent with other studies [29-31]. Some studies [32] show that brain-specific CRMP2 knockout (cKO) mice display molecular, cellular, structural and behavioural deficits. The cKO mice exhibit microtubule injury in other tissues, such as enlarged ventricles and ventricular. Loss of CRMP2 in the hippocampus leads to aberrant dendrite development and defective synapse formation in CA1 neurons. Furthermore, CRMP2 knockdown in newborn neurons results in stage-dependent defects in their development during adult hippocampal neurogenesis. On this basis, we tried to regulate CRMP2 activity with SB treatment and WT treatment in longer periods (up to 3 days) to study the cellular neuroplasticity and microtubule plasticity in PC12 cells.
We found that the expression of CRMP2 was significantly inhibited after giving SB treatment. CRMP2 protein content (Fig.3 shows) with SB treatment for 1 day (1.16±0.17, P=0.007), 2 days (2.05±0.32, P=0.000) and 3 days (1.62±0.25, P=0.000) were lower than NC group. Similarly, As Figs.4A-1, 4B-1 and 4C-1 showed the CRMP2 mRNA expression with SB treatment for 1 day (0.40±0.06, P=0.003), 2 days (0.46±0.08, P=0.002) and 3 days (0.15±0.04, P=0.000) were inhibited, compared with NC group. However, the expression of CRMP2 increased with WT treatment. The CRMP2 protein content (Fig.3 shows) with WT treatment for 1 day (0.24±0.08, P=0.035), 2 days (0.37±0.16, P=0.007) and 3 days (0.15±0.09, P=0.007) were more than NC group. Similarly, CRMP2 mRNA expression (Figs.4A-1, 4B-1 and 4C-1 show) with WT treatment for 1 day (2.42±0.11, P<0.01), 2 days (1.87±0.06, P<0.01) and 3 days (1.55±0.10, P<0.01) were also more than NC group.
The above results showed that SB and WT effectively regulated the expression of CRMP2. Next, the tubulin results of IF (Fig.2 shows) found cellular neuroplasticity was significantly inhibited with SB treatment for 1 day and 2 days, but increased at 3 days, which formed a network-like structure like the cell morphology of NC group. However, the cellular neuroplasticity was enhanced with WT treatment from 1 day to 3 days. In addition, it was difficult to find the colocalization between CRMP2 and tubulin with SB treatment at 2 days and 3 days, except for a small amount of co-expression at 1 day. However, the colocalization between CRMP2 and tubulin was clearly visible in WT group from 1 day to 3 days.
As shown in Fig.3, the tubulin protein content with SB treatment for 1 day (0.36±0.12,P=0.031) and 2 days (0.44±0.13,P=0.032) was lower than that in NC group. However, there was no significant difference in tubulin content between the SB group (0.55±0.11, P=0.305) and the NC group at 3 days. As shown in Figs.4A-2, 4B-2 and 4C-2, compared with NC group, tubulin mRNA expression with SB treatment for 1 day (0.51±0.09,P=0.004) and 2 days (0.36±0.06,P=0.001) was inhibited, followed by highly significant increases at 3 days (1.87±0.10,P=0.002). In contrast, the tubulin mRNA expression in the WT group was significantly higher at 1 day (1.82±0.12, P< 0.01), 2 days (1.93±0.11, P< 0.01) and 3 days (2.68±0.19, P< 0.01) than that in the NC group. Similarly, the level of tubulin in WT group was higher at 1 day (2.82±0.60, P=0.000), 2 days (5.40±1.21, P=0.000) and 3 days (3.34±0.58, P=0.000) than those in NC group.