- Determination of the appropriate concentrations of Fluoxetine, SB216763 and Wortmannin in PC12 cells
First, PC12 cells were cultured continuously for 3 days, and their cell activity was detected by the CCK8 assay to determine the appropriate length of continuous administration in the subsequent experiments. As shown in Fig. 1A, the NC group was cultured continuously for 3 days. Compared with the cell activity on the first day, the cell activity on the second day (93.5 ±3.5%) and the third day (71.4±4.4%) decreased over time, and there was a significant difference between the two days (P <0.000). In addition, on the 4th day of culture (results not shown), microscopic observation showed that the cells exhibited vacuoles, atrophied processes, and cell activity that decreased to approximately 20%. Other results[18] showed that the cell survival rate decreased to approximately 50%, which stimulated cell apoptosis without stimulating cell death. This indicates senescence and apoptosis during this period, which are not suitable for the experiment.
Different doses caused differences in cell activity. To investigate the appropriate doses for the different experimental groups, after the cells were inoculated and adhered to the plate, the following drugs and doses were administered for 1 day, 2 days, and 3 days: Flu(10nM, 100nM, 1μM, and 10μM), SB(1 µM, 10 µM, 100 µM, and 1000 µM) and WT (50 nM, 500 nM, 5 µM, and50 µM). The CCK8 assay was used to detect cell activity on the 1st, 2nd and 3rd days. The difference in cell activity in each group treated with different doses was analyzed. As shown in Figs. 1B, 1C and 1D, the cell activity of the Flu administration group was the highest after 10-6 mol/L administration, the SB administration group exhibited the highest activity after 10 µM administration, and the WT administration group exhibited the highest activity after 5 µM administration (all P<0.05).
- Fluoxetine improved the microtubule plasticity and increased the expression of Tubulin in the early stage of long-term administration, but aggravated deficits in microtubule plasticity in the later stage
The processes in the Flu group extended obviously on the first day but were inhibited on the third day. The intracellular expression of the Tubulin protein is important for the formation of microtubules for the cell scaffold, and the fluorescence of the protein can directly reflect changes in cell processes and cell junctions. After inoculation, PC12 cells were allowed to adhere to the plate, and then the processes gradually prolonged and extended. The fluorescence results showed that both the Flu group and NC group showed the obvious extension of processes on the first day (Fig. 2A). On the 3rd day, the processes of the NC group connected with each other to form a network-like structure. However, the processes of the Flu group did not yet exhibit the connected network structure observed in the NC group (Fig. 2C).
Merged images of CRMP2 and Tubulin fluorescence showed overlap between the CRMP2 expression region and the Tubulin expression region. There was fluorescence colocalization between the two proteins. The fluorescence colocalization in the NC group and Flu group was obvious on the first and second days but on the third day, the colocalization in the Flu group was significantly less than that in the NC group.
The Flu group exhibited increased levels of CRMP2 and Tubulin proteins on the first day, but these levels were decreased significantly on the third day. Fig. 3 shows that CRMP2 in the Flu group (0.84±0.26, P=0.004) was significantly higher than that in the NC group (0.48±0.09) on the first day, but there was no significant difference between the Flu (0.67±0.23, P=0.266) and NC groups (0.86±0.28) on the second day. On the 3rd day, the protein content (0.23±0.11, P=0.026) was lower in the Flu group than in the NC group (0.56±0.31). The Tubulin and CRMP2 protein content in the Flu group was the same after 3 days of continuous administration. The content of Tubulin in the Flu group (1.34±0.23, P=0.041) was higher than that in the NC group (0.86±0.17) on the first day, but there was no significant difference in the Tubulin content (0.73±0.29, P=0.123) between the Flu group and NC group (1.40±0.33) on the second day. On the 3rd day, the content of Tubulin (0.21±0.09, P=0.015) was lower than in the Flu group that in the NC group (0.77±0.25).
The mRNA expression of CRMP2 and Tubulin increased on the first and second day of Flu administration but decreased on the third day. As shown in Figs. 4A-1, 4B-1 and 4C-1, the expression of mRNA of CRMP2 on day 1(1.31±0.08,P=0.030) and day 2(1.60±0.07,P=0.001) in the Flu administration group was significantly higher than that on day 1(1.01±0.11) and day 2(1.00±0.07) in the NC group. However, on the third day, the CRMP2 mRNA level in the Flu group (0.46±0.04, P=0.001) was lower than that in the NC group (1.05±0.07). Similarly, as shown in Figs. 4A-2, 4B-2, 4C-2, the mRNA of Tubulin in the Flu administration group was higher than that in the NC group on the first day (1.35±0.05, P=0.017) and the second day (1.63±0.05, P=0.001). However, on the third day, the Tubulin mRNA level in the Flu group (0.64±0.07, P=0.026) was also lower than that in the NC group.
- Co-IP validates the interaction between the CRMP2 and Tubulin proteins
The immunofluorescence results showed that there was immunofluorescence colocalization of CRMP2 and Tubulin. To investigate whether there is a direct interaction between the two proteins, we performed immunoprecipitation in 1day of NC group. Fig. 5A shows IP for Tubulin, IB for CRMP2, and CRMP2 in the cell lysate bound with magnetic beads containing a Tubulin antibody. The results (anti-CRMP2) showed that there was CRMP2 in the conjugates. Fig. 5B shows IP for CRMP2, IB for Tubulin, and Tubulin in the cell lysate bound with magnetic beads containing a CRMP2 antibody. The results (anti-Tubulin) showed that Tubulin existed in the conjugates. After double verification, it was fully confirmed that there was a direct interaction between the CRMP2 and Tubulin proteins. The coexpression of the two proteins was further verified by immunofluorescence at the protein level.
- Effect of CRMP2 activity on Tubulin plasticity in PC12 cells
After determining the direct interaction between the CRMP2 and Tubulin proteins, we hypothesized that CRMP2 is involved in mediating microtubule scaffold plasticity. To answer this question, we tried to regulate CRMP2 activity with CRMP2 agonists and inhibitors for 3 days to observe the changes in Tubulin in cell scaffolds. We used the CCK8 assay to detect cell activity to determine the appropriate dose of SB216763 (a CRMP2 antagonist) and Wortmannin (a CRMP2 agonist). The results are shown in Fig. 1. The cell activity in the SB group was the highest after 10 µM administration and the cell activity in the WT administration group was the highest after 5 µM administration.
In the SB group, the processes were significantly inhibited on the 1st and 2nd days, but neurite elongation and cell junctions increased on the 3rd day. In the WT group, the processes continued to elongate, and the intercellular junctions were enhanced for 3 days. The fluorescence results (Fig. 2) showed that the development of PC12 cells was inhibited on the first and second days in the SB administration group. On the first day, the extension of the cell processes was slow. On the second day, the processes began to extend, but they were still shorter than those in the normal control group. On the 3rd day, the processes of the cells were extended, and the intercellular connections increased significantly, attaining the network-like structure of the NC group. In the WT administration group, the processes were extended, and the intercellular junctions were enhanced from the first day to the third day. In addition, the fluorescence results of the SB group showed that, except for a small amount of CRMP2 and Tubulin colocalization on the first day, it was difficult to find the colocalization on the other two days. However, the colocalization of CRMP2 and Tubulin in the WT group was clearly visible from day 1 to day 3.
The content of CRMP2 in the SB group was significantly decreased after 3 days of continuous administration, while the content of the CRMP2 protein was significantly increased in the WT group. As shown in Fig. 4, the CRMP2 protein content was significantly higher in the WT group than in the NC group on the 1st (0.24±0.08, P=0.035), 2nd (0.37±0.16, P=0.007) and 3rd (0.15±0.09, P=0.007) days in the SB group was lower than that in the NC group on the 1st, 2nd and 3rd days. In contrast, the protein content of CRMP2 on the 1st (1.16±0.17, P=0.007), 2nd (2.05±0.32, P=0.000) and 3rd (1.62±0.25, P=0.000) days. The content of the Tubulin protein in the SB group decreased on the first day and the second day, but there was no difference between the SB group and the NC group on the third day. The Tubulin content was significantly increased in the WT group after continuous administration for 3 days. As shown in Fig. 3, the content of Tubulin in the SB group was inhibited on the first day (0.36±0.12, P=0.031) and the second day (0.44±0.13, P=0.032) and was lower than that in the NC group on the first and second days. However, there was no significant difference in the content of Tubulin (0.55±0.11, P=0.305) between the SB group and the NC group on the 3rd day. The content of Tubulin in the WT group was higher than that in the NC group on the first day (2.82±0.60, P=0.000), the second day (5.40±1.21, P=0.000) and the third day (3.34±0.58, P=0.000).
The mRNA expression of CRMP2 was inhibited in the SB group for 3 days but increased in the WT group for 3 days. Figs. 4A-1, 4B-1 and 4C-1 show that the mRNA expression of CRMP2 in the SB group was significantly inhibited on day 1 (0.40±0.06, P=0.003), day 2 (0.46±0.08, P=0.002) and day 3 (0.15±0.04, P=0.000) compared with that in the NC group on day 1 (1.01±0.11), day 2 (1.00±0.07) and day 3 (1.05±0.07). In contrast, the mRNA of CRMP2 in the WT group on the first day (2.42±0.11), the second day (1.87±0.06) and the third day (1.55±0.10) was higher than that in the NC group on the first day, the second day and the third day (all P<0.01). Tubulin mRNA expression in the SB group was inhibited on the 1st and 2nd days after continuous administration for 3 days, but there was no difference between the SB group and the NC group on the 3rd day. In contrast, the mRNA expression increased in the WT group after 3 days of continuous administration. As shown in Figs. 4A-2, 4B-2 and 4C-2, compared with that on the first and second days in the NC group, the mRNA level of Tubulin in the SB administration group was also significantly inhibited on the first day (0.51±0.09, P=0.004) and the second day (0.36±0.06, P =0.001) but on the 3rd day (1.87±0.10, P=0.002), it was higher in the SB group than in the NC group. In contrast, the mRNA of Tubulin in the WT group was significantly higher on the first day (1.82±0.12), the second day (1.93±0.11) and the third day (2.68±0.19) than that in the NC group (all P< 0.01).