Overexpression of Myc-Dp40 and Myc-Dp40L170P showed different effects on the neurite outgrowth of PC12-Tet-On cells.
PC12 Tet-On cells were stably transfected with the pTRE2pur-Myc/Dp40 or pTRE2pur-Myc/Dp40L170P vector, and vector integration was tested by genomic DNA PCR (Fig. S1). To characterize the isolated clones PC12-Dp40 and PC12-Dp40L170P, we determined the minimum concentration of doxycycline to induce the overexpression of the recombinant proteins through WB using an anti-c-Myc antibody. Myc-Dp40 and Myc-Dp40L170P were overexpressed using 50 to 1000 ng/ml doxycycline in undifferentiated PC12 Tet-On cells (Fig. S2). Because higher concentrations of doxycycline did not result in an increase in recombinant protein expression, we used 100 ng/ml doxycycline to overexpress Dp40 proteins to eliminate the cytotoxic effect of doxycycline reported at 200 ng/ml [17]. With this doxycycline concentration, undifferentiated and NGF-differentiated PC12 Tet-On cells were analysed to evaluate the morphological effect of Myc-Dp40 and Myc-Dp40L170P overexpression on neurite outgrowth processes compared with those of the PC12 control cells. Myc-Dp40 and Myc-Dp40L170P overexpression did not affect the morphology of the undifferentiated PC12 cells (Fig. 1a-c), in contrast to the NGF-differentiated PC12 cells (Fig. 1d-f). The differentiation ratio (Fig. 1g) showed that the PC12-Dp40 cells (40.6% ± 2.90; P = 0.0003) had a greater number of cells with neurites than the PC12 control cells (4.96% ± 0.72), while PC12-Dp40L170P did not result in a significant difference compared with the PC12 control cells. However, a decrease in the differentiation ratio of the NGF-differentiated Dp40L170P cells (4.50% ± 1.82; P = 0.0005) was observed compared with that of the PC12-Dp40 cells. Additionally, neurite length quantification (Fig. 1h) showed that the NGF-differentiated PC12-Dp40 cells (17.3 µm ± 0.30; P = 0.0086) had longer neurites than the PC12 control cells (14.0 µm ± 0.62). However, the PC12 control cells presented longer neurites than the PC12-Dp40L170P cells (11.5 µm ± 0.052; P = 0.0359). Therefore, the differentiated PC12-Dp40L170P cells (11.5 µm ± 0.52; P = 0.006) showed lower neurite outgrowth than the PC12-Dp40 cells (17.3 µm ± 0.30). Based on these results, Myc-Dp40 expression stimulates neurite outgrowth, whereas Myc-Dp40L170P inhibits this process.
Protein expression profile of differentiated PC12-Dp40L170P cells
To understand the inhibitory mechanism of neurite outgrowth produced by Dp40L170P, we analysed the total protein extracts of the PC12 control and PC12-Dp40L170P NGF-differentiated cells for nine days by 2-DE (Fig. 2a and b). Differentially expressed protein spots with at least a 1.1-fold change were considered differentially expressed. Among the 344 protein spots detected, 14 (spots ID: 1-14) showed differential expression (Fig. 2c), 13 proteins showed upregulated expression and one showed downregulated expression, and magnified views of the differentially expressed protein spots are presented in Fig. 2c. Thus, Dp40L170P modified the protein expression profile of PC12 Tet-On cells. The 14 spots selected were excised from the 2-DE gels and analysed by MS. The details of each identified protein are summarized in Table 1. The proteins with highly upregulated expression were S100a6 (2.1-fold, pI 5.2) and α-internexin (2.0-fold, pI 5.3), which are involved in the reorganization of cytoskeletal structure and neurofilaments present in immature neurons, respectively. Only the expression of ectonucleotide pyrophosphatase (1.5-fold, pI 5.5), a protein related to cellular communication, was downregulated. The other proteins identified were related to chaperone-like activity (T-complex protein 1 subunit zeta and the endoplasmic reticulum chaperone BIP) and metabolism (phosphoglycerate mutase 1, L-lactate dehydrogenase A chain and alpha enolase). This last group of proteins usually shows upregulated expression in several proteomic analyses, probably due to cellular stress responses [18].
Myc-Dp40 and Myc-Dp40L170P overexpression modified the expression of α-internexin, VAMP, HspB1 and NF-L during the neuronal differentiation process
After MS identification, we validated the differential expression of α-internexin, one of the top two proteins with upregulated expression determined by WB in undifferentiated (Fig. 3) and differentiated (Fig. 4) PC12 control cells, PC12-Dp40 cells and PC12-Dp40L170P cells. First, we evaluated the expression of the recombinant proteins: the Myc-Dp40 and Myc-Dp40L170P proteins were overexpressed 13.3- and 7-fold in the undifferentiated PC12-Dp40 and PC12-Dp40L170P cells, respectively, showing a significant increase compared those of the PC12 control cells. In addition, Myc-Dp40 (1.9-fold) was more highly expressed than Myc-Dp40L170P in the PC12-Dp40 and PC12-Dp40L170P cells. α-Internexin, which is involved in the expression and assembly of neurofilaments in the central nervous system [19, 20], presented a 5.25-fold increase in the PC12-Dp40L170P cells compared with the PC12 control cells. However, α-internexin expression was not significantly different in the undifferentiated PC12-Dp40 cells compared with the PC12 control and PC12-Dp40L170P cells. Since a previous report [9] showed that VAMP interacts with the Dp40 protein, we decided to validate its expression. The results showed that VAMP is expressed at low levels and without a significant difference in all undifferentiated cells. Recently, it was observed that Dp71eΔ71 and mutant Dp71Δ78-79 overexpression increased the HspB1, a remodeler of the cytoskeleton, during the neuronal differentiation process of PC12 cells [15, 21]. Thus, we analysed the expression of the HspB1 protein; however, we did not detect this protein under undifferentiated conditions. In addition, NF-L expression was evaluated because it is a neuronal differentiation marker that is expressed in postmitotic neurons as part of the cytoskeletal structure. However, we did not observe a significant difference in NF-L expression in any undifferentiated PC12 cells.
In the NGF-differentiated PC12 cells (Fig. 4), Myc-Dp40 and Myc-Dp40L170P expression showed an increase of 9.8- and 4.3-fold in the PC12-Dp40 and PC12-Dp40L170P cells, respectively compared with the PC12 control cells. However, there was no significant difference between Myc-Dp40 and Myc-Dp40L170P. α-Internexin expression in the PC12-Dp40 cells did not show a significant difference compared with that in the PC12 control cells. However, its expression in the PC12-Dp40L170P cells was increased 44- and 3-fold compared with that in the PC12 control and PC12-Dp40 cells, respectively. This increase is in accordance with what we observed in the 2-DE gels in the differentiated PC12 control cells and the PC12-Dp40L170P cells. VAMP expression was increased 5.4- and 1.6-fold in the NGF-differentiated PC12-Dp40 and PC12-Dp40L170P cells, respectively, compared with that in the PC12 control cells. However, the PC12-Dp40 cells showed an expression increase of 3.3-fold compared to the PC12-Dp40L170P cells. HspB1 presented low expression and no significant difference in the differentiated cell lines (Fig. 4b). NF-L expression was not significantly different between the differentiated PC12 control and PC12-Dp40 cells. In contrast, the PC12 control cells showed an increase of 2-fold compared with the PC12-Dp40L170P cells but no significant difference compared with the PC12-Dp40 cells. All these results showed that the disruption of dystrophin Dp40 through the expression of the mutant Dp40L170P could affect the expression of neurofilaments such as α-internexin and NF-L during the neuronal differentiation process of PC12 cells.
Differential distribution of the Myc-Dp40, Myc-Dp40L170P and α-internexin proteins in PC12- Tet-On cells
Differences in the subcellular distribution of Myc-Dp40, Myc-Dp40L170P and c-Myc epitopes were evaluated through ImmF assays in the undifferentiated and NGF-differentiated PC12 Tet-On cells (Figs. 5 and 6, respectively). Myc-Dp40 protein localization did not present a significant difference between the cytoplasm (49.08% ± 2.20) and nucleus (50.92% ± 2.20; P = NS) in the undifferentiated cells, while Myc-Dp40L170P showed an increase in the nucleus (75.06% ± 1.69) compared to the cytoplasm (24.94% ± 1.69; P < 0.0001), unlike the c-Myc peptide in the PC12 control cells, which presented an increase in immunoreactivity in the cytoplasm (66.53% ± 1.58) compared to the nucleus (33.47% ± 1.580; P < 0.0001). Under NGF-differentiated conditions, in the PC12 control cells, the c-Myc-peptide was localized to the same extent in the cytoplasm (52.68% ± 2.98) and nucleus (47.32% ± 2.989; P = NS). However, Myc-Dp40 was mainly located in the cytoplasm (61.58% ± 3.00) compared to the nucleus (38.42% ± 3.00; P < 0.0001), while Myc-Dp40L170P showed higher immunoreactivity in the nucleus (62.20% ± 2.19) than in the cytoplasm (37.80% ± 2.19; P < 0.0001).
α-Internexin has been detected in the cytoplasm of postmitotic neurons of the periphery and central nervous system [22, 23]. In the undifferentiated PC12 control, PC12-Dp40 and PC12-Dp40L170P cells, α-internexin was observed in the cytoplasm. We found that the immunoreactivity of this protein was similar in the undifferentiated PC12 control (0.73% ± 0.19), PC12-Dp40 (0.83% ± 0.08; P = NS) and PC12-Dp40L170P cells (1.42% ± 0.37; P = NS). However, the NGF-differentiated PC12-Dp40L170P cells (3.03% ± 0.10) showed a higher percentage of immunoreactivity than the PC12-Dp40 (1.76% ± 0.28; P = 0.013) and PC12 control cells (1.52% ± 0.21; P = 0.003), but we did not observe a significant difference between the NGF-differentiated PC12-Dp40 (0.44% ± 0.157; P = NS) and PC12 control cells (0.36% ± 0.003). In the NGF-differentiated PC12-Dp40 cells, α-internexin was observed in the cytoplasm and along the length of neurites, while in the PC12 control and PC12-Dp40L170P cells, it was mostly distributed in the cytoplasm. Based on these results, the subcellular distributions of Myc-Dp40 and Myc-Dp40L170P were mostly cytoplasmic and nuclear, respectively. In addition, the overexpression of Myc-Dp40L170P increases α-internexin expression in the undifferentiated and NGF-differentiated PC12-Dp40L170P cells.