Dp71Δ_78‐79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1

Abstract

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71Δ78-79 dystrophin mutant is stably expressed in PC12-C11 cells. To investigate the effect of Dp71Δ78-79 overexpression on the protein profile of PC12-C11 cells, we compared the expression profiles of undifferentiated and NGF-differentiated PC12-C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein was downregulated, and five were upregulated. Dp71Δ78-79 overexpression had a greater effect on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein with the highest upregulation was HspB1. Changes in HspB1 expression were validated by Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12-C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process. Our results show that Dp71Δ78-79 affects the expression level of some proteins and that the stimulated neurite outgrowth produced by this mutant is mainly through upregulation and phosphorylation of HspB1.

Overexpression of the dystrophins Dp40 and Dp40L170P modifies neurite outgrowth and the protein expression profile of PC12 cells

“…Analysis of the mechanisms that stimulate the disruption of neuronal differentiation as a consequence of the subcellular distribution of Dp40 should be the next step. Taken together, these results suggested that the neurite outgrowth promoted by Dp40 overexpression could be carried out through a different strategy than the increase in HspB1, as was previously reported in PC12 cells that overexpress dystrophin Dp71 [15,21], possibly through VAMP expression, which is increased in the differentiated PC12-Dp40 cells but decreased in the PC12-Dp40 L170P cells.…”

“…However, previous reports have shown that overexpression of Dp71 Δ78−79 and Dp71e Δ71 stimulates neurite outgrowth in NGF-differentiated PC12 cells, and both cell lines presented a high expression of HspB1 [15,21]. Because Myc-Dp40 overexpression promotes neuronal differentiation (Fig.…”

“…The results showed that VAMP is expressed at low levels and without a signi cant difference in all undifferentiated cells. Recently, it was observed that Dp71e Δ71 and mutant Dp71 Δ78-79 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.…”

“…Total protein extracts of the NGF-differentiated PC12 control and PC12-Dp40 L170P cells were obtained and run in 2-DE gels as previously described [15]. Protein concentrations were determined by the Bradford method.…”

Dystrophin Dp71 and the Neuropathophysiology of Duchenne Muscular Dystrophy

“…Cytoskeletal reorganisation and neurotransmitter synthesis are both required for differentiation which appears a key role for Dp71 in neurones. In support of these findings, the same group generated a Dp71 mutant lacking exons 78 and 79, Dp71 Δ78-79 which they show to stimulate PC12 proliferation [149], cell differentiation [150] and neurite outgrowth through the phosphorylation of HspB1 [151].…”