Title : The participation of dystrophin Dp40 and Dp40L170P in the neuronal differentiation process of PC12 cells
Dystrophin Dp40 is the shortest dystrophin described to date. Role of this protein in the cognitive impairment described in patients with Duchenne Muscular Dystrophy (DMD) is still unclear. Dp40 is expressed in a ubiquitous manner from the same promoter for dystrophin Dp71 (Tinsley et al., 1993). This protein interacts with presinaptic proteins as VAMP2 (Vesicle-associated membrane protein), sintaxin 1A and SNAP25 (Tozawa et al., 2012). Dp40 is mainly located at the membrane and nucleus of hippocampal neurons and is expressed in post-natal stages to adult mouse brain (Fujimoto, 2014), and accumulates in the nucleus during the neuronal differentiation process of PC12 cells, while the mutant Dp40L170P promotes a nuclear distribution in undifferentiated and NGF-differentiated cells through transient transfection (Aragón et al., 2015).
To explore the role of Dp40 in neuronal differentiation, our group has obtained the PC12-Dp40, PC12-Dp40L170P and PC12-control cell lines that stably and inducible express the recombinant proteins Myc-Dp40, Myc-Dp40L170P and Myc respectively. The morphometric analysis of these clones showed that overexpression of Dp40 promotes the neurite outgrowth during the NGF-differentiation of PC12 while overexpression of Dp40L170P decreases the neurite outgrowth compared to PC12-control and PC12-Dp40 cells. As a next step, a protein expression profile through two dimensional gel electrophosis was performed comparing PC12-Dp40L170P and PC12-control in NGF-differentiated cells. Eight proteins up-regulated in PC12-Dp40L170P were identified by mass spectrometry; S100a6 and alpha-internexin (Int) neurofilament were the proteins mostly increased. It has been speculated that S100a6 regulates the actin cytoskeleton and Int is related to immature neurons (Faussone et al., 1999) and filament inclusion in neurons (Cairns et al., 2004). The expression of neurofilament light chain (NF-L) as a differentiation biomarker and HspB1 as a polymerization promoter of actin was verified. NF-L is lowly expressed in undifferentiated while in NGF-differentiated is increased in PC12-control compared to PC12-Dp40 and PC12-Dp40L170P cells. On the other hand, HspB1 was not detected in undifferentiated and is little expressed in NGF-differentiated cells. Additionally, we observed a significant increase of VAMP-1/2 in PC12-Dp40 cells compared to PC12-control and PC12-Dp40L170P NGF-differentiated cells.
Thus, our results suggest that dystrophin Dp40 could be related with the synaptic vesicle exocytosis and the mutation in the amino acid 170 may disrupt this process. In addition, mutant Dp40L170P could generate damage or immature neurons through overexpression of alpha-internexin that alter important cellular processes as neuronal differentiation.