Mutation of leucine 170 alters the subcellular distribution, neurite outgrowth and three-dimensional structure of dystrophins Dp71 and Dp40

In addition to progressive muscle wasting, one-third of patients with Duchenne muscular dystrophy (DMD) exhibit varying degrees of cognitive impairment, which are associated with mutations of the DMD gene within the region coding for dystrophins Dp71 and Dp40. A previous study reported that the deletion of leucine 3238 in dystrophin Dp427 induces cognitive impairments without muscular dystrophy. This mutation has implications for all dystrophins, including Dp71 and Dp40. This study aimed to evaluate the effect of the deletion of leucine 170 (leucine 3238 in the full-length dystrophin Dp427) on the subcellular localisation, neurite outgrowth and three-dimensional (3D) structure of Dp71 and Dp40 isoforms. PC12 Tet-On cells were transiently transfected with pTRE2pur-Myc/Dp71 or Dp40 vectors, and recombinant Myc-Dp71/Dp40 proteins were expressed by adding doxycycline. The analysis of undifferentiated and nerve growth factor- (NGF) differentiated cells was conducted by cell morphology, immunofluorescence staining and confocal microscopy. The length of the neurites of differentiated cells was obtained using ImageJ software. Analyses of 3D models were carried out by Alphafold 2.0 and TM-align tools. Our research revealed that the deletion of leucine 170 (ΔL170) disturbs the membrane localisation and promotes the cytoplasm and nuclear accumulation of Dp71 and Dp40 in undifferentiated and NGF-differentiated PC12 Tet-On cells. Furthermore, this mutation altered the neurite outgrowth of PC12 Tet-On cells. It is important to note that both the deletion of leucine 170 (ΔL170) and the mutation of leucine 170 to proline (L170P) result in alterations to the 3D structures of these dystrophins. The loss or mutation of leucine 170 disturbs the subcellular localisation, neuronal functions and 3D structures of Dp71 and Dp40. This may lead to cognitive deficits in patients with DMD.