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

César García-Cruz,Juan Pablo Reyes-Grajeda,Candelaria Merino-Jiménez,Brenda González-Assad,Cecilia Montanez,Jorge Aragón,Víctor Ceja

doi:10.1038/s41598-022-05271-2

Abstract

Dp40 is ubiquitously expressed including the central nervous system. In addition to being present in the nucleus, membrane, and cytoplasm, Dp40 is detected in neurites and postsynaptic spines in hippocampal neurons. Although Dp40 is expressed from the same promoter as Dp71, its role in the cognitive impairment present in Duchenne muscular dystrophy patients is still unknown. Here, we studied the effects of overexpression of Dp40 and Dp40L170P during the neuronal differentiation of PC12 Tet-On cells. We found that Dp40 overexpression increased the percentage of PC12 cells with neurites and neurite length, while Dp40L170P overexpression decreased them compared to Dp40 overexpression. Two-dimensional gel electrophoresis analysis showed that the protein expression profile was modified in nerve growth factor-differentiated PC12-Dp40L170P cells compared to that of the control cells (PC12 Tet-On). The proteins α-internexin and S100a6, involved in cytoskeletal structure, were upregulated. The expression of vesicle-associated membrane proteins increased in differentiated PC12-Dp40 cells, in contrast to PC12-Dp40L170P cells, while neurofilament light-chain was decreased in both differentiated cells. These results suggest that Dp40 has an important role in the neuronal differentiation of PC12 cells through the regulation of proteins involved in neurofilaments and exocytosis of synaptic vesicles, functions that might be affected in PC12-Dp40L170P.

Highlights

Dp40 is the smallest dystrophin reported to date and is transcribed from intron 62 to exon 70 of the dmd gene[1]
It has been reported that Dp40 interacts with syntaxin 1A (STX1A), vesicle-associated membrane protein 2 (VAMP2) and synaptosome-associated protein 25 (SNAP25), a group of presynaptic proteins involved in exocytosis of synaptic vesicles of the hippocampus and cortex in the mouse brain[9]
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 (Supplementary Figure S1)

Summary

Introduction

Dp40 is the smallest dystrophin reported to date and is transcribed from intron 62 to exon 70 of the dmd gene[1]. Dp40 protein shows high expression in neuronal cells and a decrease in non-neuronal cells in primary culture of the mouse hippocampus. A report showed that six patients with different degrees of cognitive deficit had a deletion of three base pairs at positions 9711–9714 in the dmd gene[12]. This deletion is located in the same residue where the punctual change from leucine to proline is located in the mutant of Dp40 ( Dp40L170P). The results of this study provide valuable information about the role of Dp40 in neurite outgrowth during neural differentiation and its participation in cognitive deficits when the Dp40 isoform is disrupted

Methods

Results

Conclusion