Tissue- and cell-specific whole-transcriptome meta-analysis from brain and retina reveals differential expression of dystrophin complexes and new dystrophin spliced isoforms.

The large DMD gene encodes a group of dystrophin proteins in brain and retina, produced from multiple promoters and alternative splicing events. Dystrophins are core components of different scaffolding complexes in distinct cell types. Their absence may thus alter several cellular pathways, which might explain the heterogeneous genotype-phenotype relationships underlying central comorbidities in Duchenne muscular dystrophy (DMD). However, the cell-specific expression of dystrophins and associated proteins (DAPs) is still largely unknown. The present study provides a first RNA-Seq-based reference showing tissue- and cell-specific differential expression of dystrophins, splice variants and DAPs in mouse brain and retina. We report that a cell type may express several dystrophin complexes, perhaps due to expression in separate cell subdomains and/or subpopulations, some of which with differential expression at different maturation stages. We also identified new splicing events in addition to the common exon-skipping events. These include a new exon within intron 51 (E51b) in frame with the flanking exons in retina, as well as inclusions of intronic sequences with stop codons leading to the presence of transcripts with elongated exons 40 and/or 41 (E40e, E41e) in both retina and brain. PCR validations revealed that the new exons may affect several dystrophins. Moreover, immunoblot experiments using a combination of specific antibodies and dystrophin-deficient mice unveiled that the transcripts with stop codons are translated into truncated proteins lacking their C-terminus, which we called N-Dp427 and N-Dp260. This study thus uncovers a range of new findings underlying the complex neurobiology of DMD.

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

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.

The dystrophin isoform Dp71eΔ71 is involved in neurite outgrowth and neuronal differentiation of PC12 cells.

The Dp71 protein is the most abundant dystrophin in the central nervous system (CNS). Several dystrophin Dp71 isoforms have been described and are classified into three groups, each with a different C-terminal end. However, the functions of Dp71 isoforms remain unknown. In the present study, we analysed the effect of Dp71eΔ71 overexpression on neuronal differentiation of PC12 Tet-On cells. Overexpression of dystrophin Dp71eΔ71 stimulates neuronal differentiation, increasing the percentage of cells with neurites and neurite length. According to 2-DE analysis, Dp71eΔ71 overexpression modified the protein expression profile of rat pheochromocytoma PC12 Tet-On cells that had been treated with neuronal growth factor (NGF) for nine days. Interestingly, all differentially expressed proteins were up-regulated compared to the control. The proteomic analysis showed that Dp71eΔ71 increases the expression of proteins with important roles in the differentiation process, such as HspB1, S100A6, and K8 proteins involved in the cytoskeletal structure and HCNP protein involved in neurotransmitter synthesis. The expression of neuronal marker TH was also up-regulated. Mass spectrometry data are available via ProteomeXchange with identifier PXD009114. SIGNIFICANCE: This study is the first to explore the role of the specific isoform Dp71eΔ71. The results obtained here support the hypothesis that the dystrophin Dp71eΔ71 isoform has an important role in the neurite outgrowth by regulating the levels of proteins involved in the cytoskeletal structure, such as HspB1, S100A6, and K8, and in neurotransmitter synthesis, such as HCNP and TH, biological processes required to stimulate neuronal differentiation.