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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.

Dp40 is ubiquitously expressed, including in the central nervous system. Dp40 mRNA and protein are detected in the early stages and postnatal stages of the mouse brain, respectively. 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 (a mutant of Dp40) during the neuronal differentiation process 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 carried out in nerve growth factor-differentiated PC12-Dp40L170P cells showed that the protein expression profile was modified compared to that of the control cells (PC12 Tet-On). The proteins with the highest upregulated expression were α-internexin and S100a6, which are involved in cytoskeletal structure. 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. HspB1 was absent in undifferentiated cells and weakly detected in all differentiated cells. These results suggest that the subcellular distribution and expression of Dp40 has an important role in the neurite outgrowth of PC12 cells through the regulation of proteins involved in neurofilaments and exocytosis of synaptic vesicles, functions that might be affected in PC12-Dp40L170P.

Sympathetic neurons of the rat superior cervical ganglion (SCG) can segregate their neurotransmitters and co-transmitters to separate varicosities of single axons. We have shown that transmitter segregation is a plastic phenomenon and that it is correlated with the strength of synaptic transmission. Here, we determined whether sympathetic dysfunction occurring in stress and hypertension was correlated with plastic changes of neurotransmitter segregation. We characterized the expression of the markers, L-glutamic acid decarboxylase of 67 kDa (GAD67) and vesicular acetylcholine (ACh) transporter (VAChT) in the SCG of cold stressed and spontaneously hypertensive rats (SHR). Considering that the SCG comprises a heterogeneous neuronal population, we explored whether the expression and segregation of neurotransmitters would also have an intraganglionic heterogeneous distribution in ganglia of stressed and hypertensive rats. Furthermore, since hypertension in SHR is detected around 8–10 weeks, we evaluated expression and segregation of ACh and GABA in adult hypertensive (12-week old (wo)) and young pre-hypertensive (6-wo) SHR. We found an increase in segregation of ACh and GABA with no change in transmitter expression in ganglia of stressed animals. In contrast, in SHR, there was an increase in GABA expression, although segregation did not vary. Segregation showed a caudo-rostral gradient in controls but not in the ganglia of stressed animals. GABA expression showed a rostro-caudal gradient in adult SHR, which was not present in young 6-wo rats. In young SHR, ACh increased and, unexpectedly, segregation of ACh and GABA was higher than in adults. Data suggest that ACh and GABA segregation increases in acute sympathetic hyperactivity like stress, but does not vary in chronic hyperactivity such as in hypertension. Changes in segregation are age-dependent and might be involved in the mechanisms underlying stress and hypertension.

International audience; PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71 Delta(78-79) dystrophin mutant is stably expressed in PC12-C11 cells. To investigate the effect of Dp71 Delta(78-79) 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. Dp71 Delta(78-79) 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 Dp71 Delta(78-79) 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.