Cellular, molecular and functional alterations in the development of central and peripheral nervous system of mdx mice, an animal model of Duchenne Muscular Dystrophy

Dp427 is a cortical cytoskeletal protein expressed by skeletal, cardiac and smooth muscles, and by some populations of central and autonomic neurons. Tissues other than muscles express also shorter dystrophin isoforms. Progressive and lethal muscle degeneration is the main characteristic of the disease. However, Duchenne Muscular Dystrophy (DMD) patients also experience different forms of cognitive impairment, neurological and autonomic disorders, altered scotopic electroretinogram and red-green color vision defects. Differently from muscles, DMD-related damages to nervous system are established during embryonic development and are not progressive after birth, suggesting a role for all dystrophins in nervous system development and differentiation. My PhD project is focused on the possible role for Dp427 in the following aspects: axonal regeneration of autonomic neurons, specifically dependent on NGF for their survival; neuritic and axonal growth of hippocampal neurons and retinal development, by using genetically dystrophic mice (mdx) and their wild-type (wt) control. Previous studies on the autonomic superior cervical ganglion (SCG) of mdx mice, demonstrated several developmental alterations of SCG neurons: in vitro experiments revealed a reduction in NGF-dependent neuritic outgrowth of mdx mouse neurons compared to the wt, and in vivo axotomy demonstrated a loss of regeneration of adrenergic axons projecting to muscular targets, containing low levels of NGF. Furthermore, protein levels of activated NGF receptor, the pTrkA receptor, and of some kinases dowstream the NGF-TrkA signaling are reduced. These data suggested that mdx mouse SGC neurons are less sensitive to NGF compared to wt and that limited amount of NGF could alter the dynamics of axonal growth and regeneration of these neurons. In order to confirm that the altered axonal regeneration previously observed in mdx mice in vivo depends on intrinsic neuronal damages, we analysed wt and mdx mouse SCG axon regeneration in vitro, without the influence of their target tissues, by using microfluidic chambers which allow to separate somas and axons in two different compartments and to treat the two sides independently. 24h after in vitro axotomy of SCG neuron axons in the presence of limited amount of NGF, we observed a significant reduction in the number of regenerated axons in mdx mouse neuron cultures compared to the wt. Moreover, co-immunolocalization of TrkA and some Dystrophin-Glycoprotein Complex proteins in the growth cone of SCG axons revealed a reduced co-localization in mdx neurons compared to the wt, leading to the hypothesis that Dp427 contributes to TrkA stabilization in growth cone. Therefore, the lack of Dp427 may cause a reduction in membrane TrkA stabilization and, consequently, reduction in the NGF intracellular signaling cascade. To verify whether the role of Dp427 in neurite growth was exclusive of NGF-dependent neurons, or could be important also for other types of neurons, we analysed the same parameters in hippocampal neurons in vitro. These central neurons are functionally affected by the lack of Dp427 in both DMD patients and animal models. Differently from the SCG neurons, we did not observe differences in neuritic growth parameters between the two genotypes. We, therefore, specifically analysed axonal growth of hippocampal neurons in the microfluidic chambers, in correlation with the activity of the neurotrophic factor BDNF. Hippocampal neurons are responsive to BDNF which increases axonal growth even if is not necessary for survival. Our results demonstrate that axonal growth is slower in the mdx genotype compared to wt, in both presence and absence of BDNF. Retina is characterized by different cell types arranged according to a complex organization, which is finely specified by sequential gene expression during development. Different isoforms of dystrophin are found in the synapses between photoreceptors and bipolar cells, in the inner and outer nuclear layers and in amacrine cells. Absence of dystrophin mainly induces the destabilization of glutamate receptors and alterations of the electroretinogram. In order to identify possible alterations in mdx mouse retinal development, we analysed wt and mdx mouse retinas at different post-natal stages: P0, P5, P10 and adults (6-7 weeks). We performed a comparative morphometric analysis on retina sections from wt and mdx mice, in order to identify possible alterations in the formation of the different layers. The obtained results show that, at P0, the entire retina and the ganglion cell layer (GCL) were significantly thicker in mdx mice compared to wt. Both these regions, as well as the outer segment of photoreceptor (ph), are significantly thinner in mdx mice compared to wt at P5, and similar between the two genotypes at later, more mature, ages. We, therefore, hypothesized a delay, and/or an unbalance, in the initial steps of retina differentiation (characterized by a highly predetermined cell body migration along the retina Y axis) in the mdx genotype compared to control. Successively, we analysed, the distribution of Hoechst stained nuclei in the inner plexiform layer (IPL) and in the GCL, and the acquisition of differentiated chemical phenotypes, by immunolabeling for cell specific markers: calretinin (retinal ganglion cells-RGCs), PKC-α (bipolar cells-BCs) and calbindin (horizontal cells-HCs). Quantitative analysis of the number of differentiated cells/mm, disclosed early differences (between P0 and P10) in the number of nuclei in the GCL and IPL, as well as in the number of immunopositive RGCs, HCs and BCs, in mdx mouse retina compared to wt. We also investigated the onset of ribbon synapse formation, the tripartite synaptic structure established among phs, BCs and HCs, by immunolabeling for its specific marker VGluT1. At a first analysis, timing of formation was similar between the two genotypes and gross abnormalities were not revealed by simple immunofluorescence analysis. However, some difference in the quality of immunolabeling observed suggests that fine ultrastructural alteration may exist between wt and mdx mice, which will deserved a deeper analysis by electron microscopy. One of the major hallmarks of retina development is the temporary establishment, at early post-natal dates (P0-P15), of a large cholinergic circuit between starburst amacrine cells and RGCs, the spontaneous cholinergic activity of which is fundamental for correct retinal differentiation and layering. We previously demonstrated that, in the SCG, Dp427 is involved in the stabilization and activity of α3β2/β4-nAChRs. In the retina a number of different nAChR has been identified, among which receptors containing α3, β2, β4 and α7 subunits. We, therefore, began to investigate for possible differences in the protein levels of these subunits at early post-natal dates (P0, P5, and P10). The obtained results indicate a temporary, but significant, reduction in the expression of α3, β4 and α7 subunits in mdx mouse retina respect to wt. Finally, a RNA seq analysis revealed the presence of genes that were differently modulated between the two genotypes at P5. In particular, 9 genes were up-regulated in mdx mice respect to wt and verified by real time RT-PCR. Among these, genes encoding proteins involved in the rescue of cells by apoptotic cell death, in the protection from erroneously folded proteins, in cell signalling and development. Only one of the down-regulated genes, Erdr1, was confirmed by real time. Interestingly, up-regulated genes were exclusive of the ciliary margin, the most anterior part of retina, source of retinal staminal cells. These data argue in favor of a role of the sole Dp427 in final stages of early post-natal differentiation of those retinal neurons expressing Dp427, possibly regulating the correct structural and functional (e.g. intracellular signaling) connection between cell cytoskeleton and extracellular matrix.