THE IN-VIVO IMPACT OF SMPX MUTATIONS DURING DEVELOPMENT AND DISEASE

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in both cardiac and skeletal muscles, as well as in the inner ear, with suggested roles as mechano-transductor. In the last decade, several mutations in SMPX, mainly nonsense and frameshift, have been associated with X-chromosomal progressive non syndromic hearing loss in humans. However, the lack of a reliable animal model has strongly limited functional studies of SMPX in vivo, thus very little information is known concerning the roles of SMPX. In the first part of this work, the zebrafish Smpx protein was localized at the level of the inner ear hair cell apical plasma membrane and specifically in the actin-rich cuticular plate. Functional studies via gene knockdown demonstrated that Smpx-deficient embryos had fewer kinocilia in the hair cells of the inner ear, that also resulted morphologically altered. In addition, Smpx-deficient hair cells were no longer able to mechanotransduce sound waves. Surprisingly, we discovered a hitherto unknown pathogenic phenotype in the skeletal muscle as indeed, slow and fast muscle fibers of Smpx-deficient larvae were evidently impacted as compared to control fibers, leading to a severe movement impairment possibly due to the accumulation of ROS species. In the second part of the work, we started a preliminary analysis in the smpxcrispant embryos generated via CRISPR/Cas9 technology, uncovering that the V-shaped patterning of Vinculin, the only known interactor of Smpx, was completely misshaped in the myotendinous junctions; additionally, we pinpointed that a small proportion of Vinculin became mislocalized within the myotome. More recently, different SMPX missense variants were identified as the genetic origin of a novel type of distal myopathy in human patients. We overexpressed the p.S78N and p.A13V pathological SMPX human forms in zebrafish finding slow muscle fibers aberrations in terms of length, and a general somite disorganization compared to those overexpressing the wild type human form. We also uncovered smpx expression in the neuromast mechanosensory hair cells of both anterior and posterior lateral line. The protein was localized throughout the cytoplasm of such cells and, as opposed to inner ear hair cells, also in the primary cilium. Loss-of-function experiments revealed that the lack of Smpx leads to fewer neuromast clusters due to the smaller size of the posterior lateral line primordium where the Smpx protein was localized as well. A significant reduction in the mechanotransduction activity of the neuromast hair cells was also observed. Eventually, a preliminary RNA-seq profiling of Smpx-deficient and control embryos revealed several dysregulated genes and signaling pathways that might account for the above-mentioned phenotypes. These results will be the starting point for the future elucidations of the pathomechanisms, associated to SMPX mutations, in the zebrafish stable knockout models that are currently under generation.