Cieri, Federica (2024) A network-biology based approach for the development of SMN-independent treatments. [Tesi di dottorato]

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Abstract

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disease caused by mutations or deletions of the Survival of Motoneuron 1 gene (SMN1), resulting in low levels of SMN protein. Current treatments for SMA, designed to restore SMN protein levels, have impressive effects but cause some side effects. Moreover, later stages of the neurodegeneration cannot be reversed only by SMN-restoration. It is, therefore, essential to better elucidate the molecular pathomechanism of the disease and to improve the current SMA therapeutic strategies, in order to significantly improve patients’ lifestyle. In this regard, since SMN protein levels have already been enhanced in treated patients, complementary SMN-independent approaches are needed. To this purpose model organisms and bioinformatics are essential tools. In SMA several altered signaling pathways regulating neurodegeneration have been identified. These signaling pathways form networks which connectivity is still not completely known. Elucidating the networks of altered signaling in SMA could be a good strategy to identify new potential SMN-independent therapeutic targets. Using a phospho-array approach on SMA mice and network-biology, 38 dysregulated proteins involved in motoneuron degeneration have been identified, and B-Raf pinpointed as major signaling hub. Analyses in SMA mice revealed that B-Raf was down-regulated in motoneurons and in the spinal cord with B-Raf being reduced at pre-symptomatic stages. These results have been confirmed in patients and are also conserved across species; a Caenorhabditis elegans SMA model, indeed, showed less expression of the B-Raf homolog lin-45. In this PhD project, the role of B-Raf/lin-45 has been characterized in vivo using a C. elegans model of SMA, demonstrating its cell autonomous neuroprotective role when smn-1 is down-regulated. This neuroprotective effect was demonstrated to be mediated by the MEK/ERK pathway. Then, using C.elegans, was investigated the role played by the others 37 proteins belonging to the network in order to choose the best target genes to be further studied. Thus, to rapidly identify modifier genes among those 37 candidates, a new C. elegans model of SMA has been set up. 16 new modifier genes among all the 37 genes tested have been identified. Using cell-specific RNAi, we demonstrated that 11 play a role in neurons and 5 of those specifically in MNs. Two of them, that act as suppressors of the neurodegeneration caused by smn-1 down-regulation, have been chosen for further studies and validation in mammals. Analysis in mammal models of SMA confirmed what had been observed in C. elegans and suggested a possible SMN-independent role of these candidates. These data unveil a neuroprotective role of a network of genes in MNs which are candidate targets for future therapies in SMA and support that the combined use of in vivo and in vitro model systems contribute significantly to achieving rapid progress in the study of a devastating disease such as SMA.

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