Unravelling the physiopathological role of sacsin, the protein mutated in the autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).

Sacsin is a large, multimodular protein encoded by the SACS gene and found only in vertebrates. It is expressed in the central nervous system, especially in Purkinje cells and corticospinal neurons. Bioinformatics’ predictions suggest that sacsin plays a role in protein quality control. However, to date sacsin physiological functions and subcellular localization are still open questions. Loss of function mutations in SACS gene cause Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS), an early-onset neurodegenerative disorder. Being sacsin function still unknown, the molecular pathogenesis leading to ARSACS has very poorly been investigated. My thesis is focused on exploring sacsin physiopathological role at the cellular level and on dissecting the pathogenesis of ARSACS. First, we examined how sacsin mutations alter the residual amount of the protein in ARSACS patient cells, since until now it has never been evaluated, leaving a hole in the genotypephenotype correlation in ARSACS. At the same time, we investigated the effects of the lack of sacsin in different models depleted of its expression (ARSACS patient fibroblasts, SACS KO HeLa cells, Sacs KO mouse embryonic fibroblasts, and primary Purkinje neurons from the Sacs KO mouse model). We found alterations in mitochondrial dynamics, namely mitochondrial hyperfusion and, by dissecting the molecular mechanism, we demonstrated that the mitochondrial fission process was indeed impaired. In line with these results, we showed that mitochondrial hyperfusion has a strong impact on mitochondrial trafficking in primary Purkinje cells that lack sacsin expression. This highlights the importance of this phenotype in the pathogenesis of ARSACS, as Purkinje neurons are the first cells to be affected. In sacsin-depleted cells, we detected a dramatic remodelling of the cytoskeletal network, particularly of intermediate filaments. Our data supports the hypothesis that sacsin may be involved in intermediate filament remodelling in an indirect manner, such as by regulating their folding. This is also suggested by the predicted chaperone-like activities of certain sacsin domains. An intermediate filament reorganization and/or accumulation could sterically hinder both transport and distribution of mitochondria in Purkinje neurons of Sacs KO mouse. On the other hand, cytoskeletal derangement could be responsible for impairing DRP1 recruitment. The cellular phenotypes that we have identified in the absence of sacsin are steps forward in understanding the pathogenetic cascade of ARSACS and shed light on potential sacsin functions.