Investigare e targettare la deregolazione del calcio in ARSACS

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset cerebellar ataxia associated with lower limb spasticity and peripheral neuropathy. ARSACS is caused by loss-of-function mutations in SACS gene, which encodes for sacsin, a huge cytosolic protein mainly expressed in neurons with the highest levels in Purkinje cells (PCs). Loss of PCs is indeed the most prominent feature of ARSACS patients and mouse models (both Sacs-/- and SacsR272C/R272C mice). In the last years several studies in cell models have been performed to understand sacsin function, that however remains largely unknown so far. We and others have identified the remodeling of the intermediate filament (IF) cytoskeleton as one of the earliest consequences of the absence of sacsin. Both vimentin (in ARSACS patient fibroblasts, SACS-/- HEK293T and SH-SY5Y cells) and neurofilaments (NFs, in different types of neurons) accumulate in the absence of sacsin, forming aberrant dense bundles. However, in the mouse models only PCs were found degenerating and, thus, causing motor abnormalities typical of ARSACS clinical spectrum. To date, no more information is known about ARSACS pathogenetic cascade and, thus, no treatments are available for ARSACS. My PhD project aimed at dissecting ARSACS pathogenesis, by studying the effects of NF accumulation specifically in PCs and how this phenotype causes degeneration. Based on the knowledge derived from these studies, we designed and carried out a pharmacological treatment in Sacs-/- mice. We demonstrated that the accumulation of non-phosphorylated NFH (npNFH) bundles in PCs is an early event in Sacs-/- mice, appearing just after birth and characterizing mainly the anterior lobules of cerebellum. To mechanistically explore the consequences of npNFH accumulation, we took advantage of Sacs-/- cerebellar cultures enriched in PCs, which recapitulate the main features observed in vivo. We discovered that mitochondrial (and also ER) trafficking to distal dendrites is altered in Sacs-/- primary PCs. The failure in transportation was not a consequence of altered mitochondrial metabolism or morphology, as we found that mitochondria manifest conserved ultrastructure, as well as unaltered ATP production and mitochondrial membrane potential, both in vivo and ex vivo in Sacs-/- PCs. To identify the missing link between NF accumulation and defective mitochondrial (and ER) distribution, we immunoprecipitated endogenous sacsin in a simpler tool, i.e. SH-SY5Y cells differentiated into neurons. In these cells we previously demonstrated aberrant NF remodelling upon depletion of sacsin by genome editing. This approach allowed us to detect some physical sacsin interactors, all related to cytoskeleton. In addition to NFs, we pulled down plectin, a large cytolinker protein interacting both with NFs and mitochondria, and myosin Va, crucial for ER transport in dendrites. This suggests that abnormal NF accumulation in the absence of sacsin may oppose to mitochondrial and ER trafficking, thus favouring their docking in proximal dendrites. To validate plectin involvement in ARSACS pathogenesis, we revealed decreased plectin levels in SACS-/- SH-SY5Y cells and in a panel of ARSACS patient fibroblasts (analysis of plectin levels in cerebellum are now ongoing). These results support the idea that sacsin may act as a scaffold for cytoskeletal proteins, mediating a connection between cytoskeleton and organelles. We thus hypothesized that defective mitochondrial and ER trafficking to PC dendrites could lead to pathologic Ca2+ deregulation in Sacs-/- PCs, leading to degeneration. In fact, both these organelles are crucial regulators of Ca2+ homeostasis. In particular, mitochondria not only provide ATP to active Ca2+ clearance systems at the plasma membrane and ER, but also exert themselves a fine shaping of Ca2+ signals in spines by accumulating Ca2+ into the matrix. In support of our hypothesis, two in vivo OMICS approaches revealed deregulation of many key players regulating Ca2+ homeostasis, which was further confirmed by increased phosphorylation of CamKIIβ and by Ca2+ imaging experiments in primary PCs. These results provided us the rationale to test a pharmacological treatment with Ceftriaxone in the Sacs-/- mouse model. Ceftriaxone is a β-lactam antibiotic able to reduce glutamate concentration in inter-synaptic space and thus to attenuate Ca2+ influxes in post-synaptic PCs. Interestingly, we proved that Ceftriaxone administration, at both pre- and post-symptomatic stages, improves motor ability and delays PC degeneration in Sacs-/- mice. This treatment may represent a future therapeutic option for diagnosed presymptomatic ARSACS patients, but also for patients with overt symptoms, which are most cases. Finally, our data revealed an early involvement of neuroinflammation in ARSACS disease progression, showing a remarkable astrocyte and microglia activation in Sacs-/- cerebellum as early as 1 month of age. Preliminary data suggest that Ceftriaxone treatment may target also this mechanism (probably via NF-kB signaling), offering neuroprotection in ARSACS by multiple mechanisms. Overall, our data improve the knowledge of ARSACS pathogenesis and offer encouraging perspectives for ARSACS disease treatment