Investigare e targettare la deregolazione del calcio in ARSACS

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset cerebellar ataxia 
\nassociated with lower limb spasticity and peripheral neuropathy. ARSACS is caused by loss-of-function 
\nmutations in SACS gene, which encodes for sacsin, a huge cytosolic protein mainly expressed in neurons 
\nwith the highest levels in Purkinje cells (PCs). Loss of PCs is indeed the most prominent feature of ARSACS 
\npatients and mouse models (both Sacs-/-
\nand SacsR272C/R272C mice). In the last years several studies in cell 
\nmodels have been performed to understand sacsin function, that however remains largely unknown so far.
\nWe and others have identified the remodeling of the intermediate filament (IF) cytoskeleton as one of the 
\nearliest consequences of the absence of sacsin. Both vimentin (in ARSACS patient fibroblasts, SACS-/-
\nHEK293T and SH-SY5Y cells) and neurofilaments (NFs, in different types of neurons) accumulate in the 
\nabsence of sacsin, forming aberrant dense bundles. However, in the mouse models only PCs were found 
\ndegenerating and, thus, causing motor abnormalities typical of ARSACS clinical spectrum. To date, no more 
\ninformation is known about ARSACS pathogenetic cascade and, thus, no treatments are available for 
\nARSACS.
\nMy PhD project aimed at dissecting ARSACS pathogenesis, by studying the effects of NF accumulation 
\nspecifically in PCs and how this phenotype causes degeneration. Based on the knowledge derived from 
\nthese studies, we designed and carried out a pharmacological treatment in Sacs-/- mice. 
\nWe demonstrated that the accumulation of non-phosphorylated NFH (npNFH) bundles in PCs is an early 
\nevent in Sacs-/- mice, appearing just after birth and characterizing mainly the anterior lobules of cerebellum. 
\nTo mechanistically explore the consequences of npNFH accumulation, we took advantage of Sacs-/-
\ncerebellar cultures enriched in PCs, which recapitulate the main features observed in vivo. We discovered 
\nthat mitochondrial (and also ER) trafficking to distal dendrites is altered in Sacs-/-
\nprimary PCs. The failure in 
\ntransportation was not a consequence of altered mitochondrial metabolism or morphology, as we found 
\nthat mitochondria manifest conserved ultrastructure, as well as unaltered ATP production and 
\nmitochondrial membrane potential, both in vivo and ex vivo in Sacs-/-
\nPCs.
\nTo identify the missing link between NF accumulation and defective mitochondrial (and ER) distribution, we 
\nimmunoprecipitated endogenous sacsin in a simpler tool, i.e. SH-SY5Y cells differentiated into neurons. In 
\nthese cells we previously demonstrated aberrant NF remodelling upon depletion of sacsin by genome 
\nediting. This approach allowed us to detect some physical sacsin interactors, all related to cytoskeleton. In 
\naddition to NFs, we pulled down plectin, a large cytolinker protein interacting both with NFs and 
\nmitochondria, and myosin Va, crucial for ER transport in dendrites. This suggests that abnormal NF 
\naccumulation in the absence of sacsin may oppose to mitochondrial and ER trafficking, thus favouring their
\ndocking in proximal dendrites. To validate plectin involvement in ARSACS pathogenesis, we revealed 
\ndecreased plectin levels in SACS-/-
\nSH-SY5Y cells and in a panel of ARSACS patient fibroblasts (analysis of 
\nplectin levels in cerebellum are now ongoing). These results support the idea that sacsin may act as a 
\nscaffold for cytoskeletal proteins, mediating a connection between cytoskeleton and organelles.
\nWe thus hypothesized that defective mitochondrial and ER trafficking to PC dendrites could lead to 
\npathologic Ca2+ deregulation in Sacs-/-
\nPCs, leading to degeneration. In fact, both these organelles are crucial 
\nregulators of Ca2+ homeostasis. In particular, mitochondria not only provide ATP to active Ca2+ clearance 
\nsystems at the plasma membrane and ER, but also exert themselves a fine shaping of Ca2+ signals in spines 
\nby accumulating Ca2+ into the matrix. In support of our hypothesis, two in vivo OMICS approaches revealed 
\nderegulation of many key players regulating Ca2+ homeostasis, which was further confirmed by increased 
\nphosphorylation of CamKIIβ and by Ca2+ imaging experiments in primary PCs.
\nThese results provided us the rationale to test a pharmacological treatment with Ceftriaxone in the Sacs-/-
\nmouse model. Ceftriaxone is a β-lactam antibiotic able to reduce glutamate concentration in inter-synaptic 
\nspace and thus to attenuate Ca2+ influxes in post-synaptic PCs. Interestingly, we proved that Ceftriaxone 
\nadministration, at both pre- and post-symptomatic stages, improves motor ability and delays PC 
\ndegeneration 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.
\nFinally, our data revealed an early involvement of neuroinflammation in ARSACS disease progression, 
\nshowing a remarkable astrocyte and microglia activation in Sacs-/-
\ncerebellum as early as 1 month of age. 
\nPreliminary data suggest that Ceftriaxone treatment may target also this mechanism (probably via NF-kB 
\nsignaling), offering neuroprotection in ARSACS by multiple mechanisms.
\nOverall, our data improve the knowledge of ARSACS pathogenesis and offer encouraging perspectives for 
\nARSACS disease treatment

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