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Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin, a huge protein highly expressed in cerebellar Purkinje cells (PCs). Patients with ARSACS, as well as mouse models, display early degeneration of PCs, but the underlying mechanisms remain unexplored, with no available treatments. In this work, we demonstrated aberrant calcium (Ca2+) homeostasis and its impact on PC degeneration in ARSACS. Mechanistically, we found pathological elevation in Ca2+-evoked responses in Sacs-/- PCs as the result of defective mitochondria and ER trafficking to distal dendrites and strong downregulation of key Ca2+ buffer proteins. Alteration of cytoskeletal linkers, which we identified as specific sacsin interactors, likely account for faulty organellar trafficking in Sacs-/- cerebellum. Based on this pathogenetic cascade, we treated Sacs-/- mice with Ceftriaxone, a repurposed drug that exerts neuroprotection by limiting neuronal glutamatergic stimulation and, thus, Ca2+ fluxes into PCs. Ceftriaxone treatment significantly improved motor performances of Sacs-/- mice, at both pre- and postsymptomatic stages. We correlated this effect to restored Ca2+ homeostasis, which arrests PC degeneration and attenuates secondary neuroinflammation. These findings disclose key steps in ARSACS pathogenesis and support further optimization of Ceftriaxone in preclinical and clinical settings for the treatment of patients with ARSACS.

Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disease caused by mutations of the mitochondrial protease AFG3L2. The SCA28 mouse model, which is haploinsufficient for Afg3l2, exhibits a progressive decline in motor function and displays dark degeneration of Purkinje cells (PC-DCD) of mitochondrial origin. Here, we determined that mitochondria in cultured Afg3l2-deficient PCs ineffectively buffer evoked Ca²⁺ peaks, resulting in enhanced cytoplasmic Ca²⁺ concentrations, which subsequently triggers PC-DCD. This Ca²⁺-handling defect is the result of negative synergism between mitochondrial depolarization and altered organelle trafficking to PC dendrites in Afg3l2-mutant cells. In SCA28 mice, partial genetic silencing of the metabotropic glutamate receptor mGluR1 decreased Ca²⁺ influx in PCs and reversed the ataxic phenotype. Moreover, administration of the β-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca²⁺ influx, improved ataxia-associated phenotypes in SCA28 mice when given either prior to or after symptom onset. Together, the results of this study indicate that ineffective mitochondrial Ca²⁺ handling in PCs underlies SCA28 pathogenesis and suggest that strategies that lower glutamate stimulation of PCs should be further explored as a potential treatment for SCA28 patients.

The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca(2+) uptake capacity. This defect is neither a consequence of global alteration in cellular Ca(2+) homeostasis nor of the reduced driving force for Ca(2+) internalization within mitochondria, since cytosolic Ca(2+) transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca(2+) uptake speed in Afg3l2(-/-) cells, indicating the presence of functional Ca(2+) uptake machinery. Our results show that the defective Ca(2+) handling in Afg3l2(-/-) cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca(2+) elevations, hampering the proper Ca(2+) diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2(-/-) MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca(2+) buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca(2+) homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.