Restoring calcium homeostasis in Purkinje cells arrests neurodegeneration and neuroinflammation in the ARSACS mouse model
Andrea Del Bondio, Fabiana Longo, Daniele De Ritis, Erica Spirito, Paola Podini, Bernard Brais, Angela Bachi, Angelo Quattrini, Francesca Maltecca
Andrea Del Bondio, Fabiana Longo, Daniele De Ritis, Erica Spirito, Paola Podini, Bernard Brais, Angela Bachi, Angelo Quattrini, Francesca Maltecca
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Research Article Cell biology Neuroscience

Restoring calcium homeostasis in Purkinje cells arrests neurodegeneration and neuroinflammation in the ARSACS mouse model

Abstract

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.

Authors

Andrea Del Bondio, Fabiana Longo, Daniele De Ritis, Erica Spirito, Paola Podini, Bernard Brais, Angela Bachi, Angelo Quattrini, Francesca Maltecca

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Figure 4

Ca2+ deregulation in Sacs–/– primary PCs and cerebellum.

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Ca2+ deregulation in Sacs–/– primary PCs and cerebellum. (A) Representat...
(A) Representative traces of cytosolic Ca2+ responses before and after KCl stimulation of Sacs–/– and WT control primary PCs. Graph shows PC Ca2+-evoked responses after stimulation with 30 mM KCl (normalized increase measured above the initial value). Data are shown as mean ± SEM; n = at least 15 from at least 5 independent experiments; Welch’s t test. ****P <0,0001. (B) Granule cell Ca2+-evoked responses after stimulation with 30 mM KCl (normalized increase measured above the initial value). Data are shown as mean ± SEM; n = at least 19 from at least 5 independent experiments; Welch’s t test. (C) Heatmap of cerebellar protein profile comparing Sacs–/– and WT controls at 5 months of age; n = 6 from 3 biological replicates. (D) g:Profiler enrichment of deregulated proteins comparing 5-month-old Sacs–/– and WT cerebellum, showing the top 10 categories for each GO: molecular function and biological process. Color bar represents number of proteins. (E–G) WB analysis showing levels of IP3R1 (E), Calbindin and PCP2 (F), and pCaMKIIβ (upper band as indicated by the arrow) and CaMKIIβ (G) in Sacs–/– and WT control cerebellum at 5 months of age with relative quantitation (normalized to calnexin). Data are shown as mean ± SEM; n = at least 4; Welch’s t test. *P < 0.05, **P < 0.01.