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 6

Postsymptomatic Ceftriaxone treatment improves motor coordination, delays PC loss, and mitigates Ca2+ deregulation in Sacs–/– cerebellum.

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Postsymptomatic Ceftriaxone treatment improves motor coordination, delay...
(A) Schematic representation of preclinical postsymptomatic Ceftriaxone administration protocol. (B) BW test performance in term of latency time to cross the beam and number of hindfoot missteps of female mice of the indicated genotypes, as well as mice that were Ceftriaxone treated and vehicle treated, at 7 months of age. Data are shown as mean ± SEM; n = at least 5; 2-way ANOVA with Tukey’s correction. *P < 0.05, ***P < 0.001, ****P < 0.0001. (C) Representative semithin sections of cerebellum of Ceftriaxone- and vehicle-treated mice of the indicated genotype, with relative quantitation of PC density at 7 months. Scale bar: 25 μm. Data are shown as mean ± SEM; n = at least 4 (10 images per sample); 2-way ANOVA with Tukey’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Arrows indicate the PC layer. (D) WB analysis showing levels of pCaMKIIβ (upper band as indicated by the arrow), CaMKIIβ, and npNFH in WT, vehicle-treated, and Ceftriaxone-treated Sacs–/– cerebellum at 7 months of age with relative quantitation (normalized to calnexin). Data are shown as mean ± SEM; n = at least 3; 2-way ANOVA with Tukey’s correction. *P < 0.05, **P < 0.01. (E) qPCR showing levels of Gfap mRNA (relative to Hprt1 mRNA) in WT, vehicle-treated, and Ceftriaxone-treated Sacs–/– cerebellum at 7 months of age. Data are shown as mean ± SEM; n = at least 4; 2-way ANOVA with Tukey’s correction. *P < 0.05, ***P < 0.001. (F) Representative images of immunofluorescence analysis showing astrocytes (GFAP, in red) in 7-month-old Ceftriaxone- and vehicle-treated Sacs–/– and WT control cerebellum. Data are shown as mean ± SEM; n = 3; 2-way ANOVA with Tukey’s correction. *P < 0.05. Scale bar: 0.2 mm.