S100B Mitigates Cytoskeletal and Mitochondrial Alterations in a Glial Cell Model of Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay

Abstract

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurological disorder caused by mutations in the SACS gene, resulting in the loss of sacsin function. Sacsin is a multidomain protein that plays key roles in chaperone regulation, protein quality control, and neurofilament dynamics. Sacsin deficiency leads to disruption of intermediate filament and mitochondrial networks. S100B, a multifunctional brain-enriched protein, exhibits protective neuroprotective functions that include chaperone activity and interactions with filament proteins and mitochondria. In this study, we used an established astroglial C6 cell model of ARSACS to investigate the potential compensatory effects of S100B on sacsin loss with respect to neurofilament integrity and mitochondrial morphological and functional hallmarks. Our results demonstrate that sacsin deletion induces S100B upregulation at both mRNA and protein levels, with the S100B protein colocalizing with perinuclear nestin aggregates and filamentous mitochondria networks. Genetic silencing and pharmacological inhibition of S100B exacerbate filament protein aggregation and mitochondrial defects, while supplementation with exogenous recombinant S100B improves ARSACS hallmarks, including decreased nestin aggregates. These findings provide evidence for functional compensation of sacsin loss by S100B in glial cells, and suggests a potential role for glial cells in ARSACS.

Keywords: ARSACS; Astroglia; Intermediate filaments; Mitochondria; Molecular chaperones; S100B; Sacsin.

Fig. 1

Sacsin deletion increases S100B levels and alters its subcellular distribution. a Representative images of C6 and C6^Sacs−/− cells showing nestin juxtanuclear accumulation (green), nucleus (blue) and mitochondria (magenta). Scale bar – 20 µm. b, c Quantitative analysis of nestin area and nestin tangles circularity, respectively. Data were collected from 6 independent experiments with a total number of 982 WT cells and 1449 KO cells analysed. Data are shown as mean ± SEM; unpaired student’s t-test: *P < 0.05, **P < 0.01, ****P < 0.0001. d, e Quantitative analysis of mitochondria aspect ratio (d) and total branch length per area (µm) (e), respectively. Data were collected from 8 independent experiments with a total number of 481 WT cells and 631 KO cells analysed. Data are shown as mean ± SEM; unpaired student’s t-test: *P < 0.05, **P < 0.01, ****P < 0.0001. f Representative western blots showing sacsin deletion and S100B overexpression levels in C6 and C6^Sacs−/−cells. g Levels of S100B mRNA are upregulated in C6^Sacs−/− (n = 6, p-value = 0.0183). Data are expressed as fold change over S100B levels in reference C6 cells. Data are represented as mean values ± SEM and statistical analysis was performed through Student’s t-test. *p < 0.05. h, i Representative images of C6 and C6^Sacs−/− cells with endogenous S100B (h) and overexpression of an exogenous S100B-cerulean construct (i), respectively. When overexpressed in the C6^Sacs−/−cells, S100B locates between IFs tangles and nucleus in C6^Sacs−/−cells, indicated by white arrow. Scale bar – 20 µm

Fig. 2

Effects of S100B modulation on nestin accumulation and mitochondrial morphology in C6^Sacs−/− cells. a Immunocytochemistry images showing nestin distribution (green), mitochondrial network (magenta) and nucleus (blue) after transfection with siRNA control or against S100B; after 48 h incubation with pentamidine (0.5 µM) and after incubation with exogenous S100B (30 µM). Scale bar – 20 µm. b Representative western blot showing inhibition of S100B with siRNA and decrease in nestin levels of expression. c Densitometric analysis of western blots normalized to GAPDH levels. Fold-change representation of S100B and Nestin comparing to C6^Sacs−/− cells transfected with siRNA control (p-values = 0.0010 and 0.0284, respectively; unpaired t-test, n = 5). d Graphical representation of the percentage of cells with nestin accumulation. Data are shown as mean ± SEM; unpaired student’s t-test: *P < 0.05, **P < 0.01. e, f Quantitative analysis of nestin area (µm²) and nestin tangle circularity, respectively, following S100B inhibition with siRNA (752 cells) or 0.5 µM pentamidine (1422 cells) or exposure to 30 µM of exogenous S100B (1055 cells). siRNA control (628 cells) was used for comparison with siRNA S100B and non-treated C6^Sacs−/− cells (2117 cells) were used as control for pentamidine and exogenous S100B. Data were obtained from at least 3 independent experiments and are shown as mean ± SEM; one-way ANOVA followed by Tukey’s test: *P < 0.05, **P < 0.01, ****P < 0.0001. g, h Quantitative analysis of mitochondria aspect ratio and total branch length per area (1/µm), respectively following S100B inhibition with siRNA (736 cells) or 0.5 µM pentamidine (428 cells) or exposure to 30 µM of exogenous S100B (398 cells). siRNA control (498 cells) was used for comparison with siRNA S100B and non-treated C6^Sacs−/− cells (636 cells) were used as control for pentamidine and exogenous S100B. Data were collected from at least 3 independent experiments. Data are shown as mean ± SEM; one-way ANOVA followed by Tukey’s test: *P < 0.05, **P < 0.01, ****P < 0.0001

Fig. 3

Pharmacological inhibition of S100B activity influences mitochondrial homeostasis and function. a Number of mitochondria per cell comparing C6 (483 cells), C6^Sacs−/− (636 cells) and C6^Sacs−/− treated with pentamidine (428 cells). Data are shown as mean ± SEM; one-way ANOVA followed by Tukey’s test: ****P < 0.0001. b-f Transcript levels of DRP1, MFN2, OPA1, TFAM and LDH in C6 and C6^Sacs−/− incubated with pentamidine or the vehicle (DMSO 0.1% v/v). Data are represented as mean ± SEM (n = 4–10 independent experiments), normalized versus reference C6 cells. Statistical analysis was performed through one-way ANOVA followed by Tukey’s test: *P < 0.05. g LDH activity in the same experimental groups. Data are represented as mean ± SEM of 7 independent experiments, normalized versus reference C6 cells. h ATP levels are represented as mean ± SEM of 5 independent experiments, normalized versus reference C6 cells. Statistical analysis was performed through one-way ANOVA followed by Tukey’s test:****p < 0.0001. i Graphical representation of ratio between JC-1 aggregates (FL2—585/42 nm) and JC-1 monomers (FL1—530/30 nm). Data are represented as mean ± SEM (n = 4 independent experiments), Statistical analysis was performed through one-way ANOVA followed by Tukey’s test:*p < 0.05