Sacsin deficiency drives early alterations in glial intermediate filament and astroglial reactivity in zebrafish

Autosomal recessive spastic ataxia of Charlevoix–Saguenay (ARSACS) is an early-onset neurodegenerative disorder [1] caused by loss-of-function mutations in sacsin [2], a multifunctional chaperone involved in proteostasis and cytoskeletal regulation, particularly of intermediate filaments (IFs) [3]. While disruption of neuronal IF organization has been well documented in ARSACS models, the role of sacsin in glial cells remains poorly defined. To date, glial reactivity in ARSACS has largely been interpreted as a secondary neuroinflammatory response to neuronal degeneration [4], raising the question of whether glial alterations may instead arise early from sacsin loss. Here, we investigated whether sacsin deficiency drives early glial changes during development. Using zebrafish as an in vivo model, which enables the study of early neurodevelopmental processes in a whole-organism context, we assessed the expression of the glial IF proteins vimentin and GFAP, along with the astrocytic and microglial stress markers s100b and aif1l, across defined developmental stages. Our aim was to determine whether sacsin loss disrupts glial IF homeostasis and promotes early glial reactivity, thereby contributing directly to ARSACS pathogenesis.

sacs^⁻/⁻ mutant and wild-type (AB^+/+) zebrafish embryos and larvae were collected daily between 60 and 180 hours post-fertilization, covering key stages of early neural and glial development. RT-qPCR was used to quantify the expression of sacs, the astrocytic marker s100b, and the microglial marker aif1l, while Western blotting was performed to assess protein levels of vimentin, a glial precursor IF protein, and GFAP, a marker of differentiated astrocytes. Expression changes were analyzed both across developmental stages and between genotypes to capture temporal patterns as well as genotype-dependent differences.

sacs transcript levels showed no genotype-dependent differences until 180 hpf, when sacs^⁻/⁻ larvae exhibited a significant reduction, coinciding with a developmental surge observed in wild-type larvae that was absent in mutants, suggesting a delayed and stage-specific reduction in sacs expression. In contrast, s100b expression was consistently elevated in sacs^⁻/⁻ larvae across developmental stages, supporting the presence of an early astroglial stress or reactive response. Although aif1l expression did not differ overall between genotypes, a modest increase at 156 hpf in mutants may indicate subtle or delayed microglial activation. At the protein level, vimentin was significantly upregulated in sacs^⁻/⁻ larvae, pointing to disrupted intermediate filament homeostasis in glial precursors. Conversely, GFAP protein levels did not show genotype-dependent differences, likely reflecting the dilution of region-specific astrocytic alterations in whole-embryo lysates.

Our findings demonstrate that sacsin deficiency induces selective and stage-dependent glial alterations during early zebrafish development. The consistent upregulation of s100b and the marked increase in vimentin suggest that sacsin loss disrupts glial intermediate filament homeostasis and promotes an early astroglial stress or reactive response. Future studies will incorporate anatomical and cell-type-specific approaches, including whole-mount immunohistochemistry, to visualize glial intermediate filament organization in vivo. Extending analyses to later developmental stages will further clarify how these early glial alterations evolve over time and contribute to disease progression.

References

1. Shimazaki, H., Takiyama, Y.. 2012. Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS): Clinical, radiological and epidemiological aspects, in: Gazulla, J., Spinocerebellar Ataxia, InTech, 156-172.
2. Bagaria, J., Bagyinszky, E., An, S.S.A., 2022. Genetics of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) and role of sacsin in neurodegeneration. Int. J. Mol. Sci. 23 (1), 552.
3. Gentil, B.J., Lai, G.T., Menade, M., Larivière, R., Minotti, S., Gehring, K., et al. 2019. Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics. FASEB J. 33 (2), 2982-2994.
4. Del Bondio, A., Longo, F., De Ritis, D., Spirito, E., Podini, P., Brais, B., et al. 2023. Restoring calcium homeostasis in Purkinje cells arrests neurodegeneration and neuroinflammation in the ARSACS mouse model. JCI Insight 8 (12), e163576.