Sacsin deletion disrupts the response of C6 rat glioblastoma cells to inflammatory cues

Abstract

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare ataxia caused by mutations in the SACS gene, which encodes for the sacsin protein. Loss of sacsin modifies the expression and distribution of intermediate filaments in neurons. Autophagy is elevated in ARSACS, but other histopathological features typical of neurodegenerative disorders have been barely studied for this disease. We aimed at analysing the alterations in neuroinflammatory and endoplasmic reticulum (ER) stress pathways upon deletion of sacsin. We deleted sacsin from C6 rat glioblastoma cells by means of a CRISPR/Cas9 approach, isolated sacsin knockout cell lines by Flow Cytometry-assisted Cell Sorting and analysed the effects of sacsin loss in the levels of intermediate filaments by western blotting. Neuroinflammation and ER stress pathways were later analysed upon stimulation by different cytokines for 20 min and 24h. C6 cells expressed high levels of sacsin, as well as the three main glial intermediate filaments: GFAP, vimentin and nestin. Removal of sacsin caused a striking increase in the protein levels of intermediate filament networks. C6 Sacs KO cells showed an impaired inflammatory response to IL-6/IL-6R stimulation, using STAT3 activation as a surrogate. BMP-2 and IL-6/IL-6R induction greatly activated the SMAD pathway in reference and knockout cells. No inflammatory response was registered upon LIF incubation. CHOP expression was highly reduced in C6 Sacs KO cells but there were no differences in the expression of calnexin and PERK between the two cell lines either in the presence or absence of cytokines. In summary, sacsin loss impairs glial intermediate filament assembly and intracellular distribution, response to inflammatory cytokines and specific ER stress pathways. These results point at a potential role for sacsin in glial cells, and could be relevant for the treatment of ARSACS, but also for a wide spectrum of human pathologies caused by disruption of intermediate filament networks.