Sacsin deletion disrupts Golgi organization and Autophagy in C6 rat glioblastoma cells

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare childhood-onset ataxia characterized by progressive cerebellar ataxia, spasticity, motor sensory neuropathy and axonal demyelination. ARSACS is caused by mutations in the SACS gene, which leads to the production of defective forms of the 520 kDa multidomain protein sacsin. C6 rat glioblastoma cells were targeted for sacsin deletion by means of CRISPR/Cas9 technique to generate an astroglial model of ARSACS. Sacsin knockout cell lines were isolated by Flow Cytometry-assisted Cell Sorting and sacsin loss was subsequently confirmed in further experiments. Intermediate filaments (IF) expression levels were analyzed upon serum starvation and incubation with IL-6 and BMP2 cytokines, while IF aggregation and their disruption on organelle organization was observed by immunocytochemistry. C6 SACS-/- cells revealed an increase in expression of IF proteins and aggregation of nestin in the juxtanuclear area. Golgi apparatus was often pushed out of the perinuclear area and disarrayed in SACS-/- cells. STAT3 and SMAD1/5 signalling was altered in C6 SACS-/- cells in response to cytokines. The expression of the alarmin protein S100B was significantly higher in C6 SACS-/- cells. The expression of the cytosolic form of the autophagy protein LC3-I was identical in both cells lines but the levels of the activated LC3-II form was reduced in C6 SACS-/- cells, indicating alterations in the autophagy flux. ER stress pathways were also analyzed for possible novel phenotypes in ARSACS cells. The expression of the Unfolded Protein Response (UPR)-related protein Bip did not differ for both C6 strains. CHOP had an increased expression in reference C6 cells in normal conditions but was unexpectedly not expressed in conditions of serum starvation. Withaferin A (WFA), a drug known to bind and disorganize the type III IF vimentin, disrupted Nestin filaments and induced Golgi dispersion similar to C6 SACS-/- cells. In conclusion, lack of sacsin protein disrupts glial ...