Sacsin deletion disrupts Golgi organization and Autophagy in C6 rat glioblastoma cells
Bibliographic record
Abstract
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 intermediate filament assembly and intracellular organelle distribution, enhances S100B expression and impairs autophagy, while being inconclusive regarding ER stress. These results suggest a potential function for sacsin in glial cells and may have implications for treating ARSACS as well as a variety of human illnesses caused by the disruption of intermediate filament networks.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.002 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.000 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.010 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".