Background

Niemann Pick type C disease (NPC) is a neurovisceral pathology caused by autosomal recessive mutations in NPC1 and NPC2 genes that result in an endosomal accumulation of sphingomyelin and non-esterified cholesterol mainly in brain cells, liver, spleen, and immune system (Berry-Kravis, 2020). Autophagy represents the main lysosomal pathway by which cells recycle raw material, degrade damaged constituents, and protect themselves by eliminating toxic organelles and peptides (Nixon, 2024). Thus, the deficiency in lipid degradation in lysosomal disorders correlates with an alteration in autophagic flux. The aim of this study was to compare autophagy induction between fibroblasts of NPC and healthy controls.

Methods

Autophagy induction was performed by serum starvation for 6 h in cultures of 3 fibroblast cell lines from apparently healthy individuals and 3 from NPC patients to determine whether there were differences in the autophagic process between controls and patients. We also tested the effect of Chloroquine 5 uM for 6 h. Autophagy induction was confirmed using the Cyto-ID autophagy detection. We analyzed the expression of cellular proliferation proteins mTOR and p-mTOR, as well as autophagic pathway proteins: LC3-II, p62, HSP70, and HSC70 by Western Blotting. The transcriptomes of both control and patient cell lines were analyzed for differential expression to identify mechanisms involved in the autophagic pathway.

Results

Treatment in the absence of serum or with chloroquine had a greater effect on the induction of autophagy in cells from patients with NPC as compared to controls; an increase in the presence of cytoplasmic autophagy vacuoles, a reduction in the expression of mTOR, a greater expression of LC3-II and an increase in the expression of p62 and HSP70 in fibroblasts from patients with NPC were confirmed.

Conclusion

NPC fibroblast cells were more prone to induction of the first phases of autophagy when treated with Chloroquine under starvation conditions compared to fibroblasts from healthy individuals, suggesting an impaired autophagy flux as demonstrated by p62 increase. This result was confirmed by the Cyto-ID, decreased mTOR expression, and increased expression of LC3-II, p62, and HSP70. Cells derived from NPC patients constitute an optimal in vitro model for studying this neurodegeneration.