Abstract
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurological disease caused by mutations in SACS, which encodes sacsin. The complex architecture of sacsin suggests that it could be a key player in cellular protein quality control system. Molecular chaperones that operate in protein folding/unfolding and assembly/disassembly patterns have been described as essential modulators of selectivity during the autophagy process. We performed RNA-sequencing analysis to generate a whole-genome molecular signature profile of sacsin knockout cells. Using data analysis of biological processes significantly disrupted due to loss of sacsin, we confirmed the presence of decreased mitochondrial function associated with increased oxidative stress, and also provided a demonstration of a defective autophagic pathway in sacsin-depleted cells. Western blotting assays revealed decreased expression of LC3 and increased levels of p62 even after treatment with the lysosomal inhibitor bafilomycin A1, indicating impairment of the autophagic flux. Moreover, we found reduced co-immunolocalization of the autophagosome marker LC3 with lysosomal and mitochondrial markers suggesting fusion inhibition of autophagic compartments and subsequent failed cargo degradation, in particular failed degradation of damaged mitochondria. Pharmacological up-regulation of autophagy restored correct autophagic flux in sacsin knockout cells. These results corroborate the hypothesis that sacsin may play a role in autophagy. Chemical manipulation of this pathway might represent a new target to alleviate clinical and pathological symptoms, delaying the processes of neurodegeneration in ARSACS.
Highlights
Spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease characterized by cerebellar ataxia, lower limb pyramidal tract signs, and axonal-demyelinating sensorimotor peripheral neuropathy[1,2,3,4]
We demonstrated that KO cells are a valid model in Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)
Loss-of-function mutations in SACS lead to ARSACS and impair the function of sacsin, a protein that shares features with molecular chaperones[11,12,14] and is implicated in mitochondrial dynamics[18,19,20], neurofilament organization and protein quality control[22]
Summary
Spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease characterized by cerebellar ataxia, lower limb pyramidal tract signs, and axonal-demyelinating sensorimotor peripheral neuropathy[1,2,3,4]. The nature and architecture of these modules suggest that sacsin is involved in protein quality control; this would be consistent with the role that other molecular chaperones are increasingly recognized to play in neurodegeneration, as key mediators of: protein homeostasis (proteostasis) in the ubiquitin-proteasome system; endoplasmic reticulum-associated degradation; and different autophagic pathways, including chaperone-mediated-, micro-, and macro-autophagy, and www.nature.com/scientificreports/. We show that mTOR-mediated autophagy is differentially regulated in sacsin-depleted cells (KO cells), and that pharmacological induction of autophagy restores a correct flux Clarification of these mechanisms, in addition to improving our biological understanding of how sacsin operates in disease conditions, might suggest that chemical manipulation of the autophagic pathways is a valid new avenue to pursue in the pharmacological approach to ARSACS
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