Functional Transcriptome Analysis in ARSACS KO Cell Model Reveals a Role of Sacsin in Autophagy

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.

Functional Network Profiles in ARSACS Disclosed by Aptamer-Based Proteomic Technology

“…In a previous work, we generated and characterized the SH-SY5Y neuroblastoma cell line and derived KO and WT clones ( 11 ). In brief, using CRISPR/Cas9 gene editing, we produced clones harboring a loss-of function mutation in SACS , and in RNAseq experiments compared transcriptomic profiles with WT clones.…”

“…In this study, capitalizing on our early work investigating the mRNA signature in neuronal models of ARSACS ( 11 ), we used a targeted proteomics assay as the starting point of an analysis aimed at identifying the neuronal pathways that are impaired in this disease. We used SOMAscan, a new aptamer-based proteomics assay that, by means of innovative and specific SOMAmer-based DNA signals, can quantitatively detect proteins in biological samples up to femtomolar concentration ( 7 , 8 ).…”

“…After assaying 1,300 SOMAmer-associated putative biomarkers in cultured skin fibroblasts from healthy controls and ARSACS patients, and in wild-type (WT) and sacsin knocked-out (KO) SH-SY5Y cells, we used functional network annotation to identify new major dysregulated biological processes in our models, namely neuroinflammation, synaptogenesis, and engulfment of cells. In particular, a short-term treatment with FCCP was used in SH-SY5Y cells to mimic the pathological phenotype related to defects in mitochondrial function and dynamics, as previously demonstrated by us and other ( 11 , 12 ).…”

A Novel CCT5 Missense Variant Associated with Early Onset Motor Neuropathy

“…A CCT5 gene mutation is associated with a human phenotype, i.e., a mutilating, distal sensory neuropathy with severe atrophy of the posterior tract of the spinal cord [ 3 , 4 ]. Diseases associated with mutations in the genes encoding proteins within the Hsp40 and Hsp70 families, as well in the gene encoding the very large chaperone, sacsin have been described associated with a variety of neurologic disorders [ 2 , 5 ].…”

Docosahexaenoic acid in ARSACS: observations in two patients

“…The pathological mechanisms underlying neurodegeneration in sacsin-mutated neurons are not fully understood. A recent study in neuronal models of sacsin depletion suggests that loss of the protein could selectively impair mitophagy, leading to accumulation of damaged mitochondria and subsequent bioenergetic dysfunction, increased levels of oxidative stress products, and peroxidation of membrane phospholipids [2]. The hypothesis of bioenergetic and autophagy dysfunction in ARSACS pathogenesis suggests a potential therapeutic role for agents targeting these pathways.…”