Integrative Organelle-Based Functional Proteomics: In Silico Prediction of Impaired Functional Annotations in SACS KO Cell Model

Abstract

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an inherited neurodegenerative disease characterized by early-onset spasticity in the lower limbs, axonal-demyelinating sensorimotor peripheral neuropathy, and cerebellar ataxia. Our understanding of ARSACS (genetic basis, protein function, and disease mechanisms) remains partial. The integrative use of organelle-based quantitative proteomics and whole-genome analysis proposed in the present study allowed identifying the affected disease-specific pathways, upstream regulators, and biological functions related to ARSACS, which exemplify a rationale for the development of improved early diagnostic strategies and alternative treatment options in this rare condition that currently lacks a cure. Our integrated results strengthen the evidence for disease-specific defects related to bioenergetics and protein quality control systems and reinforce the role of dysregulated cytoskeletal organization in the pathogenesis of ARSACS.

Keywords: ARSACS; KO models; SACS; biomarkers; lysosomes; mitochondria; omics.

Figure 1

Bioinformatic examination of mitochondria-specific proteome profiles. (A) Mitochondria-focused connectivity network displaying differentially abundant proteins (mtDEP) and their major associated diseases and functions. Red-bordered nodes were shared between proteomic and transcriptomic datasets. (B) Functional annotations and corresponding activation z-scores in the mitochondrial dataset. Red—up-regulated, blue—downregulated protein abundance. (C) Heatmap representation of shared annotations in the mitochondria-focused transcriptome (DEGs, differentially expressed genes) and proteome analyses (DEPs, differentially expressed proteins), sorted according to their z-scores. n = 3 in each experimental condition.

Figure 2

Bioinformatic examination of lysosome-specific proteome profiles. (A) Lysosome-specific connectivity network sorted according to predicted, most significantly associated pathway activation/inhibition. Red-bordered nodes represent identifiers shared between proteomic and transcriptomic datasets. (B) Functional annotations and linked activation z-scores in ARSACS KO lysosomes. (C) Heatmap of shared annotations in lysosome-focused transcriptome (DEGs, differentially expressed genes) and proteome analyses (DEPs, differentially expressed proteins). Red—up-regulated, blue—downregulated protein abundance. n = 3 in each experimental condition.

Figure 3

Heat bubble matrix representing the mitochondria-(A) and lysosome-(B) associated IDs transversally identified as dysregulated in both proteomic and transcriptomic studies. The degree of significance and fold change in differential gene expression/protein abundance are reported as size and color of a bubble, respectively.

Figure 4

Bioenergetic phenotype in SACS KO cell model of ARSACS. (A) Oxygen consumption rate (OCR) was measured in WT and KO cells using the Agilent Seahorse XF Cell Mito Stress Test. The assay was performed under basal conditions and after addition of oligomycin (2 μM), carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) (1.5 μM), and rotenone plus antimycin A (1 μM). KO cells showed an impaired energy metabolism compared with WT ones. Data refer to n = 8 and n = 5 independent measures for WT and KO cells, respectively. * p < 0.05; ** p < 0.01. (B) Oxidative stress was measured by oxidation of MitoSOX fluorescent reagent both in regular conditions (RM = regular medium) and by using antimycin A (aA) as reactive oxygen species (ROS) generator. The production of superoxide by mitochondria was elevated in KO cells. * p < 0.05. Each symbol refers to an independent replicate value obtained from a technical triplicate.

Figure 5

Autophagic flux impairment in SACS KO cells. Representative immunofluorescence images of autophagosomes (LC3, in red) and p62-cargos (in green). The fluorescent dye 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (in blue) was used as a nuclear stain. WT and sacsin KO cells were analyzed under normal conditions (regular medium—RM) and FCCP treatment (FCCP 2h), and colocalization of LC3 with p62 (yellow areas) was measured as mean ± SD of three replicates, * p < 0.05 by two-tailed Student’s t-test.

Figure 6

Representative immunofluorescence images for filament network marker, vimentin (red channel) and ER marker, and calreticulin (green channel). Blue channel refers to nuclear staining with DAPI. Scale bar = 10 µm. n = 3 independent experiments.