Altered organization of the intermediate filament cytoskeleton and relocalization of proteostasis modulators in cells lacking the ataxia protein sacsin

Abstract

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin’s physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments. To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations. Reorganization of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumulate. In ARSACS patient fibroblasts HSP70, ubiquitin and the autophagy-lysosome pathway proteins Lamp2 and p62 relocalized to the area of the vimentin accumulation. There was no overall increase in ubiquitinated proteins, suggesting the ubiquitin–proteasome system was not impaired. There was evidence for alterations in the autophagy–lysosome pathway. Specifically, in ARSACS HDFs cellular levels of Lamp2 were elevated while levels of p62, which is degraded in autophagy, were decreased. Moreover, autophagic flux was increased in ARSACS HDFs under starvation conditions. These data show that loss of sacsin effects the organization of intermediate filaments in multiple cell types, which impacts the cellular distribution of other organelles and influences autophagic activity.

Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics

“…1C shows extensive cross-bridging between the parallel arrays of NFs in the Sacs 2/2 neuron. Organelles including mitochondria were sparse within dense NF bundles, being largely excluded to the periphery, consistent with our previous observations at the light microscopic level in cultured motor neurons (8). From an ultrastructural point of view, bundles in Sacs 2/2 motor neurons are made of juxtaposed NF.…”

“…Absence of sacsin also causes bundling and juxtanuclear collapse of IFs composed of vimentin in cultured fibroblasts derived from patients' skin biopsy specimens (8). We determined the effects of sacsin UBL, SIRPT1, SacsJ, and HEPN domains on IF organization in a fibroblast line homozygous for the loss-of-function 8844delT sacsin mutation resulting in loss of sacsin expression and bundling of vimentin IFs (Supplemental Fig.…”

“…NF bundling occurred before impaired mitochondrial elongation and transport, another feature of ARSACS cells. Strikingly, bundling and collapse of the vimentin intermediate filament (IF) network and mitochondrial abnormalities also occur in ARSACS human fibroblasts in culture (8,9), suggesting a role of sacsin in IF assembly and/or maintenance in multiple cell types.…”

Autosomal Recessive Cerebellar Ataxias: Paving the Way toward Targeted Molecular Therapies

“…Several recent lines of evidence from patient cells, as well as from mouse models, suggest that sacsin acts as a chaperone or regulator for the organization of the intermediate filament cytoskeleton (Duncan et al, 2017). Sacsin deficiency thus leads to an altered organization of these intermediate filaments and-probably downstream-alterations in multiple mitochondrial functions, including mitochondrial fission (Girard et al, 2012), oxidative phosphorylation (Criscuolo et al, 2015), and oxidative stress pathways (Duncan et al, 2017). This might explain the partly ''mitochondrial clinical features'' observed in several ARSACS patients (such as, e.g., ophthalmoplegia, epilepsy, or hearing impairments).…”

Changes in Protein Glycosylation as a Result of Aptamer Interactions with Cancer Cells

“…[12] Also, a co-chaperone, which acts as a regulator of the Hsp70 chaperone machinery that may be involved in the processing of ataxia-linked proteins SACS (Sacsin), has been identified in samples interacting with aptamers A26 and A33. [13] Furthermore, a protein that plays an important role in the regulation of stress-induced apoptosis UACA (Uveal autoantigen with coiled-coil domains and ankyrin repeats) has been found in the samples incubated with A26 and A33 aptamers. [14] Essentially, all proteins present in the MCF7 protein extracts after incubation with potential therapeutic aptamers sequences and not identified in control samples were described in the literature as cancerrelated agents.…”