Effects of Cellular Pathway Disturbances on Misfolded Superoxide Dismutase-1 in Fibroblasts Derived from ALS Patients

Abstract

Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS.

Pelargonidin improves memory deficit in amyloid β25-35 rat model of Alzheimer’s disease by inhibition of glial activation, cholinesterase, and oxidative stress

“…Increased oxidative stress at initial stages of neurotoxic conditions is also associated with activation of autophagylysosomal system [55,56] that could be beneficial and protective per se. Meanwhile, Pickford et al have shown decreased autophagy in addition to accelerated Ab aggregation in the brain of mice model of AD [57].…”

The molecular pathogenesis of superoxide dismutase 1-linked ALS is promoted by low oxygen tension

“…Previously, we have utilized ALS patient-derived dermal fibroblasts as an in vitro model to study misfolding under physiological SOD1 expression levels (Keskin et al, 2016). Cells cultured in vitro are typically maintained in humidified ambient air supplemented with 5% (v/v) CO 2 , resulting in an O 2 tension of approximately 19%.…”

“…Our previous study of patient-derived fibroblasts showed that misfolded SOD1 is primarily degraded by the proteasome (Keskin et al, 2016). However, exposure to low O 2 tension did not lead to a reduction in proteasome activity in MNAC extracts (Fig EV3C).…”

“…Most of the truncated mutants lack C146 and thereby the C57-C146 disulfide bond, which is critical for SOD1 stability. These mutants are highly unstable and rapidly degraded (Birve et al, 2010, Jonsson et al, 2004, Keskin et al, 2017, Keskin et al, 2016, Sato et al, 2005). Hence, the collective genetic and biochemical evidence from patients infers that any common ALS-causing SOD1 species is disordered, lacks the C57-C146 disulfide bond and is only present in minute amounts.…”

“…However, the cytosol is strongly reducing and the C57-C146 bond could be an evolution-related Achilles heel of eukaryotic SOD1, and a critical determinant of its role in ALS. Absence of the disulfide bond promotes misfolding and aggregation of SOD1 in vitro (Chattopadhyay et al, 2008, Chattopadhyay et al, 2015, Furukawa et al, 2008, Keskin et al, 2016, Lang et al, 2012). Moreover, aggregated as well as soluble misfolded SOD1 species in the central nervous system (CNS) of SOD1 Tg models lack the bond (Bergemalm et al, 2010, Karch et al, 2009, Zetterström et al, 2013, Zetterstrom et al, 2007).…”