Adaptation to G93Asuperoxide dismutase 1 in a motor neuron cell line model of amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal disease that manifests with progressive paralysis caused by the degeneration and death of large motor neurons of the spinal cord, brainstem and motor cortex. Extensive oxidative damage to neuronal tissue is found in sporadic and familial forms of ALS (SALS and FALS) [1], but the molecular mechanisms leading to these changes remain unknown.Mutations in the gene coding for Cu,Zn superoxide dismutase (SOD1) cause 2-5% of ALS cases (FALS1)[2]. SOD1 is one of the three mammalian SOD isozymes that catalyse the dismutation of superoxide to hydrogen peroxide (H 2 O 2 ) and water, and provide defence against oxidative stress. Extensive studies in FALS1 models showed that mutations confer new toxic properties on SOD1 rather than simply reducing the clearance of superoxide radicals [3].One explanation proposed for this 'gain of toxic function' is that mutant SOD1 has enhanced or different oxidative activities from wild-type SOD1 (wtSOD1) Motor neuron degeneration in amyotrophic lateral sclerosis involves oxidative damage. Glutathione (GSH) is critical as an antioxidant and a redox modulator. We used a motor neuronal cell line (NSC-34) to investigate whether wild-type and familial amyotrophic lateral sclerosis-linked G93A mutant Cu,Zn superoxide dismutase (wt ⁄ G93ASOD1) modified the GSH pool and glutamate cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis. We studied the effect of various G93ASOD1 levels and exposure times. Mutant Cu,Zn superoxide dismutase induced an adaptive process involving the upregulation of GSH synthesis, even at very low expression levels. However, cells with a high level of G93ASOD1 cultured for 10 weeks showed GSH depletion and a decrease in expression of the modulatory subunit of GCL. These cells also had lower levels of GSH and GCL activity was not induced after treatment with the pro-oxidant tertbutylhydroquinone. Cells with a low level of G93ASOD1 maintained higher GSH levels and GCL activity, showing that the exposure time and the level of the mutant protein modulate GSH synthesis. We conclude that failure of the regulation of the GSH pathway caused by G93ASOD1 may contribute to motor neuron vulnerability and we identify this pathway as a target for therapeutic intervention.Abbreviations ALS, amyotrophic lateral sclerosis; dox, doxycycline; EGFP, enhanced green fluorescent protein; FALS, familial amyotrophic lateral sclerosis; FALS1, mutant SOD1-linked familial amyotrophic lateral sclerosis; GCL, glutamate cysteine ligase; GCLC, catalytic subunit of GCL; GCLM, modulatory subunit of GCL; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione disulfide; GST, glutathione S-transferase; Nrf2, nuclear factor erythroid 2-related factor 2; SALS, sporadic amyotrophic lateral sclerosis; SOD1, Cu,Zn superoxide dismutase; t-BHQ, tertbutylhydroquinone; wtSOD1, wild-type Cu,Zn superoxide dismutase.

Characterization of Detergent-Insoluble Proteins in ALS Indicates a Causal Link between Nitrative Stress and Aggregation in Pathogenesis

“…7B). These data suggest that the increase in nitrated proteins in G93A SOD1 cells has to be attributed to the increased oxidative/nitrative stress caused, directly or indirectly, by mutant SOD1 [25]–[28]. The L-NAME treatment was therefore effective only in G93A SOD1 cells possibly because only there oxidative/nitrative stress played a role in the formation and consolidation of the aggregates.…”

“…We therefore propose that L-NAME interferes more generally with oxidative modification-induced protein aggregation in the presence of mutant SOD1. Under this condition the reported decrease in the level of endogenous antioxidants might play a role [28], [57]. However, in this cell paradigm we could not really evaluate the effect of reduced protein aggregation on cell viability.…”

Dysregulation of Glutathione Homeostasis in Neurodegenerative Diseases

“…An intriguing study by Tartari et al using NSC-34 cells containing an inducible Gly93Ala SOD1 vector, reported that short-term expression of Gly93Ala SOD1 resulted in a 30% increase in cellular GSH, while prolonged expression (14 passages) resulted in a 30% decrease in GSH [168]. Time dependent loss of GSH may explain part of the reason for latency of ALS onset.…”

Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation

“…Both cell lines (HEK293T and NSC-34) were used to express an EGFP fusion of SOD1 variant . HEK293T is a cell line used extensively in cell biology experiments and biologics production, and NSC-34 is a mouse neuroblastoma commonly used for fALS research [33–38]. Both HEK293T and NSC-34 cells were transfected and harvested two days post-transfection for flow cytometric analysis.…”