A copper chaperone–mimetic polytherapy for SOD1-associated amyotrophic lateral sclerosis

L Mcalary,V.K Shephard,G.S.A Wright,J.J Yerbury

doi:10.1016/j.jbc.2022.101612

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons progressively and rapidly degenerate, eventually leading to death. The first protein found to contain ALS-associated mutations was copper/zinc superoxide dismutase 1 (SOD1), which is conformationally stable when it contains its metal ligands and has formed its native intramolecular disulfide. Mutations in SOD1 reduce protein folding stability via disruption of metal binding and/or disulfide formation, resulting in misfolding, aggregation, and ultimately cellular toxicity. A great deal of effort has focused on preventing the misfolding and aggregation of SOD1 as a potential therapy for ALS; however, the results have been mixed. Here, we utilize a small-molecule polytherapy of diacetylbis(N(4)-methylthiosemicarbazonato)copper(II) (CuATSM) and ebselen to mimic the metal delivery and disulfide bond promoting activity of the cellular chaperone of SOD1, the “copper chaperone for SOD1.” Using microscopy with automated image analysis, we find that polytherapy using CuATSM and ebselen is highly effective and acts in synergy to reduce inclusion formation in a cell model of SOD1 aggregation for multiple ALS-associated mutants. Polytherapy reduces mutant SOD1-associated cell death, as measured by live-cell microscopy. Measuring dismutase activity via zymography and immunoblotting for disulfide formation showed that polytherapy promoted more effective maturation of transfected SOD1 variants beyond either compound alone. Our data suggest that a polytherapy of CuATSM and ebselen may merit more study as an effective method of treating SOD1-associated ALS.

Highlights

Over 160 mutations have been identified throughout the gene encoding Cu/Zn superoxide dismutase (SOD1) that are known to cause the motor neuron disease amyotrophic lateral sclerosis (ALS) [1,2]
This analysis showed that all variants (G85R data not shown due to no activity observed under any treatment), exhibited a significant increase in the activity of SOD1-TdTomato for CuATSM, and combination therapy treated cells compared to vehicle-treated cells (Figure 4 E and G)
Considering that ALS-associated mutations in SOD1 disrupt its maturation, some therapeutic strategies have focused on catalysing proper SOD1 folding [30,41,67,68,69]

Summary

Introduction

Over 160 mutations have been identified throughout the gene encoding Cu/Zn superoxide dismutase (SOD1) that are known to cause the motor neuron disease amyotrophic lateral sclerosis (ALS) [1,2]. Application of this method to a subset of compounds showed ebselen was capable of reducing inclusion formation for both WTL and MBR SOD1 mutants. We utilized this method to investigate CuATSM and ebselen co-therapy aiming to divert nascent SOD1 from the misfolding pathway to the maturation pathway and thereby reduce mutant toxicity. We show these compounds can act in a synergistic manner to reduce SOD1 aggregation through the promotion of dimerization, disulfide formation and promoting increased levels of active SOD1. This work highlights the unexplored possibilities of mutation-specific personalized therapy for SOD1ALS and the potential use of a CCS-mimetic polytherapy targeting steps on the SOD1 PTM maturation pathway

Results

Discussion

Conflict of Interest Statement

Experimental Procedures

Declaration of interests