key: cord-0335920-kd2g7g37
authors: L., McAlary; V.K., Shephard; G.S.A., Wright; J.J., Yerbury
title: A copper chaperone-mimetic polytherapy for SOD1-associated amyotrophic lateral sclerosis
date: 2021-02-23
journal: bioRxiv
DOI: 10.1101/2021.02.22.432389
sha: 72d06f28fce06a7219fbcf34dd391535e42a28f3
doc_id: 335920
cord_uid: kd2g7g37

Amyotrophic lateral sclerosis (ALS)-associated mutations in Cu/Zn superoxide dismutase (SOD1) reduce folding stability, 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 utilise a small-molecule polytherapy of CuATSM and ebselen to mimic the metal delivery and disulfide bond promoting activity of SOD1’s cellular chaperone, the ‘copper chaperone for SOD1’ (CCS). We find that polytherapy using CuATSM and ebselen is highly effective at reducing inclusion formation in a cell model of SOD1 aggregation, reduces mutant SOD1-associated cell death, and promotes effective maturation of SOD1 beyond either compound alone. Our data suggest that a polytherapy of CuATSM and ebselen may be an effective method of treating SOD1-associated ALS.

Over 160 mutations have been identified throughout the gene encoding Cu/Zn superoxide 30 dismutase (SOD1) that are known to cause the motor neuron disease amyotrophic lateral 31 sclerosis (ALS) [1, 2] . These mutations result predominantly in amino acid substitutions found in 32 all SOD1 protein secondary structure elements, cofactor-binding sites and homodimer interface. 33 Each is thought to cause ALS by decreasing SOD1 folding stability, thereby creating a pool of 34 misfolded and aggregation-prone SOD1 [3, 4] . While there is debate as to whether proteinaceous 35 aggregates or smaller, soluble non-native oligomers are toxic [5-7] the initial event considered to 36 spark cell death is SOD1 protein misfolding [3,4, [8] [9] [10] [11] . 37 Here, we report the effect of ebselen on intracellular mutant SOD1 inclusion formation in a 77 disease-relevant cell model. To achieve this, we developed a machine learning-based image 78 analysis pipeline for accurate measurement of protein inclusion formation in large microscopy 79 data sets. Application of this method to a subset of compounds showed ebselen was capable of 80 reducing inclusion formation for both WTL and MBR SOD1 mutants. We then utilized this 81 method to investigate CuATSM and ebselen co-therapy aiming to divert nascent SOD1 from the 82 misfolding pathway to the maturation pathway and thereby reduce mutant toxicity. We show 83 these compounds can act in a synergistic manner to reduce SOD1 aggregation through the 84 promotion of dimerization, disulfide formation and copper loading. All mutants analyzed display 93 94 We, and others, have previously utilized genetically encoded fluorescent proteins as a tool to 95 investigate the inclusion formation of SOD1 in cultured cells finding that ALS-associated and de 96 novo mutations, as well as small molecules, can alter this process [3, 29, 41, [45] [46] [47] [48] . We have also 97 utilized several methods to detect fluorescent proteinaceous inclusions, including manual 98 counting [3], fluorescence intensity thresholding [29] , and cell permeabilization to release 99 soluble GFP-tagged protein [29, 41, 49] . We now sought to enhance our detection of protein 100 inclusion formation in cells for use in larger mutational or drug screens. This was accomplished 101 by exploiting advances made in the area of microscopy image analysis with the application of 102 user-assisted machine learning to accurately classify cellular phenotypes [50] .

To this end, we developed an image analysis pipeline using CellProfiler software [51] to identify 105 and segment transfected cells for measurement followed by classification in CellProfiler Analyst 106 software. Measurement parameters were chosen to append spatial data (shapes, texture, 107 granularity, radial intensity, and intensity) in order to generate cytoprofiles that were processed 108 by a random forest classifier ( Figure 1A and Supp. Fig. 1 and 2A ) [50] . Normalization of the 109 extracted cellular features demonstrated significant differences in texture, radial intensity, and 110 intensity measurements, but not shape or granularity (Supp. Fig. 2B ). This indicated that these 111 measurements were appropriate for the profiling of cells. We found that our segmentation 112 parameters correctly identified transfected cells with high accuracy (97 ± 2%) and that the 113 chosen measurements effectively facilitated accurate classification for both cells with and 114 without inclusions (95 ± 0.5% and 98 ± 0.9% accuracy respectively) ( Figure 1B) . Having established a rapid and accurate image-based method of classifying cells containing 134 inclusions, we next sought to examine the effect of ebselen and a small panel of other similar 135 small molecules on SOD1 inclusion formation. The molecules, other than ebselen, were 136 omeprazole, clopidogrel, and lipoic acid. These compounds were chosen on the basis that they 137 contained sulfur moieties that may be redox-active in a similar manner to ebselen [30] .

To this end, NSC-34 cells were transfected with SOD1 variants WT, A4V (WTL mutant), or 140 G85R (MBR mutant) and were treated with determined non-toxic concentrations of ebselen, 141 lipoic acid, omeprazole, or clopidogrel (Supp. Fig. 3) for 48 h prior to being fixed, imaged, and 142 analysed. We observed that WT SOD1 formed very few inclusions across treatments, with only 143 0.8 ± 0.3% of cells in the untreated group being classified as containing inclusions (Figure 2A) , 144 in line with previous observations by us and others [3, 29, 41, [45] [46] [47] [48] . In contrast, both A4V and 145 G85R SOD1 readily formed inclusions in this system with 27.7 ± 5.8% and 24.1 ± 5.3% cells Ebselen was found to be more effective at attenuating the inclusion formation and toxicity of 338 A4V when compared to G85R. Previous investigations have shown that SOD1 with Cu bound is 339 less susceptible to disulfide reduction [59,71,72]. Since A4V is a WTL mutant that retains Cu-340 binding capacity similar to that of WT, it would be expected that Cu-bound forms of A4V would 341 respond more favorably to ebselen-associated disulfide formation. Indeed, our data showed that 342 combination treatment resulted in a greater level of enzymatically active V148G as compared to 343 either CuATSM or ebselen alone, suggesting a strong effect of polytherapy to stabilise even the 344 most destabilizing ALS-associated mutants. In contrast, the pool of G85R that binds Cu is 345 relatively low [52,73], meaning that the synergistic effect of Cu-binding and disulfide formation 346 that we observed for both V148G and A4V would not be expected to be seen for G85R. In line 347 with this, we saw no additive or synergistic effect of CuATSM in combination with ebselen 348 against G85R expression in our cell model.

Other pharmacological chaperones, such as those targeted against tryptophan-32 in SOD1 Collectively, we have shown here that a biophysical understanding of the folding pathway of a 366 protein can be exploited to target it at key points to promote proper folding. However, the 367 strategy presented here is by no means the only one that may be pursued against SOD1-fALS. 

NSC-34 cells were plated into 6-well culture plates at a confluency of 40% and incubated 447 overnight at 37 °C in a humidified incubator with 5% atmospheric CO 2 . To overcome the effect 448 of transfection efficiency differences within assays, cells were transfected as described above 449 and incubated for 5 h at 37 °C in a humidified incubator with 5% atmospheric CO 2 . Following Following electrophoresis, total protein on the gel was quantified using a Criterion Stain Free™ 538 Imager (BioRad, Australia) prior to transferring for immunoblotting. imaged with a stain-free imager post-transfer to confirm transfer and measure total protein.

Following stain-free imaging, membranes were washed for 5 min in DH 2 O before being blocked 545 in 1× Tris-buffered saline (pH 7.4) with 0.02% Tween-20 (v/v) (TBST) and 5% skim milk (w/v) 546 at room temperature for 1 h. Following blocking, PVDF membranes were incubated in 547 polyclonal rabbit anti-GFP primary antibody (ab290 -Abcam, USA) diluted 1:5000 in TBST 548 with 5% skim milk (w/v) at 4 o C overnight with tipping. Following overnight incubation in 549 primary antibody, membranes were washed three times in TBST for 10 min per wash.

Following washing, membranes were incubated in goat anti-rabbit HRP-conjugated secondary 551 antibody (P0448 -Dako, Denmark) at a dilution of 1:5000 in TBST with 5% skim milk at room 552 temperature for 1 h with tipping. Following secondary antibody incubation, membranes were 553 washed three times in TBST prior to chemiluminescent detection of bands using SuperSignal™ 554 West Pico Plus substrate (Thermofisher, USA) and being imaged on a ChemiDoc™ MP Imaging 555 System (Biorad, Australia). Analysis and quantification was performed using ImageJ (version 556 1.53c).

In-gel zymography for SOD1 enzymatic activity 559 Cell lysates from transfected NSC-34 cells were generated as above, with the exception that lysis 560 buffer was 100 mM Tris-base (pH 7.5) with 0.1% TX-100 (v/v) and protease inhibitor. Cell 561 lysates were mixed 1:2 with 3× Native-PAGE sample buffer (240 mM Tris-HCl pH 6.8, 30% 562 glycerol (v/v), 0.03% bromophenol blue (w/v)) and loaded into Tris-glycine Native-PAGE gels 563 (4.5% stacking gel pH 8.8, 7.5 % resolving gel pH 8.8). Samples were electrophoresed for 30 564 min at 60 V and then for 2.5 h at a constant voltage of 125 V at 4 o C. Following Native-PAGE, 565 EGFP or TdTomato signal in the gel was detected using a ChemiDoc™ MP Imaging System 566 (Biorad, Australia). Gels were then subject to zymography as described previously [63] and 567 imaged using a GS-900™ Calibrated Densitometer (BioRad, Australia). Quantification of 568 fluorescence signal and enzymatic activity was performed using ImageJ (version 1.53c) [88] . 576  577  578  579  580  581  582  583  584  585  586  587  588  589  590  591  592  593  594  595  596  597  598  599  600  601  602  603  604  605  606  607  608 

Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic 659 lateral sclerosis

ALSOD: the Amyotrophic 661 Lateral Sclerosis Online Database

Susceptibility of Mutant SOD1 to Form a Destabilized 663

Monomer Predicts Cellular Aggregation and Toxicity but Not Aggregation Propensity. Front 664 Neurosci

Systematically perturbed folding 666 patterns of amyotrophic lateral sclerosis (ALS)-associated SOD1 mutants

Nonnative SOD1 trimer is toxic to 669 motor neurons in a model of amyotrophic lateral sclerosis

Large SOD1 aggregates, unlike 672 trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis

SOD1-positive aggregate 675 accumulation in the CNS predicts slower disease progression and increased longevity in a mutant 676 SOD1 mouse model of ALS

SOD1 aggregation 678 in ALS mice shows simplistic test tube behavior

Common denominator of Cu/Zn superoxide dismutase mutants 680 associated with amyotrophic lateral sclerosis: decreased stability of the apo state

Fibrillation precursor of superoxide dismutase 1 683 revealed by gradual tuning of the protein-folding equilibrium

Loss of metal ions, 686 disulfide reduction and mutations related to familial ALS promote formation of amyloid-like 687 aggregates from superoxide dismutase

Human SOD1 before harboring the catalytic 689 metal: solution structure of copper-depleted, disulfide-reduced form

The copper chaperone for 692 superoxide dismutase

Oxygen-induced maturation of SOD1: a key role for 694 disulfide formation by the copper chaperone CCS

Human superoxide 696 dismutase 1 (hSOD1) maturation through interaction with human copper chaperone for SOD1 697 (hCCS)

Zittin Potter S. Copper-zinc superoxide dismutase and amyotrophic 699 lateral sclerosis

The biophysics of superoxide dismutase-1 and 701 amyotrophic lateral sclerosis

The molecular 703 pathogenesis of superoxide dismutase 1-linked ALS is promoted by low oxygen tension

Initiation and 706 elongation in fibrillation of ALS-linked superoxide dismutase

Complete loss of post-709 translational modifications triggers fibrillar aggregation of SOD1 in the familial form of amyotrophic 710 lateral sclerosis

Decreased stability 712 and increased formation of soluble aggregates by immature superoxide dismutase do not account for 713 disease severity in ALS

Expression of wild-715 type human superoxide dismutase-1 in mice causes amyotrophic lateral sclerosis

Glutathionylation potentiates benign superoxide dismutase 1 718 variants to the toxic forms associated with amyotrophic lateral sclerosis

Destabilization of the dimer interface is a 720 common consequence of diverse ALS-associated mutations in metal free SOD1

SOD1A4Vaggregation alters ubiquitin homeostasis in a cell model of ALS

Phase 1-2 Trial of 726

Antisense Oligonucleotide Tofersen for ALS

Randomized, double-728 blind, placebo-controlled trial of arimoclomol in rapidly progressive ALS

Tryptophan 32 731 mediates SOD1 toxicity in a in vivo motor neuron model of ALS and is a promising target for small 732 molecule therapeutics

The cysteine-737 reactive small molecule ebselen facilitates effective SOD1 maturation

CuII(atsm) Increases Mutant SOD1 In Vivo but Protects Motor Neurons and Improves the 740 Phenotype of a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Chemotherapeutic agent 5-fluorouracil increases survival of SOD1 mouse model of ALS

A molecular chaperone activity of CCS restores the maturation of 746 SOD1 fALS mutants

Molecular recognition and 748 maturation of SOD1 by its evolutionarily destabilised cognate chaperone hCCS

Biological effects of CCS in the absence of SOD1 751 enzyme activation: implications for disease in a mouse model for ALS

Redox susceptibility of SOD1 mutants is 754 associated with the differential response to CCS over-expression in vivo

Overexpression of 757 CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial 758 pathology

CuII(atsm) improves the 760 neurological phenotype and survival of SOD1G93A mice and selectively increases enzymatically 761 active SOD1 in the spinal cord

CuATSM 763 efficacy is independently replicated in a SOD1 mouse model of ALS while unmetallated ATSM 764 therapy fails to reveal benefits

Copper delivery to the 766 CNS by CuATSM effectively treats motor neuron disease in SOD(G93A) mice co-expressing the 767 Copper-Chaperone-for-SOD

CuATSM Protects Against the

Vitro Cytotoxicity of Wild-Type-Like Copper-Zinc Superoxide Dismutase Mutants but not Mutants 770 That Disrupt Metal Binding

Ebselen 773 as template for stabilization of A4V mutant dimer for motor neuron disease therapy

Elucidation of the Molecular Characteristics of Wild-Type and ALS-Linked 776

Mutant SOD1 Using the NanoLuc Complementation Reporter System

Novel Selenium-779 based compounds with therapeutic potential for SOD1-linked amyotrophic lateral sclerosis

Superoxide Dismutase 1 Provide Insight into Potential Mechanisms Involved in Aberrant 783 Aggregation in Familial Amyotrophic Lateral Sclerosis

Loss of charge mutations in 785 solvent exposed Lys residues of superoxide dismutase 1 do not induce inclusion formation in 786 cultured cell models

Impaired extracellular 788 secretion of mutant superoxide dismutase 1 associates with neurotoxicity in familial amyotrophic 789 lateral sclerosis

Tryptophan residue 32 791 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection

Rapid flow cytometric 794 measurement of protein inclusions and nuclear trafficking

Scoring diverse 796 cellular morphologies in image-based screens with iterative feedback and machine learning

CellProfiler 799 3.0: Next-generation image processing for biology

Structures of 801 the G85R variant of SOD1 in familial amyotrophic lateral sclerosis

Structural 804 consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg

Phenotypic 807 heterogeneity in motor neuron disease patients with CuZn-superoxide dismutase mutations in 808 Scandinavia

Combination 810 therapy in combating cancer

Advances in developing novel therapeutic strategies for Alzheimer"s 812 disease

Emerging Developments in Targeting Proteotoxicity in 814 Neurodegenerative Diseases

SOD1A4V 816 aggregation alters ubiquitin homeostasis in a cell model of ALS

The unusually stable 818 quaternary structure of human Cu,Zn-superoxide dismutase 1 is controlled by both metal occupancy 819 and disulfide status

In-cell NMR reveals potential 821 precursor of toxic species from SOD1 fALS mutants

Decreased metallation 823 and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral 824 sclerosis

Familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide 826 dismutase are susceptible to disulfide reduction

Superoxide dismutase: Improved assays and an assay applicable to 828 acrylamide gels

Insights into the role of the unusual 830 disulfide bond in copper-zinc superoxide dismutase

Strategies for stabilizing superoxide 832 dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral 833 sclerosis

Cyclic 835 Thiosulfinates and Cyclic Disulfides Selectively Cross-Link Thiols While Avoiding Modification of 836 Lone Thiols

Strategies to promote the maturation of ALS-associated SOD1 mutants: 838 small molecules return to the fold

An intersubunit disulfide 840 bond prevents in vitro aggregation of a superoxide dismutase-1 mutant linked to familial amytrophic 841 lateral sclerosis

Prevention of transthyretin amyloid disease 843 by changing protein misfolding energetics

Calorimetric 845 analysis of thermodynamic stability and aggregation for apo and holo amyotrophic lateral sclerosis-846 associated Gly-93 mutants of superoxide dismutase

Dissociation of human 848 copper-zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular 849 basis for dimer stability

Folding of human superoxide dismutase: 851 disulfide reduction prevents dimerization and produces marginally stable monomers

Chaperone-facilitated copper binding is a 854 property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide 855 dismutase mutants

Samar Hasnain S. Ligand binding and 857 aggregation of pathogenic SOD1

The molecular 859 tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly and in the G93A-860 SOD1 mouse model of ALS

Ebselen treatment reduces noise induced hearing loss via 862 the mimicry and induction of glutathione peroxidase

Safety and efficacy of 864 ebselen for the prevention of noise-induced hearing loss: a randomised, double-blind, placebo-865 controlled, phase 2 trial

Structure of M pro from SARS-CoV-2 and 867 discovery of its inhibitors

Inhibition of TDP-43 869 accumulation by bis(thiosemicarbazonato)-copper complexes

Diacetylbis(N(4)-871 methylthiosemicarbazonato) copper(II) (CuII(atsm)) protects against peroxynitrite-induced 872 nitrosative damage and prolongs survival in amyotrophic lateral sclerosis mouse model

Proteome Homeostasis Dysfunction: A Unifying Principle in 875 ALS Pathogenesis

Randomized, double-877 blind, placebo-controlled trial of arimoclomol in rapidly progressive SOD1 ALS

Pyrimethamine decreases levels of 880 SOD1 in leukocytes and cerebrospinal fluid of ALS patients: a phase I pilot study

Antisense 883 oligonucleotides extend survival and reverse decrement in muscle response in ALS models

Is SOD1 loss of function involved in 886 amyotrophic lateral sclerosis? Brain

Neuroblastoma x spinal 888 cord (NSC) hybrid cell lines resemble developing motor neurons

Crystal Violet Assay for Determining Viability of Cultured 890 Cells

ImageJ2: ImageJ for 892 the next generation of scientific image data