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

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.

Keywords: CuATSM; amyotrophic lateral sclerosis; ebselen; protein aggregation; protein folding; superoxide dismutase 1.

Figure 1

User-assisted machine learning to determine cells containing inclusions.A, the image analysis pipeline first performs illumination correction for both DAPI and GFP channels and then segments the transfected cells for measurement. A user then identifies phenotypes in a small subset of the cell population to train the classifier for identification of the entire cell population. B, the classifier requires less than 100 to become accurate at categorizing cells into inclusion containing (blue) and those that did not contain inclusions (orange). C, correlations of the percentage of cells with inclusions in this work versus previously published examination of the same SOD1-EGFP expression constructs in NSC-34s (left) versus manual counts from McAlary et al., 2016 (3) and (right) versus saponin-permeabilized cells from Farrawell et al. (25) 2018. Error bars represent SD of the mean from at least three separate classification requests. DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced GFP; SOD1, superoxide dismutase 1.

Figure 2

Ebselen reduces inclusion formation of SOD1 ALS-associated mutants in cultured cells. NSC-34 cells expressing (A) SOD1-EGFP variants SOD1^WT, SOD1^A4V, and SOD1^G85R were treated with vehicle (black), clopidogrel (red), ebselen (orange), lipoic acid (teal), or omeprazole (pink) for 48 h, and the number of cells containing inclusions was enumerated. B, NSC-34 cells expressing SOD1-EGFP variants SOD1^C6G, SOD1^G37R, SOD1^H46R, SOD1^D90A, SOD1^E100G, SOD1^G93A, SOD1^G127X, and SOD1^V148G were all treated with increasing concentrations of ebselen, and inclusion formation was measured. Ebselen treatment decreased inclusion formation for most variants except for SOD1^G127X and SOD1^H46R. Error bars represent SD of the mean of at least three separate experiments. Statistical significance was determined using Student's t test against vehicle control (∗∗∗p < 0.001; ∗p < 0.05). ALS, amyotrophic lateral sclerosis; EGFP, enhanced GFP; SOD1, superoxide dismutase 1.

Figure 3

CuATSM and ebselen polytherapy is more effective than monotherapy at reducing WT-like SOD1 mutant inclusion formation.A, highest single agent (HSA) heatmaps of CuATSM and ebselen checkerboard treatment of NSC-34 cells expressing either SOD1^WT (top left), SOD1^A4V (top right), SOD1^G37R (middle left), SOD1^G85R (middle right), SOD1^G93A (bottom left), and SOD1^V148G (bottom right). Color scale represents the degree to which antagonism or synergy is occurring. Numbers in boxes represent mean values from three separate experiments, and a number above 10 is considered as synergistic at those concentrations. B, transfected cell counts of NSC-34 cells expressing SOD1^A4V (left) and SOD1^G85R (right) treated with vehicle DMSO (black), ebselen (10 μM; orange), CuATSM (0.5 μM; green), and ebselen/CuATSM combo (10 μM/0.5 μM; blue). Cell counts are relative to SOD1^WT transfected cells treated with the same compounds. Inset in each panel is the area under the curve measurements for each drug treatment. Error bars represent SEM of three separate experiments. Statistical significance was determined using a one-way ANOVA with Dunnet's test against DMSO-treated cells (∗∗∗p < 0.01; ∗∗p < 0.05). CuATSM, diacetylbis(N(4)-methylthiosemicarbazonato)copper(II); DMSO, dimethyl sulfoxide; SOD1, superoxide dismutase 1.

Figure 4

A combination treatment of CuATSM and ebselen is effective at rescuing SOD1 ALS-associated mutant folding.A, nonreducing SDS-PAGE AMS assay shows that treatment with ebselen but not other redox-containing compounds resulted in recombinant SOD1^A4V disulfide formation (reduced = SH, oxidized intact = SS). B, all size-exclusion chromatographies show that ebselen promotes recombinant SOD1^A4V homodimerization, whereas oxidized glutathione (GSSG) does not (D = dimers, M = monomers). C, differential SDS-PAGE migration of SOD1-EGFP from cell lysates under reducing (+β-merc) and nonreducing (−β-merc) conditions shows that the proportion of disulfide-bonded SOD1 (SS) is increased with CuATSM (green; 0.5 μM), ebselen (orange; 20 μM), and a combination treatment (blue; 0.5 μM CuATSM/20 μM ebselen) compared with vehicle control (black) for both SOD1^WT and SOD1^A4V, but SOD1^G85R remains fully reduced. D, densitometry of disulfide formation immunoblots for SOD1^WT and SOD1^A4V showed that CuATSM, ebselen, and the combination therapy were capable of promoting disulfide formation in living cells. E, native-PAGE of SOD1–TdTomato lysates shows oligomers, dimers, and monomers of SOD1 variants (top) and in-gel zymography of the same gels shows the relative activity of each species, including dimer, monomer, and mouse SOD1. F, quantification of the fluorescence signal from native-PAGE of the proportion of SOD1–TdTomato signal present for the dimer, showing that CuATSM, ebselen, or combination therapy promoted the dimerization of both SOD1^WT and SOD1^G93A, but not SOD1^A4V, SOD1^G85R, and SOD1^V148G. G, quantification of the achromatic bands from in-gel zymography, normalized to TdTomato fluorescence signal, showing that only treatment with CuATSM and the combination therapy increased the relative levels of active SOD1 for SOD1^WT, SOD1^A4V, SOD1^G93A, and SOD1^V148G (SOD1^G85R not shown because of lack of activity). Error bars represent SD of the mean of at least three separate experiments. Significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05). ALS, amyotrophic lateral sclerosis; AMS, acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; CuATSM, diacetylbis(N(4)-methylthiosemicarbazonato)copper(II); EGFP, enhanced GFP; SOD1, superoxide dismutase 1.

Supplement Figure S1

Example thumbnails of cells containing inclusions from the entire set of data used for this work, showing the heterogeneity of inclusion formation.

Supplement Figure S2

Key measurements used to generate the cytoprofiles of cells. A, heat map of the 150 features that were measured from 150 cells in each classification group (Inclusions versus No Inclusions). B, Z-normalised values from each extracted feature for both cells with inclusions (dark gray) and cells without inclusions (light gray). Data are plotted as box-whisker plots where whiskers show the 5 to 95% range, the box the 25 to 75% range and the middle bar the median values. Significance was determined using student’s t-test (∗∗∗p ≤ 0.001).

Supplement Figure S3

Determination of the non-toxic concentration of compounds to treat NSC-34 cells. A, crystal violet assay determined NSC-34 densities following treatment with either clopidogrel, ebselen, lipoic acid, or omeprazole. Above each plot is an example set of well images showing the crystal violet staining of cells at different compound concentrations. B, phase contrast imaging of NSC-34 cells at a concentration of 20 μM for each drug. Scale bar = 100 μm. Error bars represent SD of the mean from at least 3 separate biological replicates.

Supplement Figure S4

Heat map of SOD1-EGFP variants inclusion formation in a checkerboard assay of CuATSM and ebselen. Related to main text Figure 3.

Supplement Figure S5

Full immunoblot representation of the electrophoretic migration assay to determine SOD1 disulfide status using SOD1-EGFP constructs. Related to main text Figure 4 panels C and D.

Supplement Figure S6

Full Native-PAGE images from TdTomato fluorescence (left) and in-gel zymography (right). Related to main text Figure 4 panels E and F.

Supplement Figure S7

EGFP-tagged SOD1 migrates closely to mouse SOD1 in Native-gel electrophoresis. Note overexposure is to show monomer species.