Research Article pubs.acs.org/chemneuro
Kaplan−Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset Alireza Abdolvahabi,† Yunhua Shi,† Sanaz Rasouli,†,‡ Corbin M. Croom,† Amir Aliyan,§ Angel A. Martí,§,∥ and Bryan F. Shaw*,† †
Department of Chemistry and Biochemistry, Baylor University, Waco, Texas 76706, United States Institute of Biomedical Studies, Baylor University, Waco, Texas 76706, United States § Department of Chemistry, Rice University, Houston, Texas 77005, United States ∥ Department of Bioengineering, Rice University, Houston, Texas 77005, United States ‡
S Supporting Information *
ABSTRACT: Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan−Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼103 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R2 = 0.67; Pearson’s r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R2 = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R2 = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate). KEYWORDS: Superoxide dismutase, thioflavin-T fluorescence, amyotrophic lateral sclerosis
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INTRODUCTION Do SOD1 mutations cause amyotrophic lateral sclerosis (ALS)1 by uniformly increasing the intrinsic rate of amyloidogenesis of superoxide dismutase-1 (SOD1)?2,3 This simple question cannot be rigorously answered with current kinetic data. The intrinsic (in vitro) aggregation rates of only four out of >150 different ALS variants of apo-SOD1 have been determined at pH 7,4−6 with the statistical significance required to measure rates of processes that are as stochastic.2,4−6 Answering this question will help discern whether SOD1 mutations trigger ALS by increasing intrinsic rates of nucleation or propagation of SOD1 oligomers or by another mechanism, such as increasing the intrinsic cytotoxicity of oligomers or impairing maturation processes critical to preventing oligomerization.7 Quantifying intrinsic rates of SOD1 aggregation will © 2017 American Chemical Society
help answer several other questions. For example, does SOD1 cause ALS by self-assembling into a neurotoxic, amyloid-like oligomer8 or is fibrillization a primarily nonpathogenic side effect (similar to the amyloidosis of immunoglobulins in multiple myeloma)?9 Can the intrinsic rate (or free energy) of ALS-variant SOD1 amyloidogenesis explain (in part or whole) the wide variation in the age of onset or survival time of ALS patients with different SOD1 mutations?10,11 Expecting a correlation (of some magnitude) between mutant SOD1 biophysics and patient phenotype is not unrealistic.12−15 Direct Received: January 20, 2017 Accepted: March 14, 2017 Published: March 14, 2017 1378
DOI: 10.1021/acschemneuro.7b00029 ACS Chem. Neurosci. 2017, 8, 1378−1389
Research Article
ACS Chemical Neuroscience
Figure 1. Biophysical characterization of apo-SOD1 proteins. (a) Ribbon structure of the apo-SOD1 dimer (PDB: 2C9V). Location of amino acid substitutions highlighted in red. (b) Differential scanning thermograms, and (c) electrospray ionization mass spectra of WT and ALS-variant apoSOD1 proteins.
third study by Lang and co-workers measured fibrillization rates of four ALS-variants of SOD1 (at pH 6.3), and several other pseudo-ALS variants that also contain C6S, C111S, and F50E substitutions. Kinetic values in this study were reported as averages of between 6 and 198 replicates, which makes it difficult to assess the significance of rates for any given variant.2 A recent study from our group examined rates of amorphous and fibrillar aggregation at reasonable statistical power (n > 50 per variant) but only examined three ALS-variants of SOD1.4 We found that the D90A mutation affects the rate of apo-SOD1 fibrillization by a negligible amount.4 The stochastic nature of SOD1 aggregation in vitro and observations in vivo that SOD1 oligomers form in a fraction of neural cells before prion-like transmission and propagation to neighboring cells8 caused us to question whether conventional metrics express aggregation kinetics in physiologically relevant terms. For example, does a probability-based metric more realistically reflect aggregation processes in vivo compared to average rates of nucleation or elongation? We propose that the probability distribution of aggregation is a more realistic benchmark because pathogenesis might require initial aggregation of SOD1 in a small fraction of motor neurons, regardless of whether SOD1 aggregates ab initio in the vast majority of cells.8,26 To this end, we used Kaplan−Meier estimators27 to compute the probability of amyloid fibrillization among 825 identical (iterate) solutions of wild-type apo-SOD1 and nine different ALS-variants of apo-SOD1 using microtiter plates. Kaplan− Meier analysis is widely used28 to quantify the probability of an “event” in test versus control subjects, typically the death of mice or humans,29−31but has not been used to monitor stochastic chemical reactions. Here, Kaplan−Meier estimators were used to determine the risk that an isolated solution of purified ALS-variant apo-SOD1 (within a single microplate well) will aggregate into a thioflavin-T positive oligomer, relative to solutions of wild-type apo-SOD1 in other wells (both under reducing conditions).
genotype−phenotype correlations exist in some monogenic diseases,16 e.g., Pompe disease17 and favism.18 The importance of accurately quantifying the intrinsic (in vitro) rates of SOD1 fibrillization is buffeted by reports suggesting that SOD1 aggregation in transgenic ALS mice follows the same stochastic nucleation and propagation kinetics (or “simplistic test tube behavior”2) as those during in vitro experiments with recombinantly expressed SOD1.2 Quantifying rates of SOD1 aggregation in vitro is difficult because SOD1 aggregation, both amorphous and fibrillar, is stochastic (in microplate assays that involve mechanical agitation and a significant air−water interface).2,5,6 For example, identical aliquots of E100K apo-SOD1 analyzed simultaneously in the same microplate can produce fibril lag times that vary from 7 to 50 h.4 Quantifying statistically significant rates of stochastic processes requires myriad replicate measurements (ideally, n ≥ 102).19 The stochasticity associated with SOD1 aggregation does not appear to be an artifact of random or systematic experimental error (although the importance of disruptions in the air−waterinterface during mechanical agitation20 have not been explored sufficiently for SOD1 to exclude interfacial effects as a partial source of stochasticity).4 Processes of self-assembly that involve nucleation are commonly stochastic.19,21−23 The stochasticity associated with SOD1 nucleation might be caused by competing pathways of aggregation (i.e., amorphous versus fibrillar; branched fibrils versus unbranched) that have similar activation energies.4 The stochastic elongation of fibrils could be caused by random secondary nucleation24 (i.e., the random breaking of fibrils at myriad positions along a fibril axis). Previous kinetic measurements of apo-SOD1 aggregation that involved low (or variable) statistical power suggest that ALS mutations uniformly accelerate SOD1 aggregation in vitro2,11,25 and that measured aggregation rates correlate with patient life-span.11 Measurements in two of these studies involved low numbers of replicate measurements for each variant protein (i.e., 2−10 replicate assays per protein).11,25 A 1379
DOI: 10.1021/acschemneuro.7b00029 ACS Chem. Neurosci. 2017, 8, 1378−1389
Research Article
ACS Chemical Neuroscience
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RESULTS AND DISCUSSION ALS-variant SOD1 proteins examined in this study (Figure 1a) were chosen for their diverse clinical properties, their wide range in free energy of folding, and for their position as gross outliers in previous correlation plots of clinical and biophysical properties.32 For example, the L38V missense mutation presents with a short survival time after disease onset (2.4 years), G37R with a long survival time (18 years); D90A is moderate (8 years);10 E100K and D90A SOD1 are biophysically and chemically “cryptic” proteins with native structure, stability, metal affinity, and enzymatic activity similar to those of WT SOD1;33 the H46R variant has impaired metal binding;34 the A4V mutation severely destabilizes SOD1 and is the most common ALS-SOD1 mutation in North America.35 We measured fibrillization rates of SOD1 proteins in the metal-free (apo) state because (i) we wished to determine if any uniform relationship exists between a fundamental biophysical property of ALS-variant SOD1 polypeptides, here, probability of fibrillization for the bare polypeptide, and clinical phenotype in patients. The apo state is the only state that all known ALS-variants of SOD1 can uniformly populate; (ii) demetalated SOD1 is the most prone to aggregate into amorphous and fibrillar species;36,37 (iii) active-site metal binding is not required for ALS pathogenesis;38 and (iv) metalfree SOD1 is a primary constituent of aggregated SOD1 species that form in vivo.39 Prior to initiating microtiter plate fibrillization assays, all recombinantly expressed and purified SOD1 proteins were demetalated and contained ≤0.08 equiv of Zn2+ (per dimer) and ≤0.05 equiv of Cu2+ (per dimer) according to ICP-MS (Table 1). SOD1 proteins were demetalated to levels that were
All amyloid assays were performed in 10 mM TCEP. We chose this level of reducing agent (instead of 100 mM levels used in some of our previous reports4) because it more closely mimics intracellular redox potentials (intracellular glutathione can vary from 0.5 to 10 mM across subcellular loci40). We chose TCEP, over DTT, because TCEP is ∼10-fold more stable to air oxidation than DTT.41 Reduction of the disulfide bond in SOD1 has been shown to be critical in accelerating amyloid formation (more so than the dissociation of Cu2+ or Zn2+).7 All proteins were incubated in TCEP for 30 min prior to the initiation of the amyloid assay (i.e., prior to orbital gyration and fluorescence spectroscopy). We point out that after 30 min, only a fraction of proteins (e.g., ∼ 5−10% for WT apo-SOD1) is reduced, according to DSC and size-exclusion chromatography (Figure S1). This brief, incomplete reduction prior to the beginning of assays minimizes the formation of soluble oligomer seeds and allows sufficient time to pipet mixtures of SOD1, ThT, and TCEP into microplate wells, seal the plate, and load it into the fluorescence spectrophotometer. Complete reduction will occur, however, after the beginning of orbital gyration and fluorescence.7 For example, previous studies have shown that SOD1 proteins require 1.3 h to be fully reduced in the presence of 5 mM DTT at 37 °C (with orbital gyration).7 Thus, disulfide reduction kinetics (and how they might vary among mutants) is built into the assay design. The sets of individual longitudinal plots of ThT fluorescence for each replicate amyloid assay (Figure 2a) exhibited the characteristic broad range in lag time, steepness, and maximum fluorescence that have been previously reported for the fibrillization of WT and ALS-variant apo-SOD1 in microtiter plate assays.4−6 Only one variant, H46R SOD1, did not show any ThT fluorescence during the course of the assay (Figure 2a) and did not fibrillize according to TEM but remained soluble according to SDS−PAGE (Figure 2c). The absence of fibrillization for H46R SOD1 has been recently reported by a separate research group (that studied the N-terminally unacetylated human protein expressed by E. coli).42 All other ALS variants (and the WT protein) aggregated into fibrillar species (Figure 2b). The longitudinal traces with weak maximal fluorescence and late lag times have been previously shown to not be caused by a lack of protein aggregation, per se,4,43 but instead reflect the coformation of different structural types of aggregates (some of which are amorphous4,43) that exhibit weaker ThT fluorescence. For example, analysis of protein solutions before each aggregation assay and after centrifugation at the end of each assay demonstrated that SOD1 proteins aggregate into sedimentable species (except for H46R apo-SOD1, which remained mostly soluble), even in wells exhibiting weak maximal fluorescence (Figure 2c). When analyzing replicate amyloid assays (from Figure 2a) with Kaplan−Meier analysis (Figure 3a), we defined an “event” as the occurrence of fibrillization in any well of the microtiter plate, as defined by reaching half of the maximum ThT fluorescence, i.e., the inflection point of the sigmoidal plots shown in Figure 2a (i.e., t0 in eq 1). In this study, the principal read-out of Kaplan−Meier analyses is a Hazard ratio of ALSvariant fibrillization. The Hazard ratio of fibrillization is the risk that an iterate solution of ALS-variant apo-SOD1 will fibrillize relative to the risk that an iterate solution of WT apo-SOD1 will fibrillize over that same time period. Hazard ratios of fibrillization were calculated using the log rank method (similar to methods used to calculate Hazard ratios of ALS patients10). Hazard ratios >1 indicate that the mutant SOD1 protein has a
Table 1. Thermostability and Metal Content of Apo-SOD1 Proteins SOD1 variant
Tm (°C)
Zn2+ (per dimer)
Cu2+ (per dimer)
WT A4V G37R L38V H46R D90A G93R E100K E100G I113T
53.54 41.61 45.51 42.70 53.48 49.70 42.80 49.18 43.51 42.36
0.08 0.03 0.04 0.02 0.01 0.07 0.05 0.08 0.02 0.02
0.04 0.01 0.04 0.02 0.01 0.03 0.02 0.05 0.04 0.01
between 97% and 99% free of copper and zinc for all proteins studied (i.e., the extent of demetalation only varied by 2% across all mutants studied). Proteins with the highest levels of residual metal (e.g., WT SOD1) contained ∼0.08 equiv of Zn2+ per dimer and ∼0.04 equiv of Cu2+ per dimer (of a maximum of 4.0 theoretical equivalents), i.e., WT SOD1 was 97% demetalated. Proteins with the lowest levels of Cu2+ and Zn2+ (e.g., H46R: 0.01 Zn2+/dimer and 0.01 Cu2+/dimer) were 99% demetalated. Differential scanning calorimetry (DSC) and mass spectrometry confirmed that all proteins were properly folded and lacked any post-translational modifications (Figure 1b,c), with the exception of N-terminal acetylation (we express our proteins in S. cerevisiae or baker’s yeast because SOD1 is properly acetylated at the N-terminus when expressed in S. cerevisiae). 1380
DOI: 10.1021/acschemneuro.7b00029 ACS Chem. Neurosci. 2017, 8, 1378−1389
Research Article
ACS Chemical Neuroscience
Figure 2. Stochastic fibrillization of WT and ALS variant apo-SOD1. (a) ThT fluorescence assays of fibrillization for WT and ALS-variant apo-SOD1 performed in microtiter plates (37 °C, pH 7.4, and 10 mM TCEP). Each trace represents an iterate assay (ntotal = 825; the number of assays for each variant depended upon availability). The shaded area in each plot illustrates the time range corresponding to the average lag time (τ) ± one standard deviation (σ). The average lag time for each SOD1 variant is listed above the double arrow. Note: H46R apo-SOD1 did not fibrillize. (b) Transmission electron micrographs of proteins after the completion of the assay. (c) SDS−PAGE of all apo-SOD1 proteins before and after the ThT aggregation assay. Scale bars = 200 nm.
greater risk of fibrillization relative to WT SOD1; ratios = 1 demonstrate equal risk; ratios 1 (Figure 3b; Table 2). Other variants have lower probability, e.g., E100G, G37R, and H46R SOD1 have Hazard ratios 1 indicate faster progression, relative to sporadic ALS; ratios = 1 and
