Stochastic Formation of Fibrillar and Amorphous Superoxide Dismutase Oligomers Linked to Amyotrophic Lateral Sclerosis

Recent reports suggest that the nucleation and propagation of oligomeric superoxide dismutase-1 (SOD1) is effectively stochastic in vivo and in vitro. This perplexing kinetic variabilityobserved for other proteins and frequently attributed to experimental errorplagues attempts to discern how <i>SOD1</i> mutations and post-translational modifications linked to amyotrophic lateral sclerosis (ALS) affect SOD1 aggregation. This study used microplate fluorescence spectroscopy and dynamic light scattering to measure rates of fibrillar and amorphous SOD1 aggregation at high iteration (<i>n</i>_total = 1.2 × 10³). Rates of oligomerization were intrinsically irreproducible and populated continuous probability distributions. Modifying reaction conditions to mimic random and systematic experimental error could not account for kinetic outliers in standard assays, suggesting that stochasticity is not an experimental artifact, rather an intrinsic property of SOD1 oligomerization (presumably caused by competing pathways of oligomerization). Moreover, mean rates of fibrillar and amorphous nucleation were not uniformly increased by mutations that cause ALS; however, mutations did increase kinetic noise (variation) associated with nucleation and propagation. The stochastic aggregation of SOD1 provides a plausible statistical framework to rationalize how a pathogenic mutation can increase the probability of oligomer nucleation within a single cell, without increasing the mean rate of nucleation across an entire population of cells

Gibbs Energy of Superoxide Dismutase Heterodimerization Accounts for Variable Survival in Amyotrophic Lateral Sclerosis

The exchange of subunits between homodimeric mutant Cu, Zn superoxide dismutase (SOD1) and wild-type (WT) SOD1 is suspected to be a crucial step in the onset and progression of amyotrophic lateral sclerosis (ALS). The rate, mechanism, and Δ<i>G</i> of heterodimerization (Δ<i>G</i>_Het) all remain undetermined, due to analytical challenges in measuring heterodimerization. This study used capillary zone electrophoresis to measure rates of heterodimerization and Δ<i>G</i>_Het for seven ALS-variant apo-SOD1 proteins that are clinically diverse, producing mean survival times between 2 and 12 years (postdiagnosis). The Δ<i>G</i>_Het of each ALS variant SOD1 correlated with patient survival time after diagnosis (<i>R</i>² = 0.98), with more favorable Δ<i>G</i>_Het correlating with shorter survival by 4.8 years per kJ. Rates of heterodimerization did not correlate with survival time or age of disease onset. Metalation diminished the rate of subunit exchange by up to ∼38-fold but only altered Δ<i>G</i>_Het by <1 kJ mol^–1. Medicinal targeting of heterodimer thermodynamics represents a plausible strategy for prolonging life in SOD1-linked ALS

Glycerolipid Headgroups Control Rate and Mechanism of Superoxide Dismutase‑1 Aggregation and Accelerate Fibrillization of Slowly Aggregating Amyotrophic Lateral Sclerosis Mutants

Interactions between superoxide dismutase-1 (SOD1) and lipid membranes might be directly involved in the toxicity and intercellular propagation of aggregated SOD1 in amyotrophic lateral sclerosis (ALS), but the chemical details of lipid–SOD1 interactions and their effects on SOD1 aggregation remain unclear. This paper determined the rate and mechanism of nucleation of fibrillar apo-SOD1 catalyzed by liposomal surfaces with identical hydrophobic chains (RCH₂(O₂C₁₈H₃₃)₂), but headgroups of different net charge and hydrophobicity (i.e., R­(CH₂)­N⁺(CH₃)₃, RPO₄^–(CH₂)₂N⁺(CH₃)₃, and RPO₄^–). Under semiquiescent conditions (within a 96 well microplate, without a gyrating bead), the aggregation of apo-SOD1 into thioflavin-T-positive (ThT­(+)) amyloid fibrils did not occur over 120 h in the absence of liposomal surfaces. Anionic liposomes triggered aggregation of apo-SOD1 into ThT­(+) amyloid fibrils; cationic liposomes catalyzed fibrillization but at slower rates and across a narrower lipid concentration; zwitterionic liposomes produced nonfibrillar (amorphous) aggregates. The inability of zwitterionic liposomes to catalyze fibrillization and the dependence of fibrillization rate on anionic lipid concentration suggests that membranes catalyze SOD1 fibrillization by a primary nucleation mechanism. Membrane-catalyzed fibrillization was also examined for eight ALS variants of apo-SOD1, including G37R, G93R, D90A, and E100G apo-SOD1 that nucleate slower than or equal to WT SOD1 in lipid-free, nonquiescent amyloid assays. All ALS variants (with one exception) nucleated faster than WT SOD1 in the presence of anionic liposomes, wherein the greatest acceleratory effects were observed among variants with lower net negative surface charge (G37R, G93R, D90A, E100G). The exception was H46R apo-SOD1, which did not form ThT­(+) species

Kaplan–Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset

Over 150 mutations in <i>SOD1</i> (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (<i>in vitro</i>), 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 (<i>in vitro</i>) 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 <i>SOD1</i> 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 (<i>R</i>² = 0.67; Pearson’s <i>r</i> = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (<i>R</i>² = 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 (<i>R</i>² = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that <i>SOD1</i> 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)

Deamidation of Asparagine to Aspartate Destabilizes Cu, Zn Superoxide Dismutase, Accelerates Fibrillization, and Mirrors ALS-Linked Mutations

The reactivity of asparagine residues in Cu, Zn superoxide dismutase (SOD1) to deamidate to aspartate remains uncharacterized; its occurrence in SOD1 has not been investigated, and the biophysical effects of deamidation on SOD1 are unknown. Deamidation is, nonetheless, chemically equivalent to Asn-to-Asp missense mutations in SOD1 that cause amyotrophic lateral sclerosis (ALS). This study utilized computational methods to identify three asparagine residues in wild-type (WT) SOD1 (i.e., N26, N131, and N139) that are predicted to undergo significant deamidation (i.e., to >20%) on time scales comparable to the long lifetime (>1 year) of SOD1 in large motor neurons. Site-directed mutagenesis was used to successively substitute these asparagines with aspartate (to mimic deamidation) according to their predicted deamidation rate, yielding: N26D, N26D/N131D, and N26D/N131D/N139D SOD1. Differential scanning calorimetry demonstrated that the thermostability of N26D/N131D/N139D SOD1 is lower than WT SOD1 by ∼2–8 °C (depending upon the state of metalation) and <3 °C lower than the ALS mutant N139D SOD1. The triply deamidated analog also aggregated into amyloid fibrils faster than WT SOD1 by ∼2-fold (<i>p</i> < 0.008**) and at a rate identical to ALS mutant N139D SOD1 (<i>p</i> > 0.2). A total of 534 separate amyloid assays were performed to generate statistically significant comparisons of aggregation rates among WT and N/D SOD1 proteins. Capillary electrophoresis and mass spectrometry demonstrated that ∼23% of N26 is deamidated to aspartate (iso-aspartate was undetectable) in a preparation of WT human SOD1 (isolated from erythrocytes) that has been used for decades by researchers as an analytical standard. The deamidation of asparaginean analytically elusive, sub-Dalton modificationrepresents a plausible and overlooked mechanism by which WT SOD1 is converted to a neurotoxic isoform that has a similar structure, instability, and aggregation propensity as ALS mutant N139D SOD1

Examples of Cropped Leukocoric and Non-Leukocoric Pupils from a Set of 7377 Pictures of Patient Zero (and Control Children Who Were Photographed Alongside Patient).

<p>Each spiral contains: (i) cropped leukocoric pictures from Patient Zero (denoted Lk+/Rb+), (ii) non-leukocoric pupils from Patient Zero (Lk−/Rb+), and (iii) non-leukocoric pupils from healthy control subjects (Lk−/Rb−). <b>A</b>) Cropped leukocoric pupils that exhibit a gray scale (classic leukocoria); cropped leukocoric pupils with non-black and white appearance are also shown: <b>B</b>) yellow, i.e., “xanthocoria”; <b>C</b>) pink, i.e., “rhodocoria”; <b>D</b>) orange, i.e., “cirrocoria”. Many pupils in A–D contain specular reflections of cornea that appear as a white dot and are not indicative of disease.</p

Longitudinal Set of Clinical Images of the Left Retina of Patient Zero Collected with Fundus Photography and Age-Matched Leukocoria in Left Pupil.

<p>The left retina contains three tumors; one large tumor at 12 o'clock, and two smaller tumors at 6 o'clock and 9 o'clock (the two smaller tumors were treated with laser photoablation therapy which resulted in tumor eradication and exposure of the sclera). The radiation symbol denotes the point in time when proton beam radiation therapy was administered to the left eye (age of patient is listed in days).</p

Saturation-Value Scale for Quantifying Leukocoria in Photographs of Children with Retinoblastoma.

<p><b>A)</b> Sectioning the Saturation-Value plane of HSV color space into a useful scale for classifying pupillary reflexes in recreational photographs. In this proposed scale, leukocoria is divided into differing degrees of brightness and color concentration (1° being the brightest, least colored; 3° is the least bright and most colored); areas that likely represent a typical “red” or “black” pupillary reflex are indicated. Each data point labeled “Rb” refers to the average H, S, or V of all leukocoric images of one of nine patients; the superscript of each label refers to the patient number (beginning with zero); subscript text refers to right or left pupil. “PL” refers to Pseudo-Leukocoria from images of a healthy individual that were collected with one of three different camera phones; the subscript refers to the camera that was used to photograph the individual (see text). “NL” refers to Non-Leukocoric controls (average of right and left pupils) from healthy children (i.e., data contained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076677#pone-0076677-g005" target="_blank">Figure 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076677#pone-0076677-t001" target="_blank">Table 1</a>). The value “n” below each Rb, NL, and PL point refers to the number of pictures from which each average was calculated. <b>B)</b> Plot showing the average Hue of cropped pupils from panel A.</p

A Collection of ∼7,000 Digital Photographs of a Single Patient with Retinoblastoma.

<p><b>A</b>) Longitudinal frequency of photography of “Patient Zero” by parents over a three year period (i.e., from birth to 3 years old; 7377 photographs). <b>B</b>) The majority of leukocoric pictures (∼80%) were collected with this compact 7.1 megapixel Canon PowerShot SD750 camera. <b>C</b>) Digital picture of Patient Zero (i.e., child on left, exhibiting leukocoria in left eye) and a healthy playmate (i.e., child on right, exhibiting a red reflex in both eyes). <b>D</b>) Example of a digital picture of Patient Zero; right eye exhibited leukocoria, and the left eye exhibited a red reflex. Photographs in C & D were taken with Canon PowerShot SD750. Permission to include images of the healthy control child was granted by both parents.</p

Quantification of Hue and Value of right and left Leukocoric Pupils of Patient Zero and 19 Healthy Control Children.

<p><b>A</b>) Depiction of Hue as an angular quantity. <b>B</b>) Polar plots of average Hue, per pixel (angular dimension) and average Value, per pixel (radial dimension) for right eye of patient that exhibited leukocoria (red circles), and right eye from 19 healthy children (blue squares). <b>C</b>) Polar plots of average Hue, per pixel (angular dimension) and average Value, per pixel (radial dimension) for left eye of patient that exhibited leukocoria (red circles), and left eye from 19 healthy children (blue squares). <b>D</b>) Combination of data points from plots C and D.</p