Colorimetric and Longitudinal Analysis of Leukocoria in Recreational Photographs of Children with Retinoblastoma

Retinoblastoma is the most common primary intraocular tumor in children. The first sign that is often reported by parents is the appearance of recurrent leukocoria (i.e., “white eye”) in recreational photographs. A quantitative definition or scale of leukocoria – as it appears during recreational photography – has not been established, and the amount of clinical information contained in a leukocoric image (collected by a parent) remains unknown. Moreover, the hypothesis that photographic leukocoria can be a sign of early stage retinoblastoma has not been tested for even a single patient. This study used commercially available software (Adobe Photoshop®) and standard color space conversion algorithms (operable in Microsoft Excel®) to quantify leukocoria in actual “baby pictures” of 9 children with retinoblastoma (that were collected by parents during recreational activities i.e., in nonclinical settings). One particular patient with bilateral retinoblastoma (“Patient Zero”) was photographed >7, 000 times by his parents (who are authors of this study) over three years: from birth, through diagnosis, treatment, and remission. This large set of photographs allowed us to determine the longitudinal and lateral frequency of leukocoria throughout the patient's life. This study establishes: (i) that leukocoria can emerge at a low frequency in early-stage retinoblastoma and increase in frequency during disease progression, but decrease upon disease regression, (ii) that Hue, Saturation and Value (i.e., HSV color space) are suitable metrics for quantifying the intensity of retinoblastoma-linked leukocoria; (iii) that different sets of intraocular retinoblastoma tumors can produce distinct leukocoric reflections; and (iv) the Saturation-Value plane of HSV color space represents a convenient scale for quantifying and classifying pupillary reflections as they appear during recreational photography

Aggregation of Cu, Zn superoxide dismutase in amyotrophic lateral sclerosis : kinetic, mechanistic, and therapeutic approaches.

Investigating in vitro kinetics of protein aggregation using high-throughput microplate-based assays provides open venues for obtaining valuable information regarding mechanism(s) of pathogenesis of protein aggregates in neurodegenerative diseases, and facilitates development of effective therapies. In this dissertation, I use high-throughput microplate-based assays for studying the real-time kinetics of wild type and ALS-variant Cu, Zn superoxide dismutase (SOD1) aggregation in the context of amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease that is hallmarked with selective death of motor neurons, which leads to muscle paralysis, and eventually death. Mutations in SOD1 gene are believed to underlie ~ 3 % of cases of ALS via triggering the misfolding and aggregation of SOD1 protein. These SOD1 aggregates render toxicity in motor neurons via interfering with and disrupting normal functions of cells such as cytoplasmic and axonal transport or membrane integrity. In this dissertation, I first show that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of wild-type (WT) and ALS-variant apo-SOD1 by increasing the intrinsic net negative charge of the polypeptide, via acetylation of multiple lysines. In the third chapter, I measure rates of fibrillar and amorphous SOD1 aggregation at high iteration and show that rates of oligomerization were intrinsically irreproducible and populated continuous probability distributions. In the fourth chapter, I used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ~ 103 replicate amyloid assays of WT SOD1 and nine ALS variants, and showed that the probability of apo-SOD1 fibrillization is non-uniformly altered by different mutations. I found a linear correlation between the Hazard ratios of SOD1 fibrillization and those of patient survival in SOD1-linked ALS. The fifth chapter answers a very fundamental question: “how do gyrating beads accelerate amyloid fibrillization?” I found that increasing the mass in beads from non-polymeric materials (e.g., steel) increases the nucleation rate of SOD1 fibrillization, whereas hydrophobicity and surface adhesion affected rate of SOD1 fibrillization in the case of polymeric beads. In chapter six, I study the mechanism behind Hofmeister series in proteins. Chapter seven includes a project dedicated to early detection of leukocoria in children with retinoblastoma, during recreational photography

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

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

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)