Calcium ions promote superoxide dismutase 1 (SOD1) aggregation into non-fibrillar amyloid: a link to toxic effects of calcium overload in amyotrophic lateral sclerosis (ALS)?

Abstract

Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca(2+) dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca(2+). We show that at physiological pH, Ca(2+) induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca(2+) boosts the onset of SOD1 aggregation. In agreement, Ca(2+) decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca(2+) induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca(2+)-induced aggregates, thus indicating that Ca(2+) diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca(2+) levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca(2+) may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.

Keywords: Amyloid; Amyotrophic Lateral Sclerosis (Lou Gehrig's Disease); Biophysics; Calcium; Circular Dichroism (CD); Electron Microscopy (EM); Infrared Spectroscopy; Neurodegenerative Diseases; Protein Aggregation; Superoxide Dismutase (SOD).

FIGURE 1.

Effect of Ca²⁺ on the secondary structure and hydrophobicity of apo-SOD1. A, far UV CD spectra of apo-SOD1 (−Ca²⁺) and upon overnight incubation with Ca²⁺ (+Ca²⁺) Inset, plot of the intensity of the SOD1-negative band centered at 218 nm, at increasing Ca²⁺/SOD1 ratios. B, ATR-FTIR difference spectrum in the amide I region of Ca²⁺-incubated SOD1 minus the control; positive peaks denote features increased in the presence of Ca²⁺. C, ANS fluorescence emission spectra of apo-SOD1 (−Ca²⁺) and upon incubation with Ca²⁺ (+Ca²⁺), overlaid with the emission spectrum of unbound ANS. See “Materials and Methods” for further details.

FIGURE 2.

Impact of increasing Ca²⁺ concentrations on apo-SOD1 oligomerization. DLS analysis (mean size and count rate) of apo-SOD1 upon overnight incubation at 37 °C and stirring, at increasing Ca²⁺/SOD1 ratios. See “Materials and Methods” for further details. Error bars, S.D.

FIGURE 3.

Ca²⁺ enhances apo-SOD1 aggregation. A, aggregation profile of SOD1 monitored by mean light scattering intensity analysis over time, in the absence and in the presence of Ca²⁺. B, aggregation kinetics of SOD1 monitored by ThT fluorescence emission, in the absence and in the presence of Ca²⁺. −Ca²⁺, no CaCl₂; +Ca²⁺, CaCl₂/SOD1 = 2. See Table 1 for kinetic parameters and “Materials and Methods” for further details. Error bars, S.D.

FIGURE 4.

Ca²⁺ favors the formation of larger SOD1 aggregates. A, comparison of the particle size distributions of apo-SOD1 aggregates in the presence (top) and in the absence (bottom) of Ca²⁺ upon 48-h incubation at 37 °C and stirring. Note the broader distributions at lower particle diameters in the absence of Ca²⁺. B, relative distribution of total light scattering intensities arising from aggregated SOD1 species with a hydrodynamic diameter under and above 500 nm, for the Ca²⁺-incubated SOD1 and control. +Ca²⁺, CaCl₂/SOD1 = 2. See “Materials and Methods” for further details. Error bars, S.D.

FIGURE 5.

Ca²⁺ enhances apo-SOD1 aggregation also under reducing conditions. Aggregation kinetics of 80 μm apo-SOD1 with 10 mm tris(2-carboxyethyl)phosphine was monitored by ThT fluorescence emission, in the absence and in the presence of Ca²⁺. −Ca²⁺, no CaCl₂; +Ca²⁺, CaCl₂/SOD1 = 2. See Table 2 for kinetic parameters and “Materials and Methods” for further details.

FIGURE 6.

Ca²⁺ induces secondary structure changes on SOD1 aggregates. ATR-FTIR difference spectrum in the amide I region of SOD1 aggregates formed in the presence of Ca²⁺ minus in its absence. Positive peaks denote features that are enriched by the presence of Ca²⁺, whereas negative peaks denote structures preferentially formed in the absence of Ca²⁺. The analyzed aggregates were obtained at pH 7.5 after incubation under constant agitation with a Teflon bead at 37 °C for 150 h. −Ca²⁺, no CaCl₂; +Ca²⁺, CaCl₂/SOD1 = 2. See “Materials and Methods” for further details.

FIGURE 7.

Ca²⁺ influences SOD1 aggregation pathway toward non-fibrillar aggregates. Shown is dot blot analysis using conformational antibodies. Anti-amyloid oligomer (A11) and anti-amyloid fibril (OC) antibodies were used to test conformers prevalent in SOD1 aggregates formed in the presence and absence of Ca²⁺ (n = 3). −Ca²⁺, no CaCl₂; +Ca²⁺, CaCl₂/SOD1 = 2. See “Materials and Methods” for further details.

FIGURE 8.

Ca²⁺ modulates the morphology of SOD1 aggregates, as analyzed by TEM. In the absence of Ca²⁺, apo-SOD1 formed typical amyloid fibrils with diameters of ∼10 nm (top). In the presence of Ca²⁺, fibrils are less abundant; in particular, amorphous aggregates (bottom right panel, arrow) and oligomers of variable sizes (bottom panels, arrowheads) were often visualized. Analyzed aggregates derived from apo-SOD1 incubated at pH 7.5 with or without Ca²⁺ under constant agitation with a Teflon bead at 37 °C for 150 h. −Ca²⁺, no CaCl₂; +Ca²⁺, CaCl₂/SOD1 = 2. See “Materials and Methods” for further details.