Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Abstract

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.

Keywords: ALS; neurodegeneration; protein aggregation; protein misfolding; structural modeling.

Fig. 1.

SOD1 forms nonnative trimer. (A) HS-AFM analysis reveals that under stabilizing conditions (pH 3.5), SOD1 oligomer features a tripartite structure, in line with volumetric AFM measurements of dry samples, where oligomer volume is ∼71.7 nm³ and monomer volume is ∼25.3 nm³ (Fig. S1A). We observe three distinct monomers in HS-AFM recordings at a SOD1 concentration of 30 nM. The three subunits form a nonspherical compact particle, which is stable over long time periods. (Scale bar: 5 nm.) (B) In destabilizing conditions (pH 7.4), SOD1 trimers dissociate into three distinct monomers, even at the high end of physiological concentration (100 nM). (Scale bar: 5 nm.)

Fig. S1.

Volume distributions of SOD1 oligomers demonstrate trimeric stoichiometry. (A) At pH 3.5, the volume (V) distribution of SOD1 monomer is narrow, with its peak centered at 25.3 nm³ (Left), whereas the volume distribution of SOD1 oligomer is broad, with its peak centered at 71.7 nm³ (Right). Notably, the volume of oligomer is approximately triple the volume of monomer, denoting that the SOD1 oligomer is trimeric. (Left) At pH 7.4, the peak of the dimeric volume distribution is centered at 52.4 nm³. (Right) Oligomeric volume distribution, although featuring a broad tail leading up to oligomeric values, has a peak centered at 28.5 nm³, comparable to the SOD1 monomer at pH 3.5, denoting that the SOD1 oligomer is primarily dissociated at pH 7.4. Errors are ±SD. Data were obtained with regular AFM of dried samples. (B) Using AFM, under trimer-stabilizing conditions, we observe small populations of short, insoluble fibrils with a molecular mass >600 kDa: 5 μm × 5 μm (Left) and 1 μm × 1 μm (Right) images. The color bar denotes feature height. Arrows indicate fibrils. Data and images were obtained with AFM of dried samples.

Fig. 2.

Hybrid experimental/computational method leads to a model of metastable SOD1 trimer. (A) SOD1 trimer proteolytic cut sites (gray spheres, shown here on the native dimer structure) in the dimer interface and secondary structural elements suggest significant structural differences between native and trimeric structures. (B) Structural model of the metastable SOD1 trimer obtained using limited proteolysis data as constraints in several rounds of coarse-grained and atomistic DMD simulations (Fig. S2). (C) Representation of the SOD1 linear sequence, residues 1–153. Proteolytic cut sites (vertical lines) determined in limited proteolysis experiments differ significantly in SOD1 monomer (Top), dimer (Middle), and trimer (Bottom), supporting structural rearrangement during the aggregation process and SOD1 trimer formation.

Fig. S2.

Schematic diagram for structural modeling of metastable protein oligomers. The procedure for deriving a bias potential using experimental constraints from limited proteolysis is illustrated. The term {cut} is the set of residues at which a proteolytic cut is made. E₁, E₂, E₃, and E₄ are the repulsive potentials applied to (i) the residues that are the site of proteolytic cuts, (ii) residues 1 removed in sequence from cut sites, (iii) residues 2 removed in sequence from cut sites, and (iv) residues 3 removed in sequence from cut sites. E_ij are the interresidue interaction energies applied when residues are within contact range. E_Gō is the interaction energy assigned to native contacts that are in contact range. N_c is the average number of contacts made by each cut site residue.

Fig. S3.

Folding of SOD1 monomer with various values of λ. (A) λ = 0: SOD1 monomer folding features two distinct states with no folding intermediates. Transitions in energy and the radius of gyration are sharp and well-defined. Trajectories from T = 0.80 and T = 0.85 are located at the transition, and exhibit flipping between folded and unfolded structures. (B) λ = 0.66: SOD1 monomer folding features distinct intermediate states. Transitions in energy and the radius of gyration remain well-defined. Trajectories from T = 0.70, T = 0.75, and T = 0.80 are located at the transition, and exhibit flipping between states. (C) λ = 0.99: SOD1 monomer folding features distinct intermediate states, with a greater separation in energy at λ = 0.66. Transitions in energy and the radius of gyration are well-defined. Trajectories from T = 0.50 and T = 0.55 and from T = 0.75 and T = 0.80 are located at the transitions, and exhibit flipping between two respective states. C_V, heat capacity at constant volume.

Fig. S4.

Toxic epitope exposed on SOD1 trimer surface. Ensemble of SOD1 trimer models. (A) Conformational Ab C4F6, which binds several disease-associated species of SOD1 (16), selectively binds to WT SOD1 trimer at various concentrations, but does not bind to native WT type SOD1 dimer and exhibits minimal binding to WT SOD1 monomer. (B) Recently identified epitope of the C4F6 Ab (16, 18) is exposed on the surface of our SOD1 trimer model. Individual monomers are depicted in pale green, bright orange, and light blue; residues comprising the C4F6 epitope are highlighted in darker colors: forest, brown, and deep blue, respectively. Rotation angles describe the transformation from the “front” (Upper Left), such that the structures represent the “back” (Upper Right), “top” (Lower Left), and “bottom” (Lower Right). The C4F6 Ab has been shown previously to bind WT and disease-linked mutant SOD1 trimer at physiological pH (15). In addition to experimental data from limited proteolysis, the exposure of the C4F6 epitope further verifies our model, as well as providing support for the toxicity of SOD1 trimer. (C) Replicate SOD1 trimer models resulting from 10 independent repetitions of our protocol (Fig. S3) feature significant variation in tertiary and quaternary structure. Models vary in the degree of domain swapping, as well as in the amount of native tertiary structure maintained in each monomer. We note that despite differences in tertiary and quaternary structure of the 10 SOD1 trimer models, a high level of consensus on the identity of residues involved in trimeric interface interactions is maintained, with at least 77% and as much as 94% identity in interface residue identity between any two models.

Fig. 3.

Designed mutations verify the model and demonstrate control of SOD1 aggregation. Mutations to trimer interface residues designed to stabilize or destabilize SOD1 trimer but having no effect on SOD1 monomer or dimer, are shown with ΔΔG_mut for the trimer, native dimer, and native monomer structures. Aggregation time courses were measured for each mutant after incubation at physiological concentration (30 μM) and 37 °C for for 0 h (solid line), 2 h (− −), 4 h (− − •), 8 h (− • •), or 24 h (• •). The aggregation of apo-WT SOD1 is shown for comparison, with trimer (T), dimer (D), monomer (M), and large aggregate (L) peaks labeled when present. Vertical dashed lines between panels aid comparison of trimer, dimer, and monomer peaks between mutants. We find that the N65V mutation, predicted to be destabilizing to the SOD1 trimer, results in a smaller population of trimer than WT, shifting the SOD1 population toward dimer and monomer formations. The N53I mutation, also predicted to destabilize SOD1 trimer, results in no detectable SOD1 trimer but, instead, increased populations of large aggregates. The F20L mutation, predicted to stabilize SOD1 trimer, promotes trimer formation early in the aggregation process and maintains high levels of trimer throughout the experiment. Additional aggregation time courses can be found in Fig. S5. mAU, milli-absorbance units.

Fig. S5.

Designed mutations verify the model and demonstrate control of SOD1 aggregation. Mutations to trimer interface residues are designed to stabilize or destabilize SOD1 trimer but to have no effect on SOD1 monomer or dimer. ΔΔG_mut for the trimer, native dimer, and native monomer structures can be found in Fig. 3. Aggregation time courses were measured for each mutant after incubation at physiological concentration (30 μM) and 37 °C for 0 h (solid line), 2 h (− −), 4 h (− − •), 8 h (− • •), or 24 h (• •). The aggregation of apo-WT SOD1 is shown for comparison, with trimer (T), dimer (D), monomer (M), and large aggregate (L) peaks labeled when present. We find that the G108H, I99H, and P62Y mutations, predicted to be destabilizing to the SOD1 trimer, result in smaller populations of trimer than WT, shifting the SOD1 population toward dimer and monomer formations. The G147P and D101I mutations, also predicted to destabilize SOD1 trimer, result in no or very little detectable SOD1 trimer after 24 h, but instead increased populations of large aggregates, especially in G147P-SOD1. In four additional mutants that we predict to be trimer-stabilizing, H46Q-SOD1, D124Q-SOD1, P74G-SOD1, and E21Q-SOD1, we observe the formation of trimer that is overcome by a nonnative extended dimer species (Fig. S6), which (as discussed in the main text) we could not predict computationally. mAU, milli-absorbance units.

Fig. S6.

Mutations to SOD1 trimer interfaces stabilize formation of nonnative extended dimer conformations. (A) Volume distributions of (left to right) I99H, P74G, P62Y, and G108H after 24 h of aggregation at 30 μM SOD1 at 37 °C indicate dominance of a nonnative dimer species. Solid lines are Gaussian fits, with most probable volume designated. Errors are ±SD. (B) I99H-SOD1, a trimer-destabilizing mutant, exists almost exclusively as an extended dimer, as demonstrated by HS-AFM images. (C) P62Y-SOD1, a trimer-destabilizing mutant, exists as an extended dimer, as demonstrated by HS-AFM images. (D) G108H-SOD1, a trimer-destabilizing mutant, exists as an extended dimer with a small trimeric population (not shown), as demonstrated by HS-AFM images. (E) P74G-SOD1, a trimer-stabilizing mutant, exists as an extended dimer and linear or compact trimer. In B–E, the scan area is 50 nm × 50 nm. (Scale bar: B–E, 5 nm.) Dashed lines on the image represent the 1D cross-section used for height profiling below each image, with red circles indicating the initial point of measurement. Green dots on the height profiles indicate peak positions, with the distance between the two gray lines representing the distance of two adjacent subunits. Data were obtained with regular AFM imaging of dried samples.

Fig. 4.

SOD1 trimer is cytotoxic. Expression of A4V-SOD1 (positive control) and trimer-stabilizing mutant SOD1s in NSC-34 motor neuron-like cells increases the incidence of cell death compared with WT SOD1 (negative control), whereas trimer-destabilizing mutants have no effect on viability. Applied 3 d posttransfection, red stain [propidium iodide (PI)] identifies nuclei of dead cells, whereas blue stain (Hoechst) identifies nuclei of all cells. Incidence of cell death is measured as the percentage of total cells exhibiting PI staining (left to right from Upper Left): WT, 4%; P62Y, 8%; G108H, 5%; N53I, 6%; A4V, 23%; H46Q, 29%; K136H, 25%; F20L, 27%; P74G, 24%; and E21Q, 22%. (Magnification: 20×.) Cell death correlates with trimer stability with a P value of 0.0476, or a P value of 0.000966 with exclusion of the outlier P62Y, which is extremely destabilizing (Fig. S7). Elevated levels of the apoptotic marker cleaved caspase 3 in NSC-34 motor neuron hybrid cells expressing A4V-SOD1 (positive control) and trimer-stabilizing SOD1 mutants (demonstrated by Western blot, Lower Right) confirm increased cytotoxicity of these mutants compared with WT SOD1 (negative control) and trimer-destabilizing SOD1 mutants.

Fig. S7.

Stabilization of trimer correlates with cell death. The size of the SOD1 trimer population is proportional to the stability of that species in relation to other SOD1 species. We find that the stability of the SOD1 trimer is highly indicative of the incidence of cell death, with the stability of the mutants corresponding to cell death with a P value of 0.0476 (as calculated from the Fisher test) when all points are included. With removal of the outlier P62Y-SOD1, which mutation is extremely destabilizing at +62 kcal/mol (Fig. 3), the P value of the relation between SOD1 trimer stability and cell death becomes 0.001.