Toxic SOD1 trimers are off-pathway in the formation of amyloid-like fibrils in ALS

Abstract

Accumulation of insoluble amyloid fibrils is widely studied as a critical factor in the pathology of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Misfolded Cu, Zn superoxide dismutase (SOD1) was the first protein linked to ALS, and non-native SOD1 trimeric oligomers were recently linked to cytotoxicity, while larger oligomers were protective to cells. The balance between trimers and larger aggregates in the process of SOD1 aggregation is, thus, a critical determinant of potential therapeutic approaches to treat ALS. However, it is unknown whether these trimeric oligomers are a necessary intermediate for larger aggregate formation or a distinct off-pathway species competing with fibril formation. Depending on the on- or off-pathway scenario of trimer formation, we expect drastically different therapeutic approaches. Here, we show that the toxic SOD1 trimer is an off-pathway intermediate competing with protective fibril formation. We design mutant SOD1 constructs that remain in a trimeric state (super-stable trimers) and show that stabilizing the trimeric SOD1 prevents formation of fibrils in vitro and in a motor neuron-like cell model (NSC-34). Using size exclusion chromatography, we track the aggregation kinetics of purified SOD1 and show direct competition of trimeric SOD1 with larger oligomer and fibril formation. Finally, we show the trimer is structurally independent of both larger soluble oligomers and insoluble fibrils using circular dichroism spectroscopy and limited proteolysis.

Figure 1

The formation of soluble intermediates for SOD1 is thought to happen either on- or off-pathway. On-pathway, the formation of the trimer is a necessary step in the formation of larger insoluble fibrils. Off-pathway, the formation of the toxic soluble trimer directly competes with insoluble fibril formation. To see this figure in color, go online.

Figure 2

Stabilizing the trimeric form of SOD1 reduces insoluble fibril formation and increases cell death. (A) ThT expression shows a large amount of amyloid aggregation in A4V mutants (green) compared with WT (blue), and this expression reduces as the trimer is stabilized with slight stabilization by F20L (orange) and further stabilization by F20L-H46Q and H46Q-G108H (pink and purple). (B) Dot blots of the insoluble protein fraction of NSC-34 cells expressing several SOD1 mutants show a larger portion of insoluble SOD1 in WT, A4V, and trimer destabilizing mutant D101I compared with trimer stabilizing mutants (F20L-H46Q in F-H and H46Q-G108H in H-G) showing that stabilizing the trimer reduces inclusion formation. Error bars represent the standard deviation across three repeats of the assay. p-Values are calculated using a two sided T test. (C) NADH (lactate dehydrogenase assay) intensity shows a significant increase in cell death when trimers are stabilized (F20L and F20L-H46Q in F-H), and H46G-G108H in H-G) and a decrease in cell death when trimers are destabilized (D101I) compared with native mutant A4V. Error bars represent the standard deviation across triplicate wells. p-Values are calculated using a two sided T test. (D) Expression of the different mutants of human SOD1, D101I has a lower-molecular-weight human SOD1 band than all other SOD1 constructs, but two bands (human and mouse) are still visible and other human-specific antibodies still identified D101I. ^∗∗p < 5 × 10⁻², ^∗∗∗p < 10⁻⁴. To see this figure in color, go online.

Figure 3

SEC time course for A4V 1. (A) The area under the chromatograph curves is quantified for each time point (trimer = orange, monomer = blue, soluble larger oligomer = green, insoluble aggregates = purple). The different species are then plotted against each other to determine the correlation coefficients. (B) The monomer and trimer concentrations have a correlation coefficient of 0.29 (p = 1.97 × 10⁻²). (C) The monomer and aggregate (insoluble and large soluble) concentrations have a correlation coefficient of −0.46 (p = 10⁻⁵). (D) The trimer and aggregate concentrations have a strong negative correlation coefficient of −0.98 (p = 6.93 × 10⁻⁴⁸). To see this figure in color, go online.

Figure 4

Trimeric SOD1 is structurally independent from monomeric and larger soluble and insoluble aggregates. (A) Pepsin digestion of two insoluble aggregate samples (WT and A4V) as well as two trimer samples (F20L-H46Q and H46Q-G108H) show six similar cut sites (orange) as well as 10 additional cut sites (red), which are only present in the insoluble fibrils. (B) CD spectra show that SOD1 trimers (orange) have a larger negative peak at 205 nm, which is more characteristic of a helical secondary structure, while monomers (blue) and larger soluble aggregates (green) have a much smaller peak shifted to 215 nm, which is more characteristic of β-sheets. Error curves depict the standard deviation across the six SOD1 mutants for each isolated species. To see this figure in color, go online.

Figure 5

The formation of fibrils is a natural protective mechanism in cells during stress. Formation of off-pathway oligomers is a toxic by product contributing to cell death in multiple diseases. To see this figure in color, go online.