Initiation and elongation in fibrillation of ALS-linked superoxide dismutase

Results

Apo SOD1, normally dimeric, can be dissociated into monomers by treatment with a chaotrope such as guanidinium hydrochloride (Gdm·HCl) or by reduction of its intrasubunit disulfide bond (13–15), and monomerization has been implicated in SOD1 aggregation pathways (16). To determine the effect of partial monomerization, apo human WT SOD1 was incubated with agitation at 37°C, pH 7.4, in the presence of either 1 M Gdm·HCl or 50 mM DTT, both conditions known to induce partial dimer dissociation into monomers. An exponential rise in thioflavin T (TfT) fluorescence was observed after a lag time of ≈38 (± 4.2) h in the case of Gdm·HCl and 2.1 (± 0.3) h in the case of DTT. (Fig. 1A). Turbidity developed under both conditions, and electron microscopy confirmed the presence of amyloid fibrils (Fig. 1B). Other reducing agents, specifically tris(2-carboxyethyl)phosphine (TCEP) and glutathione, were also effective in promoting rapid SOD1 fibrillation (Fig. 1C). The presence of dioxygen in these reactions did not alter the kinetics, because samples prepared under anaerobic conditions showed similar lag phases [supporting information (SI) Fig. S1]. The ability of glutathione to promote fibrillation under anaerobic conditions suggests that reducing conditions in vivo such as those found in the cytoplasm are capable of promoting fibrillation.

Gdm·HCl treatment leaves the intrasubunit disulfide bond of SOD1 intact, but 50 mM DTT treatment, in our experience, generates a mixture of disulfide-intact SOD1 (SOD1^S-S) and disulfide-reduced SOD1 (SOD1^2SH). To determine the minimum concentration of DTT required for fibrillation, apo-SOD1 was incubated with 50 μM to 25 mM DTT (Fig. 1D). Surprisingly, as low as 250 μM DTT was capable of promoting the formation of amyloid fibrils. At this concentration of reducing agent, we expect only a small fraction of the protein to be reduced, suggesting that a small amount of disulfide-reduced SOD1 is sufficient to initiate fibrillation.

SOD1^S-S is a dimer, whereas SOD1^2SH is a monomer and, thus, size-exclusion chromatography (SEC) can be used to separate them. SEC on an aliquot collected during lag phase from a fibrillation reaction containing 5 mM DTT (Fig. 2A) showed the presence of dimeric SOD1^S-S eluting at 19.8 min (peak a) and monomeric SOD1^2SH eluting at 21.2 min (peak b). Treatment of SOD1 from these 2 peaks with 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS), a thiol modifying agent, followed by SDS/PAGE demonstrated that 2 extra AMS units per subunit had been added to the SOD1 in peak b, confirming the assignments of peak a as SOD1^S-S and peak b as SOD1^2SH (Fig. S2).

To test the hypothesis that the presence of only a small amount of SOD1^2SH is sufficient to initiate fibrillation of SOD1^S-S, we studied the effect of replacing the reducing agent with SEC-purified SOD1^2SH or SOD1^S-S (peaks a and b, respectively, in Fig. 2A). Because agitation alone has been shown to promote misfolding and amyloid formation in many proteins, because of repeated transient exposures to the air–water interface, we also used SOD1^S-S that had been isolated from the same reaction as the SOD1^2SH, and thus had been similarly agitated, as a control. We found that a very small amount of SOD1^2SH (5% of the overall reaction) was sufficient to induce fibrillation, whereas SEC-purified SOD1^S-S had no effect (Fig. 2B). Amyloid fibril formation is proposed to occur by the formation of 1 or more amyloid nuclei during the lag phase, which then recruit soluble protein during the elongation phase to generate mature fibrils. Our results suggest that SOD1^2SH itself can initiate amyloid formation, even in the absence of reducing agents, by forming nuclei, which then recruit SOD1^S-S to form mature fibrils. This mechanism is likely to be relevant to fALS pathogenesis in vivo because disulfide-reduced SOD1 is present in the spinal cords of transgenic ALS mice that eventually develop SOD1-rich aggregates in their terminal stages (17).

In addition to Cys-57 and Cys-146, which form the disulfide bond, each subunit of SOD1 contains 2 reduced cysteine residues, Cys-6 and Cys-111. Previous cell culture and in vitro aggregation studies suggested that these 2 nondisulfide cysteine residues might play an important role in the formation of disulfide-cross-linked SOD1 oligomers and aggregates (18–20). To understand and distinguish the roles of Cys-6 and Cys-111 from those played by the disulfide-forming residues, Cys-57 and Cys-146, we investigated the effect of mutations in these positions on the ability to initiate fibrillation in our present system (Fig. 3A). Preparations of disulfide-reduced WT SOD1 and C6A,C111S SOD1 (AS-SOD1) were generated by overnight treatment of the apoproteins with 10 mM TCEP, followed by SEC-HPLC purification. The same proteins with cysteine thiolates blocked by reaction with N-ethylmaleimide (NEM) were also tested, as were single-cysteine mutants C57S SOD1 and C146S SOD1. In the absence of reducing agents, only disulfide-reduced WT SOD1 and AS-SOD1 were capable of nucleating the conversion of soluble SOD1 into amyloid fibrils. The absolute requirement that both C57 and C146 be present and unalkylated suggests that these residues participate in the formation of amyloid nuclei by intermolecular disulfide cross-linking.

In addition to initiation, intermolecular disulfide cross-linking might be involved in fibril elongation. For example, apo-SOD1 was found to form soluble, high molecular weight, disulfide-linked structures that bind thioflavin T (TfT) when incubated for extended periods at 37 °C and pH 7.4, with no agitation (18). To probe for the presence of intermolecular disulfide cross-linking under our conditions, SOD1 fibrils grown in 5 mM DTT were denatured in Gdm·HCl with or without TCEP, and the products were analyzed by HPLC-MS (Fig. 3B). Similar amounts of monomeric SOD1 were observed in both cases (peak at 30 min), suggesting that most of the SOD in the fibrils was not cross-linked by disulfide bonds. No dimers, trimers or higher-order oligomers were detected by HPLC. Gel electrophoresis on fibrils denatured in SDS with or without the addition of TCEP confirmed the presence of similar amounts of monomeric SOD under both conditions. Low amounts of dimers and higher-order oligomers were also detected; their presence may be caused by incomplete denaturation of fibrillar SOD1 in 0.5–1% SDS (Fig. 3C). As before, the presence of TCEP had no effect on the yield of monomeric or oligomeric SOD1. HPLC-MS of AS-SOD1 fibrils formed in the presence of 5 mM DTT and denatured in Gdm·HCl, in the absence of TCEP, yielded similar amounts of monomeric SOD in both cases, showing that the presence of free thiolates at Cys-6 and Cys-111 had no effect on the outcome of the reaction (Fig. S3). These results suggest that the nuclei, whose formation likely involved intermolecular disulfide cross-linking, developed into mature fibrils by recruiting disulfide-intact SOD1 through noncovalent interactions.

In vitro studies have shown that the intrasubunit disulfide bond in FALS-SOD1 mutant proteins is more susceptible to reduction than that in WT SOD1 and thus is more likely to be present under the reducing conditions prevalent in vivo (2). We therefore tested the ability of reduced ALS-SOD1 mutant proteins to induce fibrillation of WT SOD1. Apo-SOD1^S-S was incubated with small amounts of apo-G93A^2SH, apo-G37R^2SH, and apo-D101N^2SH (Fig. 4A) in the absence of reducing agents. In each case, the reduced mutant protein, present at 5% of the overall protein concentration, initiated fibrillation with a lag time comparable with that of WT SOD1^2SH. The presence of mutant SOD1 in the fibrils was verified by Fourier transform mass spectrometry (FTMS) on the denatured, TCEP-reduced fibrils (Fig. 4B). Small amounts of reduced ALS-SOD1 mutant proteins were also found to initiate fibrillation in 1:1 mixtures of WT and mutant-SOD1 proteins (Fig. S4).

In the native, holo enzyme, each subunit of the SOD1 dimer binds 1 copper and 1 zinc ion, and their presence has a profound impact on the stability of the protein. Fully metallated WT SOD1 melts at a very high 92 °C. The absence of bound copper lowers the melting point to 78 °C, and the successive removal of zinc ions lowers the melting point further to 62 °C (1 zinc per dimer) and 52 °C (apo-SOD1) (1). We examined the effect of adding small amounts of SOD1^2SH to the different metal-bound forms of SOD1. As shown in Fig. 4C, SOD1 with 1 zinc ion per dimer fibrillated with kinetics very similar to those for apo-SOD1, whereas isolated SOD1 containing zinc ions in both subunits showed a much slower rate of elongation. The absence of an effect caused by the binding of 1 zinc ion per apo-SOD1 dimer is surprising, because 1 zinc per dimer confers considerable structural stabilization in other assays (21). In contrast, the presence of 2 copper ions per dimer, either alone or in conjunction with bound zinc, completely inhibited the formation of amyloid fibrils, showing that copper-bound, disulfide-intact forms of SOD1 are not easily recruited by SOD1 amyloid nuclei.

Discussion

The ability of a specific form of a protein to act as an initiating agent for the fibrillation of another form has been demonstrated infrequently, presumably because of experimental difficulties associated with pinpointing initiation events. The few known instances include defining the minimum peptide sequence required to initiate fibrillation in Ure2p (22) and Aβ (23) and determining the oligomeric status of nuclei and the conformation changes required for their formation in polyglutamine peptides (24) and islet amyloid polypeptide (25). Here, we have been able to demonstrate the requirements for fibril initiation and elongation in SOD1 individually. Our studies show that a specific intermediate in SOD1 maturation pathway, disulfide-reduced, monomeric apo-SOD1, can initiate the rapid conversion of more mature forms of soluble SOD1 into amyloid fibrils. These results may be especially relevant to SOD1 fibrillation mechanisms in vivo because SOD1 monomers, including disulfide-reduced SOD1, have been detected in spinal cord extracts of transgenic mice expressing fALS SOD1 mutants well before they reach terminal stages of the disease (26, 27).

We have also shown that nucleation by disulfide-reduced (monomeric) SOD1 requires the presence of cysteine thiolates at both positions 57 and 146, suggesting that the nucleus is formed by intermolecular disulfide cross-linking using these cysteines. Similar species can form in vivo despite the reducing environment of the cytoplasm as shown by the presence of disulfide-cross-linked SOD1 oligomers in spinal cord extracts from ALS SOD1 transgenic mice. Cys-146 was one of the cysteine residues involved in their formation (28). Once SOD1 nuclei form, elongation proceeds via recruitment of apo- or partially-metallated disulfide-intact SOD1 through noncovalent interactions. Although the binding of zinc offers SOD1 limited protection from being assembled into fibrils, copper alone or in conjunction with zinc prevents SOD1 from being incorporated into amyloid fibrils. Because SOD1 aggregates isolated from the spinal cords of transgenic ALS mice are copper-deficient (17), copper binding may be an essential mechanism for protection from fibrillation in vivo.

It is important to note, however, that our studies do not show that disulfide reduction is absolutely required for initiation. In the presence of Gdm·HCl, disulfide-intact, apo-SOD1 can be induced to form amyloid fibrils, albeit with a significantly longer lag phase, possibly via a mechanism involving disulfide-intact monomers.

We found that small amounts of disulfide-reduced mutants can initiate fibrillation of WT SOD1. This result supports the idea that WT SOD1 participates in disease progression and may help explain the mechanism. Transgenic mice coexpressing WT and mutant SOD1 often show an earlier onset and accelerated progression of ALS-like neurodegenerative symptoms (9, 29, 30). Because the SS bond in ALS mutant proteins is more easily reduced than that in WT SOD1 (2), the reduced forms are more likely to be present in higher quantities in vivo, allowing for earlier nucleus formation, which could be followed by efficient incorporation of the more abundant WT SOD1. This process may be particularly important for unstable mutants such as L126Z or A4V that have low steady-state concentrations in vivo (31), and might require WT protein to sustain fibril growth, but it could also occur with more stable mutants in which WT and mutant proteins are present in approximately equal amounts. Supporting this idea, we found that in 1:1 mixtures of WT and mutant proteins the fibrils incorporated both proteins.

In conclusion, our studies have allowed us to separate the processes of initiation and elongation in SOD1 fibril formation and have demonstrated that reduction of the intrasubunit disulfide bond dramatically accelerates the initiation process. Our observation of the initiation of the fibrillation of WT SOD1 by the addition of small amounts of disulfide-reduced ALS SOD1 mutant proteins provides a feasible model for initial stages of the disease process in SOD1-linked fALS.

Materials and Methods

Amyloid Formation in Vitro.

All solutions were filtered through a 0.22-μm syringe filter before use. Amyloid conversion was carried out by using 2 formats: automated and manual. Reactions contained 50 μM apo-SOD1 in 10 mM potassium phosphate, pH 7.4 with or without with various concentrations of reducing agents (DTT, TCEP, or glutathione) or 1 M Gdm·HCl, as indicated. In the automated format, 200 μL of solution was pipetted into a chamber in a 96-well plate followed by a Teflon ball (1/8-in diameter) and 2 μL of TfT from a 4 mM aqueous stock. The plate was agitated at 300 rpm (3-mm rotation diameter) in a Fluoroskan plate-reader (Thermo) at 37 °C. Fluorescence measurements were recorded every 15 min by using λ_ex = 444 nm, λ_em = 485 nm, with an integration time of 200 μs. All reactions were performed in replicates of 3 or more. Reactions carried out anaerobically were assembled in a glove box by using reagent solutions that were purged with argon and left open inside the anaerobic chamber for 1 h before use.

In the manual format, reactions of the same composition as above were performed at a final volume of 1 mL in 2-mL glass vials containing a Teflon ball (1/8-in diameter) and agitated on an orbital shaker at 250 rpm (19-mm rotation diameter). The vials were sealed with plastic caps containing a PTFE septum and purged with nitrogen. At intervals, aliquots were withdrawn by using a Hamilton gas-tight syringe through the septum. TfT fluorescence was measured by diluting 10 μL of the reaction mix into 150 μL of 40 μM TfT stock solution made in 5 mM potassium phosphate, pH 7, and fluorescence emission at 480 nm was recorded on a spectrophotometer using λ_ex = 444 nm (excitation and emission slit widths = 2 mm). Lag times were calculated by fitting TfT fluorescence data to the formula:

where lag phase was calculated as t_m − 2/k (33).

Supplementary Material