**4.2.2 Mechanisms of ApoS-S Aggregation**

284 Amyotrophic Lateral Sclerosis

Another factor in the heterogeneity of aggregate structures formed may be that apo2SH is appreciably unfolded at physiological temperatures, and therefore may sample multiple

Aggregation of the apo S-S form of SOD1 has been studied extensively *in vitro*. This is likely related to the reasonable ease of preparation and aggregation of this quite stable form of the protein (Table 1) (see section 3.3 and 3.4). A role for apoS-S in disease is supported by various *in vitro* and *in vivo* evidence for aggregation of metal-deficient SOD1 as well as evidence that mutations can promote loss of bound metals (Valentine et al. 2005; Molnar et al. 2009; Oztug Durer et al. 2009; Hwang et al. 2010; Lelie et al. 2011). Both wild-type and fALS mutant apoS-S SOD1s are predominantly folded, but mutations can significantly increase the population of folded and unfolded monomeric species (Lindberg et al. 2004; Svensson et al. 2006; Vassall et al. 2006), and increase structural fluctuations (Ding and Dokholyan 2008; Teilum et al. 2009), both of which can promote aggregation. The formation of the disulfide bond decreases the propensity of apoS-S to aggregate compared to apoSH (see section 4.1), while the absence of bound metals in apoS-S SOD1 increases its aggregation propensity compared to holoS-S (see also section

Several studies have described the *in vitro* formation of amyloid-like soluble or fibrillar aggregates for wild-type and fALS-associated apoS-S SOD1 mutants (Furukawa and O'Halloran 2005; Banci et al. 2007; Banci et al. 2008; Oztug Durer et al. 2009). Aggregation of mutant SOD1 in these studies was often accelerated by agitation, resulting in the formation of aberrant disulfide bonds between Cys6 and Cys111 (Figure 2) and removal of the free thiol groups by mutation (Banci et al. 2007; Cozzolino et al. 2008) generally diminished aggregation. The role of aberrant disulfide bond formation has been investigated extensively using fALS mutant SOD1 mice models of ALS where it appears that such bonds are observed mainly late in disease (Cozzolino et al. 2008; Karch and Borchelt 2008). These results highlight an important question: does the role of SOD1 differ at different stages of disease? Considerable evidence suggests that the answer is yes; in particular, soluble oligomers may be particularly important early in disease, whereas larger aggregates become prominent after the onset of disease symptoms (Boillee et al. 2006; Cozzolino et al. 2008; Turner and Talbot 2008). Many questions on the roles of different aggregate species and

A number of studies have reported aggregation of apoS-S SOD1 in the absence of aberrant disulfide bond formation. Formation of such amyloid-like aggregates by pWT and mutant SOD1 can be induced by agitation at neutral pH; however, in these studies the observed ease of aggregation was not correlated with disease duration (Chattopadhyay et al. 2008; Furukawa et al. 2008; Oztug Durer et al. 2009; Furukawa et al. 2010). Also, soluble, reversible aggregates induced by heat were detected by DSC (Vassall et al. unpublished data) (Stathopulos et al. 2006; Vassall et al. 2006). In these studies, mutations in a pWT background generally increased aggregate formation. Furthermore, decreased stability of fALS-associated mutant apoS-S SOD1 was correlated with increased formation of amorphous aggregates that mature into fibril structures that resemble those found in fALS

aggregation-prone conformations.

**4.2 Aggregation of ApoS-S SOD1** 

their targets remain unanswered.

patients (Stathopulos et al. 2003).

4.3).

**4.2.1 Evidence of ApoS-S SOD1 aggregation** 

A number of studies have focussed on possible molecular mechanisms of apoS-S SOD1 aggregation, often based on various structural and dynamic data. In the absence of bound metal, there is still extensive disorder in the functional Loops IV and VII, and this disorder may promote exposure of the β-barrel core and deprotection of the β-barrel edge strands (βstrands 5 and 6) (Strange et al. 2007). With these edge strands exposed, H-bonding ligands within the strands are free to interact with the edge strands of other SOD1 molecules and these aberrant contacts can lead to fibril formation (Elam et al. 2003; Nordlund and Oliveberg 2006). As an evolutionary strategy to avoid this fibrillation mechanism, the edge strands of β-proteins often contain charged "gatekeeper" residues, advantageously positioned to disfavour β-sheet extension (Otzen et al. 2000). Protection of the β-barrel edge strands in SOD1 is facilitated both by charged residues in β-strands 5 and 6 and by Loops IV and VII, which block the edge of the β-barrel in the holoS-S form (Nordlund and Oliveberg 2006). The other edge of the β-barrel is buried within the dimer interface. Therefore, the dynamics of Loop IV and VII that cause exposure of either β-strands 5 and 6, or the dimer interface, may lead to fibrillation. Solution NMR experiments of wild-type apoS-S SOD1 suggest that disorder in Loops IV and VII allows stretches of amino acids within these functional loops to form additional β-strands, thereby initiating oligomerization (Banci et al. 2010).

In addition, β-strands 4 and 5 are connected by Loop IV and β-strands 7 and 8 by Loop VII, and the disorder in these long loops appears to propagate to the flanking β-strands. Consequently, the β-sheet formed by strands 4, 5, 7 and 8 is less defined in apoS-S SOD1 compared to the β-sheet formed by strands 1-3 and 6 (Banci et al. 2003; Banci et al. 2010). Thus, metal loss and increased mobility of Loops IV and VII may result in exposure of hydrophobic residues in the β-barrel core (Tiwari et al. 2009). In addition, β-strands 1-3 have been proposed to be a nucleation site for aggregation of apoS-S as they form a continuous patch of hydrophobic residues (Nordlund and Oliveberg 2006). These 3 strands form early in the monomer folding pathway (Lindberg et al. 2004; Nordlund and Oliveberg 2006) and therefore may become exposed by partial unfolding of the apoS-S protein.

The destabilizing effects of fALS-associated mutations appear to propagate significantly in apoS-S SOD1, in particular generally weakening the dimer interface (see section 3.3) (Vassall et al. unpublished data) (Khare et al. 2006; Bystrom et al. 2010). Destabilization of the apoS-S dimer interface can increase the levels of marginally stable, apoS-S monomers. Furthermore, it was demonstrated recently that breathing motions of the wild-type apoS-S monomer result in transient formation of a higher energy species with weakened packing and a partially exposed hydrophobic core (Teilum et al. 2009). fALS-mutations induce further perturbations in this higher energy state that open up the structure of the mutant apoS-S SOD1 monomer more compared to wild type (Teilum et al. 2009) and so may further promote aggregation.

The increased structural dynamics observed for metal-free SOD1 described above, suggests that apoS-S may be predisposed to aggregate compared to the holoS-S state. Several studies have demonstrated that apoS-S SOD1 can aggregate by a variety of different mechanisms that give rise to different aggregate morphologies, including both disulphide cross-linked and non cross-linked species, amyloid-like fibrils and nonamyloid amorphous aggregates.

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 287

for holoS-S SOD1 to arrange into an aggregation-prone state and/or form the necessary contacts required for aggregation. It is likely that dimer dissociation and/or metal loss from SOD1 occur during this lag phase and may be important triggers of aggregation (Hwang et al. 2010). Furthermore, various fALS-associated mutations appear to decrease the length of the lag phase, perhaps due to weakened metal binding and/or a weakened dimer interface,

These results suggest that fully mature SOD1 is not devoid of the ability to aggregate, as it could give rise to native-like aggregation-prone species via loss of metal, dimer dissociation, or local structural openings, promoted by mutation. Such aggregation may be highly relevant to fALS toxicity, since holo S-S is generally the most highly abundant form of SOD1

(Crow et al. 1997; Khare et al. 2004; Tiwari et al. 2009).

Fig. 4. Many forms of SOD1 may be relevant to ALS toxicity.

aggregates formed *in vitro* (Stathopulos et al. 2003).

SOD1 can exist in many forms *in vivo*, which is illustrated in Figure 4. Each monomer is depicted as a grey sphere that is smaller when metals are bound and/or the disulfide is formed. The presence of Cu and Zn is shown by orange and green spheres, respectively; and S-S and 2SH indicate disulfide oxidized and reduced species, respectively. The difference in SOD1 conformation prior to aggregation may largely influence the morphology of the aggregates formed. Images and schematic representation of possible aggregate

morphologies are shown in the centre on the right. Panels A, C and D are Atomic Force Microscopy images of SOD1 aggregates formed *in vitro* (Broom et al, unpublished data) (Hwang et al. 2010) and panel B is an electron microscopy image obtain from of SOD1

*in vivo* (Valentine et al. 2005).

### **4.3 Aggregation of SOD1 from the holoS-S state 4.3.1 Evidence of aggregation from the holoS-S state**

While the highly stable, native holoS-S form of SOD1 (see section 3.5) generally appears to be much less susceptible to aggregation than other forms of the protein (Stathopulos et al. 2003; Valentine et al. 2005), there is evidence that a number of SOD1 mutants can give rise to aggregation from the holoS-S form. Hwang et al. found that prolonged incubation of both pWT and fALS-associated holoS-S SOD1 mutants at physiological temperature and pH results in changes in metal binding and/or dimerization, diminished specific dismutase activity, and the nucleated formation of low levels of amorphous aggregates (Hwang et al. 2010). Furthermore, these experiments show that, although the aggregated SOD1 demonstrated some metal loss, there was still a significant amount of metal bound, indicating that complete metal loss was not essential for aggregation. Although both pWT and mutant holoS-S SOD1 were observed to aggregate, in general the holoS-S SOD1 mutants lose specific activity quicker, and aggregate more rapidly, and to a greater extent, than pWT. Importantly, the aggregates formed from holoS-S SOD1 in this study exhibited similar structural, dye-binding, and immunological characteristics as the aggregates found in fALS patients (Hwang et al. 2010). In contrast, other studies have reported that SOD1 does not aggregate from the holoS-S form (Chattopadhyay et al. 2008), or requires extremely destabilizing conditions with agitation to promote fibrilization (Oztug Durer et al. 2009). The differences between these findings may be related to the different experimental conditions for studying SOD1 aggregation, such as length of incubation, frequency of sampling, and methods for monitoring aggregation.

### **4.3.2 Mechanisms of holoS-S aggregation**

Immature forms of SOD1 can form amyloid fibrils far more readily than holoS-S (Banci et al. 2007; Furukawa et al. 2008; Oztug Durer et al. 2009), and this difference in aggregation tendency is likely related to the very high stability and rigidity of holoS-S compared to the less mature forms (Stathopulos et al. 2003; Rumfeldt et al. 2006; Stathopulos et al. 2006; Svensson et al. 2006; Vassall et al. 2006; Furukawa et al. 2008; Kayatekin et al. 2008; Kayatekin et al. 2010; Vassall et al. 2011). Highly disordered, predominantly unfolded, proteins tend to favour the formation of amyloid (as may be the case for apo forms of SOD1), whereas more structured proteins favour formation of amorphous aggregates (as for holoS-S) (Munishkina et al. 2004). Measurements of global thermodynamic stability have shown that, owing to the high stability of the holo form, destabilizing mutations will in general cause very small increases in the population of unfolded protein (Rumfeldt et al. 2006; Stathopulos et al. 2006); these increases are unlikely to account for SOD1 aggregation in ALS. Aggregation may alternatively arise from native-like, locally unfolded states (Chiti and Dobson 2009; Hwang et al. 2010) (Figure 1B) which appear to be enhanced in holoS-S SOD1 by some fALS-associated mutations (Shipp et al. 2003; Hough et al. 2004; Banci et al. 2005; Museth et al. 2009). Ultimately, it is likely that some sort of relatively rare/slow structural change is required to bring about aggregation from holoS-S SOD1 (Hwang et al. 2010), in contrast to the apo2SH form, which aggregates readily for some fALS-associated mutant SOD1s (Vassall et al. 2011).

SOD1 aggregation arising from the holoS-S form appears to occur through a nucleationdependent mechanism that is characterized by a lag phase (i.e. slow nucleation) followed by fast aggregate growth (Hwang et al. 2010). The lag phase corresponds to the time required

While the highly stable, native holoS-S form of SOD1 (see section 3.5) generally appears to be much less susceptible to aggregation than other forms of the protein (Stathopulos et al. 2003; Valentine et al. 2005), there is evidence that a number of SOD1 mutants can give rise to aggregation from the holoS-S form. Hwang et al. found that prolonged incubation of both pWT and fALS-associated holoS-S SOD1 mutants at physiological temperature and pH results in changes in metal binding and/or dimerization, diminished specific dismutase activity, and the nucleated formation of low levels of amorphous aggregates (Hwang et al. 2010). Furthermore, these experiments show that, although the aggregated SOD1 demonstrated some metal loss, there was still a significant amount of metal bound, indicating that complete metal loss was not essential for aggregation. Although both pWT and mutant holoS-S SOD1 were observed to aggregate, in general the holoS-S SOD1 mutants lose specific activity quicker, and aggregate more rapidly, and to a greater extent, than pWT. Importantly, the aggregates formed from holoS-S SOD1 in this study exhibited similar structural, dye-binding, and immunological characteristics as the aggregates found in fALS patients (Hwang et al. 2010). In contrast, other studies have reported that SOD1 does not aggregate from the holoS-S form (Chattopadhyay et al. 2008), or requires extremely destabilizing conditions with agitation to promote fibrilization (Oztug Durer et al. 2009). The differences between these findings may be related to the different experimental conditions for studying SOD1 aggregation, such as length of incubation, frequency of

Immature forms of SOD1 can form amyloid fibrils far more readily than holoS-S (Banci et al. 2007; Furukawa et al. 2008; Oztug Durer et al. 2009), and this difference in aggregation tendency is likely related to the very high stability and rigidity of holoS-S compared to the less mature forms (Stathopulos et al. 2003; Rumfeldt et al. 2006; Stathopulos et al. 2006; Svensson et al. 2006; Vassall et al. 2006; Furukawa et al. 2008; Kayatekin et al. 2008; Kayatekin et al. 2010; Vassall et al. 2011). Highly disordered, predominantly unfolded, proteins tend to favour the formation of amyloid (as may be the case for apo forms of SOD1), whereas more structured proteins favour formation of amorphous aggregates (as for holoS-S) (Munishkina et al. 2004). Measurements of global thermodynamic stability have shown that, owing to the high stability of the holo form, destabilizing mutations will in general cause very small increases in the population of unfolded protein (Rumfeldt et al. 2006; Stathopulos et al. 2006); these increases are unlikely to account for SOD1 aggregation in ALS. Aggregation may alternatively arise from native-like, locally unfolded states (Chiti and Dobson 2009; Hwang et al. 2010) (Figure 1B) which appear to be enhanced in holoS-S SOD1 by some fALS-associated mutations (Shipp et al. 2003; Hough et al. 2004; Banci et al. 2005; Museth et al. 2009). Ultimately, it is likely that some sort of relatively rare/slow structural change is required to bring about aggregation from holoS-S SOD1 (Hwang et al. 2010), in contrast to the apo2SH form, which aggregates readily for some fALS-associated

SOD1 aggregation arising from the holoS-S form appears to occur through a nucleationdependent mechanism that is characterized by a lag phase (i.e. slow nucleation) followed by fast aggregate growth (Hwang et al. 2010). The lag phase corresponds to the time required

**4.3 Aggregation of SOD1 from the holoS-S state 4.3.1 Evidence of aggregation from the holoS-S state** 

sampling, and methods for monitoring aggregation.

**4.3.2 Mechanisms of holoS-S aggregation** 

mutant SOD1s (Vassall et al. 2011).

for holoS-S SOD1 to arrange into an aggregation-prone state and/or form the necessary contacts required for aggregation. It is likely that dimer dissociation and/or metal loss from SOD1 occur during this lag phase and may be important triggers of aggregation (Hwang et al. 2010). Furthermore, various fALS-associated mutations appear to decrease the length of the lag phase, perhaps due to weakened metal binding and/or a weakened dimer interface, (Crow et al. 1997; Khare et al. 2004; Tiwari et al. 2009).

These results suggest that fully mature SOD1 is not devoid of the ability to aggregate, as it could give rise to native-like aggregation-prone species via loss of metal, dimer dissociation, or local structural openings, promoted by mutation. Such aggregation may be highly relevant to fALS toxicity, since holo S-S is generally the most highly abundant form of SOD1 *in vivo* (Valentine et al. 2005).

Fig. 4. Many forms of SOD1 may be relevant to ALS toxicity.

SOD1 can exist in many forms *in vivo*, which is illustrated in Figure 4. Each monomer is depicted as a grey sphere that is smaller when metals are bound and/or the disulfide is formed. The presence of Cu and Zn is shown by orange and green spheres, respectively; and S-S and 2SH indicate disulfide oxidized and reduced species, respectively. The difference in SOD1 conformation prior to aggregation may largely influence the morphology of the aggregates formed. Images and schematic representation of possible aggregate morphologies are shown in the centre on the right. Panels A, C and D are Atomic Force Microscopy images of SOD1 aggregates formed *in vitro* (Broom et al, unpublished data) (Hwang et al. 2010) and panel B is an electron microscopy image obtain from of SOD1 aggregates formed *in vitro* (Stathopulos et al. 2003).

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 289

may be similar to the roles of other globular, oligomeric proteins in misfolding diseases such as: transthyretin in familial amyloidotic polyneuropathy and senile systemic amyloidosis, lysozyme in hereditary non-neuropathic systemic amyloidosis, immunoglobulin light chain in monoclonal protein systemic amyloidosis, prion protein in Kreutzfeld Jakob, and serpins in serpinopathies (Ohnishi and Takano 2004; Harrison et al. 2007). In these diseases mutations are generally destabilizing, but the extent of destabilization of monomer versus subunit interfaces varies widely. The role of SOD1 in disease may be further complicated by the potential aberrant enzymatic activity of misfolded and/or aggregated species which could cause oxidative damage. In addition, it is worth considering the different roles of various types of SOD1 aggregate structures, or contributions of aberrant activity and the effects of these on other cellular components, at different stages throughout the disease course of ALS. For these reasons, it is important that future studies continue to consider the possible roles of multiple forms of SOD1 mutants in modulating the formation of different aggregate structures (Figure 4). A combination of further *in vitro* and *in vivo* studies of folding and aggregation will be critical for untangling the role of toxic aggregation in the

We thank the ALS Society of Canada, Muscular Dystrophy Canada, and Canadian Institutes of Health Research for their financial support. Furthermore, we would like to express our appreciation to all the members of the Meiering group for their insightful and meaningful discussions, in particular Aron Broom, Martin Smith, Ming Sze Tong and

Alvarez, B., Demicheli, V., Duran, R., Trujillo, M., Cervenansky, C., Freeman, B. A. and Radi,

Andersen, P. M. (2006). "Amyotrophic lateral sclerosis associated with mutations in the CuZn superoxide dismutase gene." *Curr Neurol Neurosci Rep* 6(1): 37-46. Armstrong, R. A., Nochlin, D. and Bird, T. D. (2000). "Neuropathological heterogeneity in

Arnesano, F., Banci, L., Bertini, I., Martinelli, M., Furukawa, Y. and O'Halloran, T. V. (2004).

Bai, Y., Englander, J. J., Mayne, L., Milne, J. S. and Englander, S. W. (1995). "Thermodynamic

Baldwin, A. J., Knowles, T. P., Tartaglia, G., Fitzpatrick, A., Devlin, G., Shammas, S.,

Proteins and the Phenomenon of Amyloid Formation." *J Am Chem Soc*.

formation of histidinyl radical." *Free Radic Biol Med* 37(6): 813-22.

R. (2004). "Inactivation of human Cu,Zn superoxide dismutase by peroxynitrite and

Alzheimer's disease: a study of 80 cases using principal components analysis."

"The unusually stable quaternary structure of human Cu,Zn-superoxide dismutase 1 is controlled by both metal occupancy and disulfide status." *J Biol Chem* 279(46):

parameters from hydrogen exchange measurements." *Methods Enzymol* 259: 344-56.

Waudby, C. A., Mossuto, M. F., Gras, S. L., Christodoulou, J., Anthony-Cahill, S. J., Barker, P. D., Vendruscolo, M. and Dobson, C. M. (2011). "Metastability of Native

syndrome of ALS.

Sarah M. Sullivan.

**7. References** 

**6. Acknowledgements** 

*Neuropathology* 20(1): 31-7.

47998-8003.
