**4.1.2 Mechanisms of Apo2SH SOD1 aggregation**

Intriguing structural variations in apoSH amyloid and non-amyloid aggregates have been reported and may be related to the different disease durations for different SOD1 mutants. Structural diversity has been observed in the amyloid fibrils formed by different SOD1 mutants (Furukawa et al. 2010). In addition, *in vitro* aggregation experiments without agitation showed that different apo2SH SOD1 mutants may form different sized, small (~40 nm - 1000 nm), soluble, non-amyloid aggregates (Vassall et al. 2011). These soluble species may be particularly relevant to fALS toxicity, as oligomeric protein aggregates have been implicated as key neurotoxic species in many other neurodegenerative diseases (Caughey and Lansbury 2003; Soto and Estrada 2008). The variations in structural characteristics for apo2SH SOD1s aggregates may indicate that different mutants favour distinct aggregation pathways, depending on the aggregation-prone conformations that mutants may adopt.

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 285

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.

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

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

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 non-

**4.2.2 Mechanisms of ApoS-S Aggregation** 

2010).

protein.

promote aggregation.

amyloid amorphous aggregates.

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-prone conformations.

### **4.2 Aggregation of ApoS-S SOD1 4.2.1 Evidence of ApoS-S SOD1 aggregation**

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 4.3).

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 their targets remain unanswered.

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 patients (Stathopulos et al. 2003).
