**4.1 Aggregation of Apo2SH SOD1**

282 Amyotrophic Lateral Sclerosis

pre-organize the Cu-binding site (Banci et al. 2003). Together these studies show that metal binding significantly modulates the efficiency of the SOD1 folding pathway by stabilizing both the monomer and dimer and decreasing the rate of unfolding. However, non-native metal binding may force the protein to be kinetically trapped in a partially folded, aggregation prone conformation that is more stable than the unfolded state (Nordlund et al. 2009). Together these results show that the kinetic unfolding and refolding mechanism of

**3.8 fALS mutations and modifications have complex effects on the folding and** 

It is evident that fALS-associated mutants have different and complex effects on the stability of SOD1, and these effects depend on the form of SOD1 being studied. For example, mutations that have a large effect on holoS-S SOD1 stability, such as metal binding mutants, tend to have a much smaller effect on the more immature forms of the protein (Valentine et al. 2005). Also, the effects of mutations are more pronounced, but to varying extents for different mutations, with decreasing stability in immature forms of SOD1 (Vassall et al. 2006; Vassall et al. 2011). It is important to understand how both the equilibrium and kinetic folding pathways of all forms of SOD1 are altered by fALS-associated mutations to untangle the complexity of SOD1 aggregation. Certain mutations may have a large effect on the thermodynamic stability of the protein, through weakened metal binding or by decreasing the stability of the dimer interface, while having a smaller effect on the kinetic stability of SOD1. Other mutations may only subtly alter the thermodynamic stability of the native state, exerting their effects by altering the kinetic stability of SOD1 by decreasing the rate of folding, or by increasing the rate of unfolding (Rumfeldt et al. 2006). These effects can increase the equilibrium and/or transient population of folded or unfolded monomeric

In addition, fALS mutations may alter the susceptibility of SOD1 to post-translational modifications which will tend to decrease protein stability. These include not only enhanced metal loss and disulfide reduction (resulting in higher population of immature species), but also other modifications, in particular ones that are enhanced under oxidizing cellular conditions, which may occur late in disease. Examples include cysteine oxidation (Gruzman et al. 2007; Karch et al. 2009; Bosco et al. 2010), glutathionylation (Proctor et al. 2011), tryptophan oxidation (Elam et al. 2003), and glycation (Meiering 2008). While there is relatively little quantitative data on the effects of these modifications on stability, there is evidence that they can be destabilizing. Such modifications may play different roles at different disease stages; for example, oxidative modifications may become more pronounced as the disease progresses and contribute to rapid disease progression by

Numerous experimental studies have characterized the aggregation of multiple forms of SOD1. These are described below, focussing mainly on *in vitro* studies, and their implications for disease. It is important to note, however, that it is not yet known what forms of SOD1 are involved in human disease: neither the disulfide bond status nor metal content of SOD1 in aggregates is known. Evidence supports the population of multiple

SOD1 is highly dependent on metal binding.

species that are prone to aggregate (see section 4).

enhancing formation of toxic aggregates (Karch et al. 2009).

**4. SOD1 aggregation** 

**stability of SOD1** 

### **4.1.1 Evidence of Apo2SH SOD1 aggregation**

Aggregation of the marginally stable apoSH form of SOD1 has been suggested in a number of studies to be particularly important in ALS pathogenesis. Studies from multiple groups have reported that wild-type, pWT and fALS-associated mutant apoSH SOD1 are all predisposed to aggregate (Lindberg et al. 2004; Furukawa and O'Halloran 2005; Hornberg et al. 2007; Chattopadhyay et al. 2008; Furukawa et al. 2008; Oztug Durer et al. 2009; Vassall et al. 2011). Additional support for the biological significance of apoSH aggregation is that fALS-associated mutants expressed in insect cells tend to be more metal deficient and disulfide reduced compared to wild type; this finding was proposed to be a consequence of enhanced opening of the mutant SOD1 structures, exposing the disulfide bond to the reducing environment of the cytosol (Tiwari and Hayward 2003). Furthermore, murine models of fALS have revealed the presence of aggregated disulfide reduced SOD1 species (Jonsson et al. 2006; Zetterstrom et al. 2007). Based on what is known about the relationship between protein stability and aggregation propensity (see section 1.2.1), and on the relatively low stability and the expanded, fluctuating structure of apoSH SOD1 (see section 3.1), it appears that aggregation of this form of SOD1 could be significant under cellular conditions (Furukawa et al. 2008). Consistent with this idea, *in vitro* agitation of apo2SH SOD1 results in amyloid fibril formation (Chattopadhyay et al. 2008; Oztug Durer et al. 2009; Furukawa et al. 2010). Although the agitation phenomenon is poorly understood at a molecular level it may favour amyloid fibril formation over pathways to other morphologies. Agitation may enhance aggregation in general by promoting the formation of aggregation-prone species at air-solution or solid-solution interfaces, as well as increase the rate of aggregation by distributing aggregation nuclei more efficiently and causing preformed aggregates to break and create new nucleation sites (Sicorello et al. 2009). Again, it should be noted that since agitation can promote aggregation of many proteins (Rousseau et al. 2008; Mahler et al. 2009; Sicorello et al. 2009), and ALS is not classified as an amyloid disease (Kerman et al. 2010), it is not yet clear how agitation-induced aggregation *in vitro* is related to aggregation in disease.
