Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Abstract

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.neurodegeneration | protein aggregation | protein misfolding | ALS | structural modeling P rotein misfolding and aggregation are linked to cell death and disease progression in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In these diseases and others, the formation of amyloid plaques, often observed post mortem, has long been thought to play a role in neurodegeneration, but toxicity has never been confirmed (1-3). Recent research has shown that small, soluble oligomers, rather than insoluble amyloids, are likely to be the cytotoxic species causing neurodegeneration (4-14). These small, soluble oligomers undergo aberrant interactions with cell machinery and activate cell death pathways, but their exact stoichiometry is not known and their properties have yet to be characterized. Recently, metastable soluble Cu,Zn superoxide dismutase (SOD1) oligomers have been identified that contain an epitope associated with disease-linked species of SOD1, mutants of which are implicated in a subset of ALS (15-18). Size exclusion chromatography (SEC) of these oligomers revealed a size range of two to four monomers, consistent with previous findings of potentially cytotoxic SOD1 oligomers (19)(20)(21).Knowledge of the structures of these species would not only allow for definitive testing of their toxicity but could potentially lead to an understanding of disease mechanism and therapeutic strategies against diseases for which no ...

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

“…The dissociation rates of unmodified and glutathionylated T2D/A4V–SOD1 are similar to the respective values for T2D-SOD1 (16 times slower than A4V–SOD1), indicating an overall decrease in dimer dissociation consistent with our results from size exclusion chromatography. Because dimer dissociation and subsequent metal loss induce the structural distortions that promote protein aggregation, the decrease we find in dimer dissociation upon phosphomimetic T2D mutation suggests that phosphorylation at T2 may inhibit the formation of toxic SOD1 oligomers (Proctor et al, 2016). …”

“…Since cytotoxicity in motor neuron-like cells is found to correlate with the propensity of forming trimeric SOD1 (Proctor et al, 2016), we study the effect of T2D mutation on the stability of toxic SOD1 trimers. We use structural models of the trimer proposed by Proctor et al (Proctor et al, 2016) to computationally evaluate the change in free energy upon mutation (ΔΔG mut ) using the Eris molecular design suite (Yin et al, 2007a, b).…”

“…We use structural models of the trimer proposed by Proctor et al (Proctor et al, 2016) to computationally evaluate the change in free energy upon mutation (ΔΔG mut ) using the Eris molecular design suite (Yin et al, 2007a, b). The T2D mutation destabilizes the SOD1 trimer structure (ΔΔG mut = 2.9 kcal/mol) (Figure S5), while the A4V mutation stabilizes the trimer (ΔΔG mut = −4.8 kcal/mol), in accordance with experimental observations (Redler et al, 2014).…”

“…SOD1 aggregates have been discovered in the ventral horns of spinal cords from patients with familial or sporadic cases of ALS, indicating a contribution of non-genetic factors to disease progression (Shibata et al, 1996; Shibata et al, 1994). Recent studies have further implicated non-native SOD1 oligomers, instead of large aggregates, as the toxic species in cellular assays (Jonsson et al, 2004; Luchinat et al, 2014; Proctor et al, 2016; Zetterstrom et al, 2007). Indeed, several disease mutants (e.g.…”

“…Indeed, several disease mutants (e.g. A4V and G93A) destabilize SOD1 and promote formation of a trimeric species (Proctor et al, 2016; Redler et al, 2014). Aberrant post-translational modifications such as glutathionylation, a result of oxidative stress (Barber et al, 2006; Townsend, 2007), also increase the rate of SOD1 oligomerization as well as the exposure of a toxicity-associated epitope (Bosco et al, 2010; Redler et al, 2014; Redler et al, 2011).…”