Article

ALS-Causing SOD1 Mutations Promote Production of Copper-Deficient Misfolded Species

Campbell Family Institute for Cancer Research, Ontario Cancer Institute/University Health Network, Department of Biochemistry, University of Toronto, TMDT 4-305, 101 College Street, Toronto, Ontario, Canada M5G 1L7.
Journal of Molecular Biology (Impact Factor: 4.33). 06/2011; 409(5):839-52. DOI: 10.1016/j.jmb.2011.04.027
Source: PubMed

ABSTRACT

Point mutations scattered throughout the sequence of Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis (ALS) cases. SOD1 is a homodimer in which each subunit binds one copper atom and one zinc atom. Inclusions containing misfolded SOD1 are seen in motor neurons of SOD1-associated ALS cases. The mechanism by which these diverse mutations cause misfolding and converge on the same disease is still not well understood. Previously, we developed several time-resolved techniques to monitor structural changes in SOD1 as it unfolds in guanidine hydrochloride. By measuring the rates of Cu and Zn release using an absorbance-based assay, dimer dissociation through chemical cross-linking, and β-barrel conformation changes by tryptophan fluorescence, we established that wild-type SOD1 unfolds by a branched pathway involving a Zn-deficient monomer as the dominant intermediate of the major pathway, and with various metal-loaded and Cu-deficient dimers populated along the minor pathway. We have now compared the unfolding pathway of wild-type SOD1 with those of A4V, G37R, G85R, G93A, and I113T ALS-associated mutant SOD1. The kinetics of unfolding of the mutants were generally much faster than those of wild type. However, all of the mutants utilize the minority pathway to a greater extent than the wild-type protein, leading to greater populations of Cu-deficient intermediates and decreases in Zn-deficient intermediates relative to the wild-type protein. The greater propensity of the mutants to populate Cu-deficient states potentially implicates these species as a pathogenic form of SOD1 in SOD1-associated ALS and provides a novel target for therapeutic intervention.

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    • "At higher concentrations (>20 M) Zn 2+ produces a voltagedependent inhibition of NMDAR currents, probably by binding at the Mg 2+ -blocking site inside the pore. Similar to Cu 2+ , other than inducing conformational changes in mutant and wild-type SOD1 (Ip et al., 2011; Homma et al., 2013), Zn 2+ dyshomeostasis can greatly affects synaptic function particularly at the central synapse where Zn 2+ modulation has been fully demonstrated (Sensi et al., 2011). We have investigated the modulatory effects that a brief exposure to extracellular Zn 2+ has on NMDAR-mediated neurotoxicity in cortical cultures carrying the SOD1G93A mutation (Nutini et al., 2011b). "
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    ABSTRACT: Amyotrophic lateral sclerosis (ALS) is now recognized as a multisystem disorder, in which the primary pathology is the degeneration of motor neurons, with cognitive and/or behavioral dysfunctions that constitutes the non-motor manifestations of ALS. The combination of clinical, neuroimaging, and neuropathological data, and detailed genetic studies suggest that ALS and frontotemporal dementia (FTD) might form part of a disease continuum, with pure ALS and pure FTD at the two extremes. Mutations in the superoxide dismutase 1 (SOD1) gene were the first genetic mutations linked to the insurgence of ALS. Since that discovery numerous animal models carrying SOD1 mutations have been created. Despite their limitations these animal models, particularly the mice, have broaden our knowledge on the system alterations occurring in the ALS spectrum of disorders. The present review aims at providing an overview of the data obtained with the SOD1 animal models first and foremost on the cortical and subcortical regions, the cortico-striatal and hippocampal synaptic plasticity, dendritic branching and glutamate receptors function.
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    • "As mentioned above, amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein [17], [20], the human PrP and its pathogenic mutants [16], [21], [22], [36], and human SOD1 pathogenic mutants [18], [23], [24] have the tendency to form fibril deposits in a variety of tissues and thereby cause Alzheimer disease, prion disease, and ALS, respectively, while the rabbit PrP [13], [25], [26] and hen egg white lysozyme [27] do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In the present study, we demonstrated that macromolecular crowding dramatically promoted fibril formation of amyloidogenic proteins, such as GSK-3β phosphorylated human Tau protein, the human PrP and its pathogenic mutants E196K and D178N, and pathological human SOD1 mutant A4V, but remarkably inhibited aggregation of some non-amyloidogenic proteins, such as the rabbit PrP and hen egg white lysozyme. "
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    ABSTRACT: Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins. As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l. We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.
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    • "Increased unfolding rates and accessibility of either on or off-folding pathway intermediates may increase the accessibility of transient protein species that can initiate aggregation (Dobson 2003; Wang et al. 2008). Furthermore, it has been proposed that fALS-associated mutations, even those far from the metal binding sites, promote increased levels of Cu-deficient intermediates along the holoS- S SOD1 unfolding pathway (Ip et al. 2010). Cu-deficient intermediates are lower in stability and therefore may have a higher tendency to aggregate. "

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