Disulphide-reduced superoxide dismutase-1 in CNS of transgenic amyotrophic lateral sclerosis models

Department of Medical Biosciences, Umeå University, Umeå, Sweden.
Brain (Impact Factor: 9.2). 03/2006; 129(Pt 2):451-64. DOI: 10.1093/brain/awh704
Source: PubMed


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease afflicting the voluntary motor system. More than 100 different mutations in the ubiquitously expressed enzyme superoxide dismutase-1 (SOD1) have been associated with the disease. To search for the nature of the cytotoxicity of mutant SOD1s, amounts, enzymic activities and structural properties of the protein as well as the CNS histopathology were examined in multiple transgenic murine models. In order to generate the ALS phenotype within the short lifespan of the mouse, more than 20-fold increased rates of synthesis of mutant SOD1s appear to be required. The organs of transgenic mice expressing human wild-type SOD1 or either of the G93A and D90A mutant proteins showed high steady-state protein levels. The major proportion of these SOD1s in the CNS were inactive due to insufficient Cu charging and all contained subfractions with a reduced C57-C146 intrasubunit disulphide bond. Both G85R and the truncated G127insTGGG mutant showed low steady-state protein levels, lacked enzyme activity and had no C57-C146 disulphide bond. These mutants were also enriched in the CNS relative to other organs, suggesting inefficient recognition and degradation of misfolded disulphide-reduced SOD1 in susceptible tissues. In end-stage disease, despite 35-fold differences in levels of mutant SOD1s, similar amounts of detergent-resistant aggregates accumulated in the spinal cord. Small granular as well as larger more diffuse human SOD1 (hSOD1)-inclusions developed in all strains, the latter more pronounced in those with high hSOD1 levels. Widespread vacuolizations were seen in the strains with high levels of hSOD1 but not those with low, suggesting these alterations to be artefacts related to high hSOD1 levels and not to the ALS-causing cytotoxicity. The findings suggest that the motoneuron degeneration could be due to long-term exposure to misfolded aggregation-prone disulphide-reduced SOD1, which constitutes minute subfractions of the stable mutants and larger proportions of the unstable mutants.

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Available from: Karin Sixtensdotter Graffmo, Mar 22, 2014
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    • "Also recently, even in the presence of the disulfide bond, destabilization of apo-SOD1 was found to allow the disulfide bond to be shuffled among four Cys residues, resulting in the formation of disulfide cross-linked oligomers in vitro [9]. While disulfide cross-linking of mutant SOD1 would be a secondary event for formation of the insoluble aggregates [10], abnormal accumulation of disulfide-reduced SOD1 was observed in fALS model mice [11]. Despite this, a C57S/C146S double mutation, by which the conserved disulfide bond cannot form, did not affect toxicity of mutant SOD1 toward cultured cells [12] [13]. "
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    ABSTRACT: Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (ALS). A pathological hallmark of the familial ALS is the formation of mutant SOD1 aggregates, leading to the proposal that SOD1 gains toxicities through protein misfolding triggered by mutations. Nevertheless, molecular requirements for mutant SOD1 to acquire pathogenicity still remain obscure. Here, we show that Cys residues in SOD1 are essential to exerting toxicities of SOD1 in a C. elegans model. Exogenous expression of wild-type as well as pathogenic mutant SOD1 fused with a fluorescent protein in C. elegans resulted in the accumulation of disulfide-reduced SOD1 and retarded the worm's motility. In contrast, little effects of exogenously expressed SOD1 on the motility were observed when all four Cys residues in SOD1 were replaced with Ser. Taken together, we propose that deregulation of Cys chemistry in SOD1 proteins is involved in the pathogenesis of SOD1-related ALS. Copyright © 2015. Published by Elsevier Inc.
    Biochemical and Biophysical Research Communications 06/2015; 463(4). DOI:10.1016/j.bbrc.2015.06.084 · 2.30 Impact Factor
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    • "From these, most SOD1- linked ALS variants are susceptible to unfolding and loss of posttranslational modifications [21] [22]. Indeed, loss of metal ions makes SOD1 aggregation-prone in vitro and in disease models insoluble SOD1 has a very low metallation [23] [24]. SOD1-linked ALS is not a loss-of-function disorder since transgenic animal models expressing ALS-SOD1 variants along with endogenous wild type SOD1 still develop the disease. "
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    ABSTRACT: Calcium deregulation is a central feature among neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Calcium accumulates in the spinal and brain stem motor neurons of ALS patients triggering multiple pathophysiological processes which have been recently shown to include direct effects on the aggregation cascade of superoxide dismutase 1 (SOD1). SOD1 is a Cu/Zn enzyme whose demetalated form is implicated in ALS protein deposits, contributing to toxic gain of function phenotypes. Here we undertake a combined experimental and computational study aimed at establishing the molecular details underlying the regulatory effects of Ca2 + over SOD1 aggregation potential. Isothermal titration calorimetry indicates entropy driven low affinity association of Ca2 + ions to apo SOD1, at pH 7.5 and 37 °C. Molecular dynamics simulations denote a noticeable loss of native structure upon Ca2 + association that is especially prominent at the zinc-binding and electrostatic loops, whose decoupling is known to expose the central SOD1 β-barrel triggering aggregation. Structural mapping of the preferential apo SOD1 Ca2 + binding locations reveals that among the most frequent ligands for Ca2 + are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. Calcium interactions thus diminish gatekeeping roles of these residues, by shielding repulsive interactions via stacking between aggregating β-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in ALS inclusions. Interestingly, many fALS mutations occur at these positions, disclosing how Ca2 + interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic ALS pathomechanisms.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 11/2014; 1854(2):118-126. DOI:10.1016/j.bbapap.2014.11.005 · 2.75 Impact Factor
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    • "Transgenic SOD1G93A mice are principally used in ALS research, followed by SOD1G37R, SOD1G85R, and SOD1G86R mice. All of these models exhibit slightly different time courses of the disease and associated neurodegenerative processes depending on the SOD1 mutation site, related enzymatic activity, transgene copy number and genetic background (Jonsson et al., 2006; Turner and Talbot, 2008; Van Den Bosch, 2011). SOD1G93A proteins, for example, are about 10 times more active than the native SOD1 proteins while in contrast, SOD1G85R mutant proteins are almost inactive. "
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    ABSTRACT: Mutations in the gene that encodes Cu/Zn-superoxide dismutase (SOD1) are the cause of approximately 20% of familial forms of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While ALS symptoms appear in adulthood, spinal motoneurons exhibit functional alterations as early as the embryonic and postnatal stages in the murine model of ALS, the SOD1 mice. Monoaminergic - i.e., dopaminergic (DA), serotoninergic (5-HT), and noradrenergic (NA) - pathways powerfully control spinal networks and contribute significantly to their embryonic and postnatal maturation. Alterations in monoaminergic neuromodulation during development could therefore lead to impairments in the motoneuronal physiology. In this study, we sought to determine whether the monoaminergic spinal systems are modified in the early stages of development in SOD1 mice. Using a post-mortem analysis by high performance liquid chromatography (HPLC), monoaminergic neuromodulators and their metabolites were quantified in the lumbar spinal cord of SOD1 and wild-type (WT) mice aged one postnatal day (P1) and P10. This analysis underscores an increased content of DA in the SOD1 lumbar spinal cord compared to that of WT mice but failed to reveal any modification of the other monoaminergic contents. In a next step, we compared the efficiency of the monoaminergic compounds in triggering and modulating fictive locomotion in WT and SOD1 mice. This study was performed in P1-P3 SOD1 mice and age-matched control littermates using extracellular recordings from the lumbar ventral roots in the in vitro isolated spinal cord preparation. This analysis revealed that the spinal networks of SOD1(G93A) mice could generate normal locomotor activity in the presence of NMA-5-HT. Interestingly, we also observed that SOD1 spinal networks have an increased sensitivity to NA compared to WT spinal circuits but exhibited similar DA responses.
    Frontiers in Neural Circuits 07/2014; 8:77. DOI:10.3389/fncir.2014.00077 · 3.60 Impact Factor
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