Detection method of the adjacent motor neuronal death in an in vitro co-culture model of familial ALS-associated Cu/Zn superoxide dismutase

Mutations in Cu/Zn-superoxide dismutase (SOD1) gene have been identified in familial amyotrophic lateral sclerosis. Motor neuron degeneration subsequently spreads to contiguous neurons of the motor systems. We developed an in vitro disease model with motor neuron-neuroblastoma hybrid cells (VSC4.1) constitutively expressing a mutant (G93A) SOD1. The extracellular effect upon adjacent motor neurons was determined using the substratum culture insert. The viability of VSC 4.1 was lowered by 26% in a co-culture of VSC 4.1 and G93A, which was reversed by Trolox, an antioxidant. This in vitro disease model confirmed the extracellular toxicity of the mutant SOD1 cells on the adjacent neurons by generating oxidative stress.     

SUMO-1 modification increases human SOD1 stability and aggregation

The mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) cause approximately 20% cases of familial amyotrophic lateral sclerosis (FALS), characterized by selective loss of motor neurons. Mutant SOD1 forms inclusions in tissues from FALS patients. However, the precise mechanism of the accumulation of mutant SOD1 remains unclear. Here we show that human SOD1 is a substrate modified by SUMO-1. A conversion of lysine 75 to an arginine within a SUMO consensus sequence in SOD1 completely abolishes SOD1 sumoylation. We further show that SUMO-1 modification, on both wild-type and mutant SOD1, increases SOD1 steady state level and aggregation. Moreover, SUMO-1 co-localizes onto the aggregates formed by SOD1. These findings imply that SUMO-1 modification on lysine 75 may participate in regulating SOD1 stability and its aggregation process. Thus, our results suggest that sumoylation of SOD1 may be involved in the pathogenesis of FALS associated with mutant SOD1.     

Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu-Zn, Zn-Zn and as-isolated wild-type enzymes

Human Cu-Zn superoxide dismutase (SOD1) protects cells from the effects of oxidative stress. Mutations in SOD1 are linked to the familial form of amyotrophic lateral sclerosis. Several hypotheses for their toxicity involve the mis-metallation of the enzyme. We present atomic-resolution crystal structures and biophysical data for human SOD1 in three metallation states: Zn-Zn, Cu-Zn and as-isolated. These data represent the first atomic-resolution structures for human SOD1, the first structure of a reduced SOD1, and the first structure of a fully Zn-substituted SOD1 enzyme. Recombinantly expressed as-isolated SOD1 contains a mixture of Zn and Cu at the Cu-binding site. The Zn-Zn structure appears to be at least as stable as the correctly (Cu-Zn) metallated enzyme. These data raise the possibility that in a cellular environment with low availability of free copper, Zn-Zn may be the preferred metallation state of SOD1 prior to its interaction with the copper chaperone.     

Structural switching of Cu,Zn-superoxide dismutases at loop VI: insights from the crystal structure of 2-mercaptoethanol-modified enzyme

Cu,Zn SOD1 (superoxide dismutase 1) is implicated in FALS (familial amyotrophic lateral sclerosis) through the accumulation of misfolded proteins that are toxic to neuronal cells. Loop VI (residues 102–115) of the protein is at the dimer interface and could play a critical role in stability. The free cysteine residue, Cys¹¹¹ in the loop, is readily oxidized and alkylated. We have found that modification of this Cys¹¹¹ with 2-ME (2-mercaptoethanol; 2-ME-SOD1) stabilizes the protein and the mechanism may provide insights into destabilization and the formation of aggregated proteins. Here, we determined the crystal structure of 2-ME-SOD1 and find that the 2-ME moieties in both subunits interact asymmetrically at the dimer interface and that there is an asymmetric configuration of segment Gly¹⁰⁸ to Cys¹¹¹ in loop VI. One loop VI of the dimer forms a 3₁₀-helix (Gly¹⁰⁸ to His¹¹⁰) within a unique β-bridge stabilized by a hydrogen bond between Ser¹⁰⁵-NH and His¹¹⁰-CO, while the other forms a β-turn without the H-bond. The H-bond (H-type) and H-bond free (F-type) configurations are also seen in some wild-type and mutant human SOD1s in the Protein Data Bank suggesting that they are interconvertible and an intrinsic property of SOD1s. The two structures serve as a basis for classification of these proteins and hopefully a guide to their stability and role in pathophysiology.     

The structure of holo and metal-deficient wild-type human Cu,Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis

Cu, Zn superoxide dismutase (SOD1) forms a crucial component of the cellular defence against oxidative stress. Zn-deficient wild-type and mutant human SOD1 have been implicated in the disease familial amyotrophic lateral sclerosis (FALS). We present here the crystal structures of holo and metal-deficient (apo) wild-type protein at 1.8A resolution. The P21 wild-type holo enzyme structure has nine independently refined dimers and these combine to form a "trimer of dimers" packing motif in each asymmetric unit. There is no significant asymmetry between the monomers in these dimers, in contrast to the subunit structures of the FALS G37R mutant of human SOD1 and in bovine Cu,Zn SOD. Metal-deficient apo SOD1 crystallizes with two dimers in the asymmetric unit and shows changes in the metal-binding sites and disorder in the Zn binding and electrostatic loops of one dimer, which is devoid of metals. The second dimer lacks Cu but has approximately 20% occupancy of the Zn site and remains structurally similar to wild-type SOD1. The apo protein forms a continuous, extended arrangement of beta-barrels stacked up along the short crystallographic b-axis, while perpendicular to this axis, the constituent beta-strands form a zig-zag array of filaments, the overall arrangement of which has a similarity to the common structure associated with amyloid-like fibrils.     

Retrograde axonal transport and motor neuron disease

Transport of material between extensive neuronal processes and the cell body is crucial for neuronal function and survival. Growing evidence shows that deficits in axonal transport contribute to the pathogenesis of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we review recent data indicating that defects in dynein-mediated retrograde axonal transport are involved in ALS etiology. We discuss how mutant copper-zinc superoxide dismutase (SOD1) and an aberrant interaction between mutant SOD1 and dynein could perturb retrograde transport of neurotrophic factors and mitochondria. A possible contribution of axonal transport to the aggregation and degradation processes of mutant SOD1 is also reviewed. We further consider how the interference with axonal transport and protein turnover by mutant SOD1 could influence the function and viability of motor neurons in ALS.     

Mapping superoxide dismutase 1 domains of non-native interaction: roles of intra- and intermolecular disulfide bonding in aggregation

Superoxide dismutase 1 (SOD1) proteins harboring mutations linked to familial amyotrophic lateral sclerosis (FALS) uniformly show heightened potential to form high molecular weight structures. Here, we examine the domains of SOD1 that are involved in forming these structures (aggregates) and study the role of intra- and intermolecular disulfide bonds. An analysis of disease mutations identified to date reveals a non-random distribution with predominant occurrence at residues within highly conserved beta-strands or at highly conserved residues in loop domains. Using a cell transfection assay for aggregation, we determined that no single domain in SOD1 is indispensable in the formation of sedimentable aggregates, suggesting multiple potential motifs in the protein mediate non-native interactions. By a cell-free aggregation assay, analysis of transgenic mouse tissues, and mutagenesis approaches, we found evidence that redox conditions may modulate SOD1 aggregation; reduction of the native intramolecular disulfide bonds may predispose SOD1 to unfolding and aggregation, whereas non-native intermolecular disulfide linkages may help stabilize aggregates in vivo. The results suggest a possible mechanism for diversity in the structures formed by different SOD1 mutants, and define a potential contribution of redox conditions to SOD1 aggregation.     

Biochemical properties and in vivo effects of the SOD1 zinc-binding site mutant (H80G)

This study presents the initial characterization of transgenic mice with mutations in a primary zinc-binding residue (H80), either alone or with a G93A mutation. H80G;G93A superoxide dismutase 1 (SOD1) transgenic mice developed paralysis with motor neuron loss, and ubiquitin inclusion-type rather than mitochondrial vacuolar pathology. Unlike G93A SOD1-related disease, the course was not accelerated by over-expression of copper chaperone for SOD1. H80G SOD1 transgenic mice did not manifest disease at levels of SOD1 transgene expressed. The H80G mutation altered certain biochemical parameters of both human wild-type SOD1 and G93A SOD1. The H80G mutation does not substantially change the age-dependent accumulation of G93A SOD1 aggregates and hydrophobic species in spinal cord. However, both H80G;G93A SOD1 and H80G SOD1 lack dismutase activity, the ability to form homodimers, and co-operativity with copper chaperone for SOD1, indicating that their dimerization interface is abnormal. The H80G mutation also made SOD1 susceptible to protease digestion. The H80G mutation alters the redox properties of SOD1. G93A SOD1 exists in either reduced or oxidized form, whereas H80G;G93A SOD1 and H80G SOD1 exist only in a reduced state. The inability of SOD1 with an H80G mutation to take part in normal oxidation-reduction reactions has important ramifications for disease mechanisms and pathology in vivo.     

Do individually ventilated cage systems generate a problem for genetic mouse model research?

Technological developments over recent decades have produced a novel housing system for laboratory mice, so‐called ‘individually ventilated cage’ (IVC) systems. IVCs present a cage environment which is different to conventional filter‐top cages (FILTER). Nothing is known about the consequences of IVC housing on genetic mouse models, despite studies reporting IVC‐mediated changes to the phenotypes of inbred mouse strains. Thus, in this study, we systematically compared the established behavioural phenotype of a validated mouse model for the schizophrenia risk gene neuregulin 1 (TM Nrg1 HET) kept in FILTER housing with Nrg1 mutant mice raised in IVC systems. We found that particular schizophrenia‐relevant endophenotypes of TM Nrg1 HETs which had been established and widely published using FILTER housing were altered when mice were raised in IVC housing. IVCs diminished the schizophrenia‐relevant prepulse inhibition deficit of Nrg1 mutant males. Furthermore, IVC housing had a sex‐dependent moderate effect on the locomotive phenotype of Nrg1 mice across test paradigms. Behavioural effects of IVC housing were less prominent in female mice. Thus, transferring the breeding colony of mouse mutants from FILTER to IVC systems can shift disease‐relevant behaviours and therefore challenge the face validity of these mice. Researchers facing an upgrade of their mouse breeding or holding facilities to IVC systems must be aware of the potential impact this upgrade might have on their genetic mouse models. Future publications should provide more details on the cage system used to allow appropriate data comparison across research sites.     

Immunoreactivity of the phosphorylated axonal neurofilament H subunit (pNF-H) in blood of ALS model rodents and ALS patients: evaluation of blood pNF-H as a potential ALS biomarker

Levels of neurofilament subunits, potential biomarkers of motor axon breakdown, are increased in amyotrophic lateral sclerosis (ALS) patient's CSF but data on blood are not available. We measured blood levels of the phosphorylated axonal form of neurofilament H (pNF-H) by ELISA in transgenic rodent models of superoxide dismutase 1 (SOD1) ALS, and in 20 ALS patients and 20 similar aged controls monthly for 4 months. All symptomatic rodent ALS models showed robust levels of blood pNF-H, while control rodents or mice transgenic for unmutated SOD1 showed no detectable blood pNF-H. Average pNF-H levels in the G93A SOD1 mouse progressively increased from day 74 through death (day ∼130). Median blood pNF-H level in ALS patients was 2.8-fold higher than controls ( p  < 0.001). Median ALSFRS-R declined a median of 0.8 pt/month ( p  < 0.001); higher baseline pNF-H level appeared to be associated with faster ALSFRS-R decline over 4 months ( p  = 0.087). The median rate of decline in ALSFRS-R was 1.9 pt/month in patients with baseline pNF-H levels above the median pNF-H value of 0.53 ng/mL; ALSFRS-R declined at a median of 0.6 pt/month in patients below this level. The pNF-H levels were relatively stable month to month in individual patients, raising questions regarding the molecular pathogenesis of ALS. Baseline control human pNF-H levels were higher in men than women and increased minimally over time. These data suggest that blood pNF-H can be used to monitor axonal degeneration in ALS model rodents and support further study of this protein as a potential biomarker of disease prognosis in ALS patients.