Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1(WT) dimers, increasing the equilibrium dissociation constant K(d) to ~10-20 μM, comparable to that of the aggressive A4V mutant. SOD1(A4V) is further destabilized by glutathionylation, experiencing an ~30-fold increase in K(d). Dissociation kinetics of glutathionylated SOD1(WT) and SOD1(A4V) are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1(I112T) has a modestly increased dissociation rate but no change in K(d) when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.     

ALS-causing SOD1 mutations promote production of copper-deficient misfolded species

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.     

Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species

Cu,Zn superoxide dismutase (SOD1) is a dimeric metal-binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to properties of the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are > 95% folded at neutral pH and 37 °C, A4V, L38V, G93A, and L106V ranged from 50% to ∼ 90% unfolded. The reduction of the disulfide bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range, where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the native dimeric state.     

Genotype-Property Patient-Phenotype Relations Suggest that Proteome Exhaustion Can Cause Amyotrophic Lateral Sclerosis

Late-onset neurodegenerative diseases remain poorly understood as search continues for the perceived pathogenic protein species. Previously, variants in Superoxide Dismutase 1 (SOD1) causing Amyotrophic Lateral Sclerosis (ALS) were found to destabilize and reduce net charge, suggesting a pathogenic aggregation mechanism. This paper reports analysis of compiled patient data and experimental and computed protein properties for variants of human SOD1, a major risk factor of ALS. Both stability and reduced net charge correlate significantly with disease, with larger significance than previously observed. Using two independent methods and two data sets, a probability < 3% (t-statistical test) is found that ALS-causing mutations share average stability with all possible 2907 SOD1 mutations. Most importantly, un-weighted patient survival times correlate strongly with the misfolded/unfolded protein copy number, expressed as an exponential function of the experimental stabilities (R ² = 0.31, p = 0.002), and this phenotype is further aggravated by charge (R ² = 0.51, p = 1.8 x 10−5). This finding suggests that disease relates to the copy number of misfolded proteins. Exhaustion of motor neurons due to expensive protein turnover of misfolded protein copies is consistent with the data but can further explain e.g. the expression-dependence of SOD1 pathogenicity, the lack of identification of a molecular toxic mode, elevated SOD1 mRNA levels in sporadic ALS, bioenergetic effects and increased resting energy expenditure in ALS patients, genetic risk factors affecting RNA metabolism, and recent findings that a SOD1 mutant becomes toxic when proteasome activity is recovered after washout of a proteasome inhibitor. Proteome exhaustion is also consistent with energy-producing mitochondria accumulating at the neuromuscular junctions where ALS often initiates. If true, this exhaustion mechanism implies a complete change of focus in treatment of ALS towards actively nursing the energy state and protein turnover of the motor neurons.     

A neurotoxic peripherin splice variant in a mouse model of ALS

Peripherin, a neuronal intermediate filament (nIF) protein found associated with pathological aggregates in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and of transgenic mice overexpressing mutant superoxide dismutase-1 (SOD1G37R), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. Mouse peripherin is unique compared with other nIF proteins in that three peripherin isoforms are generated by alternative splicing. Here, the properties of the peripherin splice variants Per 58, Per 56, and Per 61 have been investigated in transfected cell lines, in primary motor neurons, and in transgenic mice overexpressing peripherin or overexpressing SOD1G37R. Of the three isoforms, Per 61 proved to be distinctly neurotoxic, being assembly incompetent and inducing degeneration of motor neurons in culture. Using isoform-specific antibodies, Per 61 expression was detected in motor neurons of SOD1G37R transgenic mice but not of control or peripherin transgenic mice. The Per 61 antibody also selectively labeled motor neurons and axonal spheroids in two cases of familial ALS and immunoprecipitated a higher molecular mass peripherin species from disease tissue. This evidence suggests that expression of neurotoxic splice variants of peripherin may contribute to the neurodegenerative mechanism in ALS.     

Local unfolding of Cu, Zn superoxide dismutase monomer determines the morphology of fibrillar aggregates

Aggregation of Cu, Zn superoxide dismutase (SOD1) is often found in amyotrophic lateral sclerosis patients. The fibrillar aggregates formed by wild type and various disease-associated mutants have recently been found to have distinct cores and morphologies. Previous computational and experimental studies of wild-type SOD1 suggest that the apo-monomer, highly aggregation prone, displays substantial local unfolding dynamics. The residual folded structure of locally unfolded apoSOD1 corresponds to peptide segments forming the aggregation core as identified by a combination of proteolysis and mass spectroscopy. Therefore, we hypothesize that the destabilization of apoSOD1 caused by various mutations leads to distinct local unfolding dynamics. The partially unfolded structure, exposing the hydrophobic core and backbone hydrogen bond donors and acceptors, is prone to aggregate. The peptide segments in the residual folded structures form the "building block" for aggregation, which in turn determines the morphology of the aggregates. To test this hypothesis, we apply a multiscale simulation approach to study the aggregation of three typical SOD1 variants: wild type, G37R, and I149T. Each of these SOD1 variants has distinct peptide segments forming the core structure and features different aggregate morphologies. We perform atomistic molecular dynamics simulations to study the conformational dynamics of apoSOD1 monomer and coarse-grained molecular dynamics simulations to study the aggregation of partially unfolded SOD1 monomers. Our computational studies of monomer local unfolding and the aggregation of different SOD1 variants are consistent with experiments, supporting the hypothesis of the formation of aggregation "building blocks" via apo-monomer local unfolding as the mechanism of SOD1 fibrillar aggregation.     

Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni2+- and other metal-ion-substituted wild-type copper–zinc superoxide dismutases

The dimeric Cu–Zn superoxide dismutase (SOD1) is a particularly interesting system for biological inorganic chemical studies because substitutions of the native Cu and/or Zn ions by a nonnative metal ion cause minimal structural changes and result in high enzymatic activity for those derivatives with Cu remaining in the Cu site. The pioneering NMR studies of the magnetically coupled derivative Cu₂Co₂SOD1 by Ivano Bertini and coworkers are of particular importance in this regard. In addition to Co²⁺, Ni²⁺ is a versatile metal ion for substitution into SOD1, showing very little disturbance of the structure in Cu₂Ni₂SOD1 and acting as a very good mimic of the native Cu ion in Ni₂Zn₂SOD1. The NMR studies presented here were inspired by and are indebted to Ivano Bertini’s paramagnetic NMR pursuits of metalloproteins. We report Ni²⁺ binding to apo wild-type SOD1 and a time-dependent Ni²⁺ migration from the Zn site to the Cu site, and the preparation and characterization of Ni₂Ni₂SOD1, which shows coordination properties similar to those of Cu₂Cu₂SOD1, namely, an anion-binding property different from that of the wild type and a possibly broken bridging His. Mutations in the human SOD1 gene can cause familial amyotrophic lateral sclerosis (ALS), and mutant SOD1 proteins with significantly altered metal-binding behaviors are implicated in causing the disease. We conclude by discussing the effects of the ALS mutations on the remarkable stabilities and metal-binding properties of wild-type SOD1 proteins and the implications concerning the causes of SOD1-linked ALS.     

Determination of the oligomeric states of human and rat monoamine oxidases in the outer mitochondrial membrane and octyl beta-D-glucopyranoside micelles using pulsed dipolar electron spin resonance spectroscopy

Human monoamine oxidase A (hMAOA) is considered to be unique among mammalian MAOs in having a non-conservative Glu-X-Lys mutation (X being 151 in MAOAs and 142 in MAOB's), which is suggested to be the reason for its monomeric structure. This hypothesis has been tested in this work. A pargyline based nitroxide spin labeled irreversible inhibitor (ParSL) was used as a MAO active site specific spin probe to measure intersubunit distances in detergent (octyl beta-d-glucopyranoside, OGP) purified and OMM bound forms by a pulsed dipolar ESR spectroscopic (PDS) technique. In a parallel approach, the covalent flavin cofactor present in the MAO active sites was reduced to its respective anionic flavin semiquinone and used for measuring inter-flavin distances in detergent purified samples. The measured interspin distances are within 0.1-0.3 nm of those estimated from the available dimeric crystal structures of human MAOB and rat MAOA and show that all human and rat MAOs exist as dimers in the OMM. In the OGP micelle, however, human and rat MAOAs exist only partially (相似文献   

Domain motions accompanying Tet repressor induction defined by changes of interspin distances at selectively labeled sites.

To investigate internal movements in Tet repressor (TetR) during induction by tetracycline (tc) we determined the interspin distances between pairs of nitroxide spin labels attached to specific sites by electron paramagnetic resonance (EPR) spectroscopy. For this purpose, we constructed six TetR variants with engineered cysteine pairs located in regions with presumed conformational changes. These are I22C and N47C in the DNA reading head, T152C/Q175C, A161C/Q175C and R128C/D180C near the tc-binding pocket, and T202C in the dimerization surface. All TetR mutants show wild-type activities in vivo and in vitro. The binding of tc results in a considerable decrease of the distance between the nitroxide groups attached to both I22C residues in the TetR dimer and an increase of the distance between the N47C residues. These opposite effects are consistent with a twisting motion of the DNA reading heads. Changes of the spin-spin interactions between nitroxide groups attached to residues near the tc-binding pocket demonstrate that the C-terminal end of alpha-helix 9 moves away from the protein core upon DNA binding. Alterations of the dipolar interaction between nitroxide groups at T202C indicate different conformations for tc and DNA-bound repressor also in the dimerization area. These results are used to model structural changes of TetR upon induction.     

A new zebrafish model produced by TILLING of SOD1-related amyotrophic lateral sclerosis replicates key features of the disease and represents a tool for in vivo therapeutic screening

Mutations in the superoxide dismutase gene (SOD1) are one cause of familial amyotrophic lateral sclerosis [ALS; also known as motor neuron disease (MND)] in humans. ALS is a relentlessly progressive neurodegenerative disease and, to date, there are no neuroprotective therapies with significant impact on the disease course. Current transgenic murine models of the disease, which overexpress mutant SOD1, have so far been ineffective in the identification of new therapies beneficial in the human disease. Because the human and the zebrafish (Danio rerio) SOD1 protein share 76% identity, TILLING (‘targeting induced local lesions in genomes’) was carried out in collaboration with the Sanger Institute in order to identify mutations in the zebrafish sod1 gene. A T70I mutant zebrafish line was characterised using oxidative stress assays, neuromuscular junction (NMJ) analysis and motor function studies. The T70I sod1 zebrafish model offers the advantage over current murine models of expressing the mutant Sod1 protein at a physiological level, as occurs in humans with ALS. The T70I sod1 zebrafish demonstrates key features of ALS: an early NMJ phenotype, susceptibility to oxidative stress and an adult-onset motor neuron disease phenotype. We have demonstrated that the susceptibility of T70I sod1 embryos to oxidative stress can be used in a drug screening assay, to identify compounds that merit further investigation as potential therapies for ALS.KEY WORDS: MND, ALS, SOD1, Zebrafish     

Adaptive immune neuroprotection in G93A-SOD1 amyotrophic lateral sclerosis mice

Background

Innate neuroimmune dysfunction is a pathobiological feature of amyotrophic lateral sclerosis (ALS). However, links, if any, between disease and adaptive immunity are poorly understood. Thus, the role of T cell immunity in disease was investigated in human G93A superoxide dismutase 1 (SOD1) transgenic (Tg) mice and subsequently in ALS patients.

Methods and Findings

Quantitative and qualitative immune deficits in lymphoid cell and T cell function were seen in G93A-SOD1 Tg mice. Spleens of Tg animals showed reductions in size, weight, lymphocyte numbers, and morphological deficits at terminal stages of disease compared to their wild-type (Wt) littermates. Spleen sizes and weights of pre-symptomatic Tg mice were unchanged, but deficits were readily seen in T cell proliferation coincident with increased annexin-V associated apoptosis and necrosis of lymphocytes. These lymphoid deficits paralleled failure of Copolymer-1 (COP-1) immunization to affect longevity. In addition, among CD4⁺ T cells in ALS patients, levels of CD45RA⁺ (naïve) T cells were diminished, while CD45RO⁺ (memory) T cells were increased compared to age-matched caregivers. In attempts to correct mutant SOD1 associated immune deficits, we reconstituted SOD1 Tg mice with unfractionated naïve lymphocytes or anti-CD3 activated CD4⁺CD25⁺ T regulatory cells (Treg) or CD4⁺CD25⁻ T effector cells (Teff) from Wt donor mice. While naive lymphocytes failed to enhance survival, both polyclonal-activated Treg and Teff subsets delayed loss of motor function and extended survival; however, only Treg delayed neurological symptom onset, whereas Teff increased latency between disease onset and entry into late stage.

Conclusions

A profound and progressive immunodeficiency is operative in G93A-SOD1 mice and is linked to T cell dysfunction and the failure to elicit COP-1 neuroprotective immune responses. In preliminary studies T cell deficits were also observed in human ALS. These findings, taken together, suggest caution in ascribing vaccination outcomes when these animal models of human ALS are used for study. Nonetheless, the abilities to improve neurological function and life expectancy in G93A-SOD1 Tg mice by reconstitution with activated T cells do provide opportunities for therapeutic intervention.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Disease Mechanisms in ALS: Misfolded SOD1 Transferred Through Exosome-Dependent and Exosome-Independent Pathways

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neuromuscular degenerative disorder with a poorly defined etiology. ALS patients experience motor weakness, which starts focally and spreads throughout the nervous system, culminating in paralysis and death within a few years of diagnosis. While the vast majority of clinical ALS is sporadic with no known cause, mutations in human copper-zinc superoxide dismutase 1 (SOD1) cause about 20 % of inherited cases of ALS. ALS with SOD1 mutations is caused by a toxic gain of function associated with the propensity of mutant SOD1 to misfold, presenting a non-native structure. The mechanisms responsible for the progressive spreading of ALS pathology have been the focus of intense study. We have shown that misfolded SOD1 protein can seed misfolding and aggregation of endogenous wild-type SOD1 similar to amyloid-β and prion protein seeding. Our recent observations demonstrate a transfer of the misfolded SOD1 species from cell to cell, modeling the intercellular transmission of disease through the neuroaxis. We have shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell, either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or in association with vesicles which are released into the extracellular environment. Furthermore, once misfolding of wild-type SOD1 has been initiated in a human cell culture, it can induce misfolding in naïve cell cultures over multiple passages of media transfer long after the initial misfolding template is degraded. Herein we review the data on mechanisms of intercellular transmission of misfolded SOD1.     

Male-Specific Differences in Proliferation,Neurogenesis, and Sensitivity to Oxidative Stress in Neural Progenitor Cells Derived from a Rat Model of ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and the loss of large motor neurons in the spinal cord and brain stem. A clear genetic link to point mutations in the superoxide dismutase 1 (SOD1) gene has been shown in a small group of familial ALS patients. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Here we present male-specific effects of the mutant SOD1 transgene on proliferation, neurogenesis, and sensitivity to oxidative stress in rat neural progenitor cells (rNPCs). E14 pups were bred using SOD1^G93A transgenic male rats and wild-type female rats. The spinal cord and cortex tissues were collected, genotyped by PCR using primers for the SOD1^G93A transgene or the male-specific Sry gene, and cultured as neurospheres. The number of dividing cells was higher in male rNPCs compared to female rNPCs. However, SOD1^G93A over-expression significantly reduced cell proliferation in male cells but not female cells. Similarly, male rNPCs produced more neurons compared to female rNPCs, but SOD1^G93A over-expression significantly reduced the number of neurons produced in male cells. Finally we asked whether sex and SOD1^G93A transgenes affected sensitivity to oxidative stress. There was no sex-based difference in cell viability after treatment with hydrogen peroxide or 3-morpholinosydnonimine, a free radical-generating agent. However, increased cytotoxicity by SOD1^G93A over-expression occurred, especially in male rNPCs. These results provide essential information on how the mutant SOD1 gene and sexual dimorphism are involved in ALS disease progression.     

Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease in adults, is characterized by the selective degeneration and death of motor neurons leading to progressive paralysis and eventually death. Approximately 20% of familial ALS cases are associated with mutations in SOD1, the gene encoding Cu/Zn-superoxide dismutase (CuZnSOD). Previously, we reported that overexpression of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD or SOD2) attenuates cytotoxicity induced by expression of the G37R-SOD1 mutant in a human neuroblastoma cell culture model of ALS. In the present study, we extended these earlier findings using several different SOD1 mutants (G93C, G85R, and I113T). Additionally, we tested the hypothesis that mutant SOD1 increases mitochondrial-produced superoxide (O(2) (*)) levels and that SOD2 overexpression protects neurons from mutant SOD1-induced toxicity by reducing O(2) (*) levels in mitochondria. In the present study, we demonstrate that SOD2 overexpression markedly attenuates the neuronal toxicity induced by adenovirus-mediated expression of all four SOD1 mutants (G37R, G93C, G85R, or I113T) tested. Utilizing the mitochondrial-targeted O(2) (*)-sensitive fluorogenic probe MitoSOX Red, we observed a significant increase in mitochondrial O(2) (*) levels in neural cells expressing mutant SOD1. These elevated O(2) (*) levels in mitochondria were significantly diminished by the overexpression of SOD2. These data suggest that mitochondrial-produced O(2) (*) radicals play a critical role in mutant SOD1-mediated neuronal toxicity and implicate mitochondrial-produced free radicals as potential therapeutic targets in ALS.     

Expression of mutant SOD1 in astrocytes induces functional deficits in motoneuron mitochondria

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration resulting in paralysis and eventual death. ALS is regarded as a motoneuron-specific disorder but increasing evidence indicates non-neuronal cells play a significant role in disease pathogenesis. Although the precise aetiology of ALS remains unclear, mutations in the superoxide dismutase (SOD1) gene are known to account for approximately 20% of familial ALS. We examined the influence of SOD1(G93A) expression in astrocytes on mitochondrial homeostasis in motoneurons in a primary astrocyte : motoneuron co-culture model. SOD1(G93A) expression in astrocytes induced changes in mitochondrial function of both SOD1(G93A) and wild-type motoneurons. In the presence of SOD1(G93A) astrocytes, mitochondrial redox state of both wild-type and SOD1(G93A) motoneurons was more reduced and mitochondrial membrane potential decreased. While intra-mitochondrial calcium levels [Ca(2+)](m) were elevated in SOD1(G93A) motoneurons, changes in mitochondrial function did not correlate with [Ca(2+)](m). Thus, expression of SOD1(G93A) in astrocytes directly alters mitochondrial function even in embryonic motoneurons, irrespective of genotype. These early deficits in mitochondrial function induced by surrounding astrocytes may increase the vulnerability of motoneurons to other neurotoxic mechanisms involved in ALS pathogenesis.     

Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease and it is the most common adult onset neurodegenerative disorder affecting motor neurons. There is currently no effective treatment for ALS and our understanding of the pathological mechanism is still far away from prevention and/or treatment of this devastating disease. Amyloid precursor protein (APP) is a transmembrane protein that undergoes processing either by β-secretase or α-secretase, followed by γ-secretase. In the present study, we show that APP levels, and aberrant phosphorylation, which is associated with enhanced β-secretase cleavage, are increased in SOD1^G93A ALS mouse model. Fluorescence resonance energy transfer (FRET) analysis suggests a close interaction between SOD1 and APP at hippocampal synapses. Notably, SOD1^G93A mutation induces APP-SOD1 conformational changes, indicating a crosstalk between these two signaling proteins. Inhibition of APP processing via monoclonal antibody called BBS that blocks APP β-secretase cleavage site, resulted in reduction of mutant SOD1^G93A levels in animal and cellular models of ALS, significantly prolonged life span of SOD1^G93A mice and diminished inflammation. Beyond its effect on toxic mutant SOD1^G93A, BBS treatment resulted in a reduction in the levels of APP, its processing product soluble APPβ and pro-apoptotic p53. This study demonstrates that APP and its processing products contribute to ALS pathology through several different pathways; thus BBS antibody could be a promising neuroprotective strategy for treatment of this disease.     

Transduction of familial amyotrophic lateral sclerosis-related mutant PEP-1-SOD proteins into neuronal cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the selective death of motor neurons. Mutations in the SOD1 gene are responsible for a familial form of ALS (FALS). Although many studies suggest that mutant SOD1 proteins are cytotoxic, the mechanism is not fully understood. To investigate the role of mutant SOD1 in FALS, human SOD1 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce in-frame PEP-1-SOD fusion proteins (wild type and mutants). The expressed and purified PEP-1-SOD fusion proteins were efficiently transduced into neuronal cells. Neurones harboring the A4V, G93A, G85R, and D90A mutants of PEP-1-SOD were more vulnerable to oxidative stress induced by paraquat than those harboring wild-type proteins. Moreover, neurones harboring the mutant SOD proteins had lower heat shock protein (Hsp) expression levels than those harboring wild-type SOD. The effects of the transduced SOD1 fusion proteins may provide an explanation for the association of SOD1 with FALS, and Hsps could be candidate agents for the treatment of ALS.     

An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase

Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is a neurodegenerative disease characterized by motor neuron degeneration, paralysis and death. One cause of this disease is mutations in the Cu,Zn superoxide dismutase (SOD1) gene. As mutant SOD1 acquires a toxic property that kills motor neurons, by reducing the mutant protein the disease progression may be slowed or prevented. While mutant SOD1 is toxic, the wild-type SOD1 is indispensable for motor neuron health. Therefore, the ideal therapeutic strategy would be to inhibit selectively the mutant protein expression. Previously we have demonstrated that RNA interference (RNAi) can selectively inhibit some mutant SOD1 expression. However, more than 100 SOD1 mutants can cause ALS and all mutants cannot be inhibited selectively by RNAi. To overcome this obstacle, we have designed a replacement RNAi strategy. Using this strategy, all mutants and wild-type genes are inhibited by RNAi. The wild-type SOD1 function is then replaced by designed wild-type SOD1 genes that are resistant to the RNAi. Here we demonstrate the concept of this strategy.