SOD1 and amyotrophic lateral sclerosis: mutations and oligomerization

There are about 100 single point mutations of copper, zinc superoxide dismutase 1 (SOD1) which are reported (http://alsod.iop.kcl.ac.uk/Als/index.aspx) to be related to the familial form (fALS) of amyotrophic lateral sclerosis (ALS). These mutations are spread all over the protein. It is well documented that fALS produces protein aggregates in the motor neurons of fALS patients, which have been found to be associated to mitochondria. We selected eleven SOD1 mutants, most of them reported as pathological, and characterized them investigating their propensity to aggregation using different techniques, from circular dichroism spectra to ThT-binding fluorescence, size-exclusion chromatography and light scattering spectroscopy. We show here that these eleven SOD1 mutants, only when they are in the metal-free form, undergo the same general mechanism of oligomerization as found for the WT metal-free protein. The rates of oligomerization are different but eventually they give rise to the same type of soluble oligomeric species. These oligomers are formed through oxidation of the two free cysteines of SOD1 (6 and 111) and stabilized by hydrogen bonds, between beta strands, thus forming amyloid-like structures. SOD1 enters the mitochondria as demetallated and mitochondria are loci where oxidative stress may easily occur. The soluble oligomeric species, formed by the apo form of both WT SOD1 and its mutants through an oxidative process, might represent the precursor toxic species, whose existence would also suggest a common mechanism for ALS and fALS. The mechanism here proposed for SOD1 mutant oligomerization is absolutely general and it provides a common unique picture for the behaviors of the many SOD1 mutants, of different nature and distributed all over the protein.     

Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in the familial form of amyotrophic lateral sclerosis

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (fALS), and aggregation of mutant SOD1 has been proposed to play a role in neurodegeneration. A growing body of evidence suggests that fALS-causing mutations destabilize the native structure of SOD1, leading to aberrant protein interactions for aggregation. SOD1 becomes stabilized and enzymatically active after copper and zinc binding and intramolecular disulfide formation, but it remains unknown which step(s) in the SOD1 maturation process is important in the pathological aggregation. In this study we have shown that apoSOD1 without disulfide is the most facile state for formation of amyloid-like fibrillar aggregates. fALS mutations impair either zinc binding, disulfide formation, or both, leading to accumulation of the aggregation-prone, apo, and disulfide-reduced SOD1. Moreover, we have found that the copper chaperone for SOD1 (CCS) facilitates maturation of SOD1 and that CCS overexpression ameliorates intracellular aggregation of mutant SOD1 in vivo. Based on our in vivo and in vitro results, we propose that facilitation of post-translational modifications is a promising strategy to reduce SOD1 aggregation in the cell.     

Exploring the cause of aggregation and reduced Zn binding affinity by G85R mutation in SOD1 rendering amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disorder is characterized by the degeneration of upper and lower motor neuron. ALS occurs due to various notably prominent missense mutations, in gene encoding Cu‐Zn superoxide dismutase (SOD1) thereby leading to aggregation, dysfunction and reduced Zn binding affinity. In this study, one such mutation, G85R was explored in comparison with wild type SOD1, using discrete molecular dynamics (DMD). Accordingly, the conformational changes were significantly observed in mutant SOD1, through various geometrical parameters, which substantiated the difference in conformational deviation, flexibility and compactness, thus stipulating a root cause for aggregation. Followed by, analysis of essential dynamics further authenticated the cause behind the protein dysfunction. In particular, the high content of beta sheet with structural deviations, down to dysfunction was established in mutant as compared to wild type, while passing through secondary structure analysis. Subsequently, the deviation of distance in Zn binding residues was distinctly portrayed in mutant as compared to wild type, thus confirming the cause of reduced Zn binding affinity. In addition, the steered molecular dynamics analysis also authenticated the above results indicating the reduced Zn binding affinity in the mutant as compared to that of the wild type. Hence, this work revealed the theoretical mechanism to unravel the mutational effects of cofactor dependent protein. Proteins 2017; 85:1276–1286. © 2017 Wiley Periodicals, Inc.     

A limited role for disulfide cross-linking in the aggregation of mutant SOD1 linked to familial amyotrophic lateral sclerosis

One of the mechanisms by which mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis (fALS) is proposed to involve the accumulation of detergent-insoluble, disulfide-cross-linked, mutant protein. Recent studies have implicated cysteine residues at positions 6 and 111 as critical in mediating disulfide cross-linking and promoting aggregation. In the present study, we used a panel of experimental and disease-linked mutations at cysteine residues of SOD1 (positions 6, 57, 111, and 146) in cell culture assays for aggregation to demonstrate that extensive disulfide cross-linking is not required for the formation of mutant SOD1 aggregates. Experimental mutants possessing only a single cysteine residue or lacking cysteine entirely were found to retain high potential to aggregate. Furthermore we demonstrate that aggregate structures in symptomatic SOD1-G93A mice can be dissociated such that they no longer sediment upon ultracentrifugation (i.e. appear soluble) under relatively mild conditions that leave disulfide bonds intact. Similar to other recent work, we found that cysteines 6 and 111, particularly the latter, play interesting roles in modulating the aggregation of human SOD1. However, we did not find that extensive disulfide cross-linking via these residues, or any other cysteine, is critical to aggregate structure. Instead we suggest that these residues participate in other features of the protein that, in some manner, modulate aggregation.     

SOD1 gene mutations in patients with amyotrophic lateral sclerosis: Potential of method of molecular modeling

Molecular modeling is a promising method for assessing protein structures that is capable of presenting an energetically beneficial protein conformation with atomic precision. This method is of great importance for studying molecular interactions and confirming the pathogenic significance of the changes in protein structures caused by particular mutations. In this study, we used molecular modeling to assess mutations in the SOD1 gene in patients with amyotrophic lateral sclerosis (ALS), a severe neurodegenerative disorder characterized by the loss of spinal and cerebral motor neurons. The product of SOD1 is a cytosolic dimeric enzyme Cu/Zn superoxide dismutase (SOD1) responsible for the detoxification of cellular superoxide radicals. We showed that all eight revealed coding-point mutations of the gene led to moderate or significant changes in SOD1 protein energy. The mutation His49Arg increased protein energy, and the reconstruction of the respective model indicated the spatial destabilization of the molecule and abnormal interactions with the metal ion inside the active center. Conversely, the other seven mutations (Gly17Ala, Leu85Val, Asn87Ser, Asp91Ala, Ser106Leu, Glu134Gly, and Leu145Phe) led to a decrease in protein energy and an increase in the spatial stability of SOD 1, which is usually accompanied by an increased tendency for the inert mutant molecule to misfold and demonstrate cellular aggregation. Therefore, the results of the in silico analysis of the SOD1 gene mutations confirms that ALS belongs to the class of the so-called conformational diseases of the central nervous system, a characteristic feature of which is the formation of cytotoxic, insoluble protein inclusions in neurons.     

The effect of leukaemia inhibitory factor on SOD1 G93A murine amyotrophic lateral sclerosis

Before potential therapeutic strategies for the treatment of amyotrophic lateral sclerosis (ALS) can be advanced to human clinical trials, there is a need to assess them in an animal model that best resembles the disease process. SOD1 G93A mice have close resemblance to familial ALS (fALS) and have been used in this study to evaluate the therapeutic potential of leukaemia inhibitory factor (LIF). LIF action was investigated by assessing three delivery methods: (1) daily subcutaneous injection; (2) through LIF rods placed adjacent to hind limb skeletal muscle and (3) continuous intrathecal infusion. The effect on disease progression was assessed by semi-quantitative and quantitative functional measurements, and histologically on the survival of motor neurons and number of reactive astrocytes. The results show that LIF had no beneficial effects when administered using the three methods of drug delivery. These results suggest that further evaluation of LIF in this transgenic model is required to fully characterize its' therapeutic potential.     

Superoxide dismutase mutations of familial amyotrophic lateral sclerosis and the oxidative inactivation of calcineurin

Approximately 10% of all familial cases of amyotrophic lateral sclerosis (fALS) are linked to mutations in the SOD1 gene, which encodes the copper/zinc superoxide dismutase (CuZnSOD). Recently, wild-type CuZnSOD was shown to protect calcineurin, a calcium/calmodulin-regulated phosphoprotein phosphatase, from inactivation by reactive oxygen species. We asked whether the protective effect of CuZnSOD on calcineurin is affected by mutations associated with fALS. For this, we monitored calcineurin activity in the presence of mutant and wild-type SOD. We found that the degree of protection against inactivation of calcineurin by different SOD mutants correlates with the severity of the phenotype associated with the different mutations, suggesting a potential role for calcineurin-SOD1 interaction in the etiology of fALS.     

Structural instability and Cu-dependent pro-oxidant activity acquired by the apo form of mutant SOD1 associated with amyotrophic lateral sclerosis

Cu,Zn-superoxide dismutase (SOD1) is a cytosolic antioxidant enzyme, and its mutation has been implicated in amyotrophic lateral sclerosis (ALS), a disease causing a progressive loss of motor neurons. Although the pathogenic mechanism of ALS remains unclear, it is hypothesized that some toxic properties acquired by mutant SOD1 play a role in the development of ALS. We have examined the structural and catalytic properties of an ALS-linked mutant of human SOD1, His43Arg (H43R), which is characterized by rapid disease progression. As revealed by circular dichroism spectroscopy, H43R assumes a stable β-barrel structure in the Cu(2+),Zn(2+)-bound holo form, but its metal-depleted apo form is highly unstable and readily unfolds or misfolds into an irregular structure at physiological temperature. The conformational change occurs as a two-state transition from a nativelike apo form to a denatured apo form with a half-life of ～0.5 h. At the same time as the denaturation, the apo form of H43R acquires pro-oxidant potential, which is fully expressed in the presence of Cu(2+) and H(2)O(2), as monitored with a fluorogenic probe for detecting pro-oxidant activity. Comparison of d-d absorption bands suggests that the Cu(2+) binding mode of the denatured apo form is different from that of the native holo form. The denatured apo form of H43R is likely to provide non-native Cu(2+) binding sites where the Cu(2+) ion is activated to catalyze harmful oxidation reactions. This study raises the possibility that the structural instability and the resultant Cu-dependent pro-oxidant activity of the apo form of mutant SOD1 may be one of the pathogenic mechanisms of ALS.     

肌萎缩侧索硬化患者SOD1基因突变检测及突变与临床表型的关系

应用PCR技术结合DNA直接测序方法对8例临床确诊为家族性肌萎缩侧索硬化(Familiar amyotrophic lateral sclerosis,FALS)家系的先证者进行铜锌超氧化物歧化酶基因(SOD1)的突变筛查,在3例先证者中检出2种SOD1基因突变,其中,2例携带了位于4号外显子的错义突变Cys111Tyr(c.332G>A),另1例携带了位于5号外显子的错义突变Gly147Asp(c.440G>A),这2种突变在中国ALS患者中属首次报道。该结果扩大了中国FALS患者的SOD1基因突变谱,对研究中国FALS患者SOD1基因突变特点和分布规律有一定帮助。分析携带这2个突变患者的临床特点,提示Cys111Tyr突变导致的临床表型相对温和,而Gly147Asp突变可导致病情进展较快。该结果有待在更多的病例中进行证实。     

Arylsulfanyl pyrazolones block mutant SOD1-G93A aggregation. Potential application for the treatment of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease currently without a cure. Mutations in copper/zinc superoxide dismutase 1 (SOD1) have been implicated in the pathophysiology of this disease. Using a high-throughput screening assay expressing mutant G93A SOD1, two bioactive chemical hit compounds (1 and 2), identified as arylsulfanyl pyrazolones, were identified. The structural optimization of this scaffold led to the generation of a more potent analogue (19) with an EC₅₀ of 170 nM. To determine the suitability of this class of compounds for further optimization, 1 was subjected to a battery of pharmacokinetic assays; most of the properties of 1 were good for a screening hit, except it had a relatively rapid clearance and short microsomal half-life stability. Compound 2 was found to be blood-brain barrier penetrating with a brain/plasma ratio = 0.19. The optimization of this class of compounds could produce novel therapeutic candidates for ALS patients.     

Mutant SOD1 linked to familial amyotrophic lateral sclerosis,but not wild-type SOD1, induces ER stress in COS7 cells and transgenic mice

Mutations in a Cu, Zn-superoxide dismutase (SOD1) cause motor neuron death in human familial amyotrophic lateral sclerosis (FALS) and its mouse model, suggesting that mutant SOD1 has a toxic effect on motor neurons. However, the question of how the toxic function is gained has not been answered. Here, we report that the mutant SOD1s linked to FALS, but not wild-type SOD1, aggregated in association with the endoplasmic reticulum (ER) and induced ER stress in the cDNA-transfected COS7 cells. These cells showed an aberrant intracellular localization of mitochondria and microtubules, which might lead to a functional disturbance of the cells. Motor neurons of the spinal cord in transgenic mice with a FALS-linked mutant SOD1 also showed a marked increase of GRP78/BiP, an ER-resident chaperone, just before the onset of motor symptoms. These data suggest that ER stress is involved in the pathogenesis of FALS with an SOD1 mutation.     

Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1

Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathological hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular weight aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degradation. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys(6)- and Cys(111)-disulfide cross-linked form and targeted it for proteasomal degradation.     

Lack of effect of methylene blue in the SOD1 G93A mouse model of amyotrophic lateral sclerosis

Background

Methylene blue (MB) is a drug with a long history and good safety profile, and with recently-described features desirable in a treatment for ALS.

Methodology/Principal Findings

We tested oral MB in inbred high-copy number SOD1 G93A mice, at 25 mg/kg/day beginning at 45 days of age. We measured disease onset, progression, and survival. There was no difference in disease onset between MB-treated mice and controls, although subgroup analysis showed a modest but statistically significant delay in disease onset in MB-treated female mice only (control 122±10.2 versus MB 129±10.0 days). MB-treated mice of both sexes spent more time in less severe stages of disease, and less time in later, more severe stages of disease. There was a non-significant trend to longer survival in MB-treated animals (control males reached endpoint at 161±14.1 days, versus 166±10.0 days for MB-treated animals, and control females reached endpoint at 171±6.2 days versus 173±13.4 days for MB-treated animals).

Conclusions/Significance

In spite of a strong theoretical rationale, MB had no significant effects on onset or survival in the inbred SOD1 G93A mouse model of ALS.     

Structural consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg

The His46Arg (H46R) mutant of human copper-zinc superoxide dismutase (SOD1) is associated with an unusual, slowly progressing form of familial amyotrophic lateral sclerosis (FALS). Here we describe in detail the crystal structures of pathogenic H46R SOD1 in the Zn-loaded (Zn-H46R) and metal-free (apo-H46R) forms. The Zn-H46R structure demonstrates a novel zinc coordination that involves only three of the usual four liganding residues, His 63, His 80, and Asp 83 together with a water molecule. In addition, the Asp 124 "secondary bridge" between the copper- and zinc-binding sites is disrupted, and the "electrostatic loop" and "zinc loop" elements are largely disordered. The apo-H46R structure exhibits partial disorder in the electrostatic and zinc loop elements in three of the four dimers in the asymmetric unit, while the fourth has ordered loops due to crystal packing interactions. In both structures, nonnative SOD1-SOD1 interactions lead to the formation of higher-order filamentous arrays. The disordered loop elements may increase the likelihood of protein aggregation in vivo, either with other H46R molecules or with other critical cellular components. Importantly, the binding of zinc is not sufficient to prevent the formation of nonnative interactions between pathogenic H46R molecules. The increased tendency to aggregate, even in the presence of Zn, arising from the loss of the secondary bridge is consistent with the observation of an increased abundance of hyaline inclusions in spinal motor neurons and supporting cells in H46R SOD1 transgenic rats.     

Structures of the G85R variant of SOD1 in familial amyotrophic lateral sclerosis

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.     

Increased oxidative stress in patients with amyotrophic lateral sclerosis and the effect of edaravone administration

Objectives and methods: Compared to age-matched healthy controls (n?=?55), patients with amyotrophic lateral sclerosis (ALS) (n?=?26) showed increased oxidative stress as indicated by a significantly increased percentage of oxidized coenzyme Q10 (%CoQ10) in total plasma coenzyme Q10, a significantly decreased level of plasma uric acid, and a significantly decreased percentage of polyunsaturated fatty acids in total plasma free fatty acids (FFA). Therefore, the efficacy of edaravone, a radical scavenger, in these ALS patients was examined.

Results and discussion: Among 26 ALS patients, 17 received edaravone (30?mg/day, one to four times a week) for at least 3 months, and 13 continued for 6 months. Changes in revised ALS functional rating scale (ALSFRS-R) were significantly smaller in these patients than in edaravone-untreated ALS patients (n?=?19). Edaravone administration significantly reduced excursions of more than one standard deviation from the mean for plasma FFA levels and the contents of palmitoleic and oleic acids, plasma markers of tissue oxidative damage, in the satisfactory progress group (ΔALSFRS-R?≥?0) as compared to the ingravescent group (ΔALSFRS-R?<??5). Edaravone treatment increased plasma uric acid, suggesting that it is an effective scavenger of peroxynitrite. However, edaravone administration did not decrease %CoQ10. Therefore, combined treatment with agents such as coenzyme Q10 may further reduce oxidative stress in ALS patients.     

FUS and TARDBP but not SOD1 interact in genetic models of amyotrophic lateral sclerosis

Mutations in the SOD1 and TARDBP genes have been commonly identified in Amyotrophic Lateral Sclerosis (ALS). Recently, mutations in the Fused in sarcoma gene (FUS) were identified in familial (FALS) ALS cases and sporadic (SALS) patients. Similarly to TDP-43 (coded by TARDBP gene), FUS is an RNA binding protein. Using the zebrafish (Danio rerio), we examined the consequences of expressing human wild-type (WT) FUS and three ALS-related mutations, as well as their interactions with TARDBP and SOD1. Knockdown of zebrafish Fus yielded a motor phenotype that could be rescued upon co-expression of wild-type human FUS. In contrast, the two most frequent ALS-related FUS mutations, R521H and R521C, unlike S57Δ, failed to rescue the knockdown phenotype, indicating loss of function. The R521H mutation caused a toxic gain of function when expressed alone, similar to the phenotype observed upon knockdown of zebrafish Fus. This phenotype was not aggravated by co-expression of both mutant human TARDBP (G348C) and FUS (R521H) or by knockdown of both zebrafish Tardbp and Fus, consistent with a common pathogenic mechanism. We also observed that WT FUS rescued the Tardbp knockdown phenotype, but not vice versa, suggesting that TARDBP acts upstream of FUS in this pathway. In addition we observed that WT SOD1 failed to rescue the phenotype observed upon overexpression of mutant TARDBP or FUS or upon knockdown of Tardbp or Fus; similarly, WT TARDBP or FUS also failed to rescue the phenotype induced by mutant SOD1 (G93A). Finally, overexpression of mutant SOD1 exacerbated the motor phenotype caused by overexpression of mutant FUS. Together our results indicate that TARDBP and FUS act in a pathogenic pathway that is independent of SOD1.