Journal of the Neurological Sciences 336 (2014) 1–7

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Review article

Mitochondrial defects in transgenic mice expressing Cu,Zn Superoxide Dismutase mutations, the role of Copper Chaperone for SOD1 Marjatta Son ⁎, Jeffrey L. Elliott Department of Neurology and Neurotherapeutics, UT Southwestern Medical Center in Dallas, Dallas, TX, USA

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Article history: Received 7 August 2013 Received in revised form 23 October 2013 Accepted 4 November 2013 Available online 13 November 2013 Keywords: Cu,Zn Superoxide Dismutase Neurodegeneration Mitochondria Copper Chaperone for SOD1 Transgenic mouse Mutation

a b s t r a c t Several hypotheses have been proposed for the mechanisms underlying mutant Cu,Zn Superoxide Dismutase‐ related Amyotrophic Lateral Sclerosis. These include aggregation pathology, mitochondrial dysfunctions, oxidative stress, and glutamate‐mediated excitotoxicity. Mitochondrial disease may be a primary event in neurodegeneration, contributing to oxidative stress and apoptosis, or it may be caused by other cellular processes. Mitochondrial structural abnormalities have been detected in the skeletal muscle, lymphoblast and central nervous system of Amyotrophic Lateral Sclerosis patients. The cause or even the extent of mitochondrial defects in spinal cord and brain of patients with Cu,Zn Superoxide Dismutase mutations is difficult to determine because of rapid mitochondrial deterioration in autopsy samples. The focus of this review is how abnormalities in Cu, Zn Superoxide Dismutase redox states, folding and metallation contribute to mitochondrial deficiencies, investigating the differences in mitochondrial defects observed among transgenic mice expressing various Cu,Zn Superoxide Dismutase mutations. © 2013 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . 2. SOD1 and its activation by CCS . . . . . . . . . . 3. CCS-independent maturation of SOD1 . . . . . . . 4. Mitochondrial import of SOD1 and CCS . . . . . . 5. ALS linked SOD1 mutants . . . . . . . . . . . . 6. The effect of CCS on different groups of SOD1 mutants 7. The effects of SOD1 mutants on mitochondria . . . 8. Conclusion . . . . . . . . . . . . . . . . . . . Conflict of interest disclosure . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Mutations in the Cu,Zn Superoxide Dismutase (SOD1) gene account for the second most common form of autosomal dominant Familial Amyotrophic Lateral Sclerosis (FALS), a neurodegenerative disorder characterized by motor neuron loss leading to death of affected individuals. About 5–10% of ALS cases are familial and about 15–20% of FALS cases associated with mutations in SOD1. The number of SOD1 ⁎ Corresponding author at: Department of Neurology and Neurotherapeutics, UT Southwestern Medical Center in Dallas, 5323 Harry Hines Boulevard, Dallas, TX 753908813, USA. Tel.: +1 214 633 1879. E-mail address: [email protected] (M. Son). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.11.004

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mutations linked to ALS is about 170 (http://www.alsod.org). Mutations exist throughout the various domains, indicating that any alterations in SOD1 structure may have disease manifesting consequences. While the loss of SOD1 in knockout mice does not lead to ALS, overexpression of SOD1 mutations, and even over-expression of wild type (WT) human SOD1 at very high levels in transgenic mice lead to an ALS phenotype [1–3]. This suggests that “a gain of toxic function” is the cause for SOD1 related ALS, while “loss of function” might play a modifying role [4]. The findings that high levels of WT human SOD1 can cause ALS-like syndromes, indicate that oxidatively damaged and misfolded WT SOD1 might have similar effects than SOD1 mutants [5]. ALS phenotype is characterized by the formation of aggregates and mitochondrial abnormalities depending on the specific structural and

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functional change in the mutant SOD1 and its steady state levels. Profound mitochondrial vacuolar pathology has been detected in transgenic mice expressing high levels of G93A or G37R SOD1, whereas the pathology in G85R, H46R or L126Z SOD1 transgenic mice is mostly characterized by SOD1 positive aggregates and inclusions [6–11]. 2. SOD1 and its activation by CCS SOD1 catalyzes the disproportionation of superoxide anion to hydrogen peroxide and oxygen in a reaction mediated through the cyclic reduction and oxidation of the bound copper ion [12]. Human wild type SOD1 is a 153 amino acid, 32 kDa homodimeric enzyme, where each subunit folds into β‐barrel. Each SOD1 monomer binds one copper and one zinc ion and contains one disulfide bond. Human SOD1 has four cysteine residues, at positions 6, 57, 111 and 146. C57 and C146 form an intramolecular disulfide bond that plays an important role in SOD1 activation. C6 and C111 do not normally form disulfide bonds. C6 is buried within the interior of the β-barrel, whereas C111 is exposed on the surface near the dimer interface [13]. C111 is vulnerable to oxidative damage contributing to pathogenic aggregation [14,15]. The corresponding residue is serine in murine SOD1 that normally is more stable than human SOD1. However, transgenic mice expressing murine mutant (G86R) SOD1 also develop motor neuron disease characterized by inclusions. These findings indicate that several amino acid residues, besides C111, can contribute to the toxicity of mutant SOD1 [11,16]. Nascent human SOD1 acquires zinc by an unknown mechanism. The zinc-containing apo-SOD1 pool is enzymatically inactive. The Copper Chaperone for SOD1 (CCS) is involved in SOD1 maturation and activation by delivering copper to SOD1 and catalyzing the formation of SOD1 intramolecular disulfide bond [12,17,18]. Human CCS is a 54 kDa homodimeric protein; it consists of three domains, which are all involved in the activation of SOD1. According to the most recent in vitro model for human CCS–dependent human SOD1 activation, domain 1 is involved in copper uptake and delivery [18]. Domain 2 of human CCS is 47% identical to human SOD1, however it cannot bind copper [19]. CCS interacts with SOD1 by its SOD1‐like domain 2 forming CCS–SOD1 heterodimers. Domain 3, which is partially homologous to prolyl cis‐trans isomerase, has a CXC (C244, C246) dual motif that is required for disulfide transfer to SOD1 and for the formation of the SOD1 intramolecular disulfide bond [12]. CCS dependent maturation has several steps: fully mature, coppermetallated CCS recognizes and binds to a disulfide reduced, copper deficient form of SOD1, CCS transfers copper to SOD1, followed by formation of an intermolecular disulfide bond between CCS C244 and SOD1 C57, formation of an intramolecular disulfide bond between SOD1 C57 and C146, and finally dimerization of the SOD1 monomer to an active homodimer [18]. CCS can catalyze the oxidation of each SOD1 monomer only in the presence of oxygen [12]. The C57 and C146 residues of SOD1 can be slowly oxidized by oxygen alone; disulfide formation is greatly accelerated by copper‐bound CCS [20]. Mutations or oxidative modifications hinder these posttranslational processes resulting in metal‐deficient, disulfide‐reduced and monomeric forms of SOD1 that may lead to fibrils and aggregates relevant in FALS toxicity [21]. CCS also protects SOD1 from misfolding. This CCS chaperoning function requires physical interaction between SOD1 and CCS domain 2, but not disulfide oxidation or copper loading [22,23]. 3. CCS-independent maturation of SOD1 Activation of wild type human SOD1 by disulfide oxidation and copper insertion can also be achieved via CCS-independent pathways [24]. In CCS knockout mice about 15% of SOD1 activity remains, indicating that CCS is important but not totally essential for mammalian SOD1 to acquire copper and have activity [25]. In an human patient a homozygous R163W mutation in CCS domain 2 reduced CCS levels, obstructed CCS binding to SOD1, reduced SOD1 activity but not SOD1 levels,

possibly impaired copper homeostasis, and caused death at 45 months [26]. Human SOD1 can acquire copper and form the intramolecular disulfide via CCS-independent pathways that involve glutathione [27,28]. In contrast to CCS-dependent activation of SOD1, CCSindependent activation does not require oxygen and is possible even under hypoxic conditions [24]. The dual pathways allow human SOD1 activity to be maintained over a wide range of oxygen conditions, which is important in multicellular organism, where oxygen tensions range from near atmospheric to hypoxic [20,29]. 4. Mitochondrial import of SOD1 and CCS SOD1 is predominantly a cytosolic protein but is also found in other cellular compartments, including the mitochondria [30–32]. The import of nuclear coded mitochondrial proteins has been studied in yeast and in human cells [31,33–36]. The disulfide relay system involving Mia40 and Erv1 drives the import of cysteine‐rich proteins destined into mitochondrial intermembrane space (IMS) and appears to be critical for CCS and SOD1 localization within the mitochondria. Human homologs of Mia40 (CHCHD4) and Erv1 (augmenter of liver regeneration, ALR) can replace yeast Mia40 and Erv1, implying functional homology of the mammalian and yeast pathways [36]. The disulfide relay system is connected to the electron transport chain in mitochondria via Erv1, which shuttles electrons from Mia40 via cytochrome c, to Complex IV (cytochrome c oxidase; COX), to oxygen, and to cytochrome c peroxidase [37,38]. The newly synthesized, reduced and unfolded proteins enter the IMS, where Mia40 forms transient intermolecular disulfide bonds with them. After intramolecular disulfide bonds are formed in the imported proteins, they are trapped within IMS, because folded proteins are not able to cross the outer membrane. The Mia40 import mechanism can also function without the formation of disulfide bonds with the target proteins [39]. In agreement, in human cells expressing redox defective mutant Mia40 some reduced forms of target proteins can still be imported [36]. Along with Mia40, glutathione is involved in the oxidative folding of several substrates by promoting disulfide reshuffling [34,37]. Mia40 mediates directly the localization of CCS into the IMS, whereas the localization of wild type SOD1 is dependent on CCS. Disulfide bonds are transferred from Erv1 via Mia40 to CCS, which then introduces the disulfide bond and copper into SOD1, triggering folding and retention of SOD1 in the IMS [34,35,40]. As the content of CCS is 15–30‐fold less than that of SOD1 in mammalian cells, CCS likely is the limiting factor that effects the mitochondrial distribution of SOD1 [34,35,41]. In agreement, over-expression of CCS in transgenic mice increases the amount of SOD1 in mitochondria [10]. However, some mitochondria localization of SOD1 occurs via CCS-independent pathways both in yeast and in mammalian cells [31,39]. In CCS deficient yeast mitochondria, reduced SOD1 variants can be imported into IMS via Mia40 and Mitochondrial Inner Membrane Organizing System (MINOS) mediated mechanisms [39]. MINOS is a conserved large protein complex in both yeast and mammalian mitochondrial inner membrane, which plays a role in both the maintenance of mitochondrial structure and the coordination of protein import [42]. 5. ALS linked SOD1 mutants Several different human SOD1 mutations have been expressed in transgenic mice and rats [43]. The pathogenic SOD1 mutations have been grouped based on their positions in the structure. The first group, β-barrel mutants, includes G93A and G37R SOD1 that can interact with CCS, bind copper, form intramolecular disulfide bonds and mature to enzymatically active homodimers. Both G93A SOD1 and G37R SOD1 transgenic mice display in their spinal cords mitochondrial vacuoles and also SOD1 positive aggregates [6,7,44]. Even very high amounts of human wild type SOD1 can lead to mitochondrial vacuolization in the neurons of transgenic mice [45]. G93A SOD1 mutant is partially

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Table 1 Disease characteristics of transgenic mice expressing β-barrel mutant (G93A or G37R SOD1), metal-binding region mutant (G86R, H80G or H80G;G93A SOD1) or truncation mutant (L126Z SOD1) either alone or in the context of CCS over-expression. Mitochondrial Genotype

Lifespan

Paralysis

Aggregates

Vacuoles

COX level

Redox State of SOD1

G93A CCS/G93A G37R CCS/G37R G86R CCS/G86R L126Z CCS/L126Z H80G CCS/H80G G93A;H80G CCS/G93A;H80G WT SOD1 CCS/WTSOD1

242 d 36 d 270 d 32 d 113 d 115 d 230 d 230 d N2 y N2 y 513 d 505 d N2 y N2 y

+ + + + + + + + − − + + − −

+++ − +++ − + + + + − − + + − −

+ +++ + +++ − − − − − − − − − −

↓ ↓↓↓ ↓ ↓↓↓ Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal

Oxidized NNreduced Oxidized Nreduced Oxidized NNreduced Oxidized Nreduced All reduced All reduced All reduced All reduced All reduced All reduced All reduced All reduced Oxidized ≫reduced Oxidized NNNreduced

Aggregates, mitochondrial vacuoles and COX levels, and SOD1 redox states observed in the spinal cords of the transgenic mice in the endstage of the disease [10,11,22,52,70]. + observed; not observed; + some; +++ very many; ↓ decreased; ↓↓↓ much decreased; N more; NN much more; NNN very much more.

destabilized especially in the metal binding region, which may affect the intermolecular CCS–SOD1 interactions [46]. A fraction of mutant SOD1 is in reduced and monomeric form in the spinal cord of G93A SOD1 transgenic mice and rats [47,48]. The second group, the metal binding region mutants, have SOD1 mutations in the metal binding ligands themselves or in the electrostatic and zinc loop elements that are associated with metal binding, resulting in copper and/or zinc deficiency [49]. They have increased propensity to exist in monomeric form and have their disulfide bonds reduced. Transgenic mice expressing metal binding region SOD1 mutations develop motor neuron defects characterized by SOD1 containing aggregates and ubiquinated inclusions. They also have mitochondrial abnormalities; however they do not display large mitochondrial vacuoles. G85R SOD1 and its murine equivalent G86R SOD1 belong to the metal binding region mutants [8,16]. The metal binding region SOD1 mutants are more unstable than β-barrel mutants. G85R SOD1 and G86R SOD1 transgenic mouse lines express low steady state levels of the mutant protein, some of them only a fraction of the endogenous SOD1 levels, however, it is enough to cause paralysis. G85R SOD1 and G86R SOD1 do not have detectable levels of dismutase activity and cannot normally interact with CCS [11,16,50]. Mice expressing the H46R single mutant, H46R;H48Q double mutant, and quadruple H46R;H48Q;H63G;H120G SOD1 mutant that disrupts all four copper ligands have been developed [9,51]. CCS is unable to convert these SOD1 mutants to their mature forms because they lack normal copper binding sites. SOD1 with mutations in a primary zinc binding residue (H80), either alone or in the context of a G93A mutation, belongs to metal binding region mutants [52]. H80G mutation compromises the normally compact structure of SOD1, and fundamentally alters certain biochemical parameters of SOD1. SOD1 variants harboring H80G mutation or H80G;G93A mutation lack dismutase activity, and are unable to form SOD1 homodimers, indicating that they cannot interact normally with CCS. The third group is truncation mutants, where 28 amino acids from the C-terminal end are missing [53–55]. In the spinal cords of end stage mutant L126Z SOD1 transgenic mice there are relatively high amounts of SOD1 positive aggregates, even if the mice express very low levels of the mutant L126Z SOD1 variant. The SOD1 truncation mutants lack the C146 required for formation of the disulfide bond and therefore CCS cannot interact normally with them [11]. Metal binding region and truncation mutants that exist as reduced monomers or oligomers are toxic at much lower concentrations than

the β-barrel mutants in SOD1 transgenic mice [55]. Even in the βbarrel mutants, a fraction of the disulfide bonds are unstable or remain reduced (Table 1) [11,22]. Wild type human SOD1 can also become misfolded and toxic, when it is oxidatively modified [3,56,57]. Mutations of SOD1 can disturb the compact SOD1 dimeric structure exposing free sulfhydryl groups, hydrophobic regions or ionic charges, which are prone to attach onto protein complexes and membranes in mitochondria as well as in other subcellular organelles [48,52]. SOD1 mutants representing β-barrel, metal binding region and truncation mutants differ in their stability, their copper-binding capacities and in their ability to interact with CCS. However, regardless of their copper binding abilities, all SOD1 mutants were found to lead to an age-dependent accumulation of copper in the spinal cords of transgenic mice. Intracellular copper dyshomeostasis might exacerbate oxidative stress, and interfere with several cellular pathways in these mice [58]. Wild type SOD1 is normally most abundant in cytosol but it is also widely distributed in several subcellular organelles including mitochondria [30], nucleus [59], and peroxisomes [59,60], but very little or none in endoplasmic reticulum [59]. The import of SOD1 into both mitochondrial IMS and peroxisomes has been shown to be dependent on CCS [60,61]. Over-expression of SOD1 in transgenic mice leads to its pathogenic accumulation in several subcellular organelles, including endoplasmic reticulum and Golgi, and even extracellularly [62]. Over-expression of mutant SOD1 exacerbates and accelerates the disease. It might also magnify certain aspects of the disease, because over-expression of SOD1 changes its subcellular distribution. The disease time course and severity generally correlate with the expression levels of the mutant protein. Several lines of G93A SOD1 have been created. The pathological changes of the lowest expressing G93ASOD1 line, which survives for more than 13 months, resemble closest to the changes seen in human patients [63]. The pathology is characterized by inclusions with only minimal mitochondrial vacuoles. In contrast, the distinctive pathological feature of highest expressing G93ASOD1 line, with lifespan of 3.5–4 months, is mitochondrial vacuolization [1]. In the moderate level expressing G93ASOD1 line, which lives for about 8 months, the disease features mostly inclusions with some mitochondrial vacuoles [10,64,65]. Several transgenic mouse lines with metal binding region or truncation mutations express the mutant proteins at low steady state levels, often equal or less than the endogenous SOD1 levels. In human patients the most unstable mutants somewhat correlate with the most rapid disease progression [66]. Co-expression of human WT SOD1 in transgenic mice has been found to aggravate the disease caused by unstable human SOD1 mutants, including A4V SOD1 that alone does not lead to

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ALS in transgenic mice but is very toxic in humans [67,68]. The reason might be that the mutant protein is stabilized via heterodimerization with human WT SOD1, and might be protected from degradation. Human SOD1 has cysteine at position 111, whereas mouse SOD1 has serine. Cysteine 111 is very vulnerable to oxidative damage, and it is thought to play a role in mitochondrial accumulation [14,15]. These findings indicate that even human WT SOD1 might contribute to the toxicity in mutant SOD1 patients, most of which are heterozygotes. 6. The effect of CCS on different groups of SOD1 mutants Deletion of CCS in G93A, G37R or G85R SOD1 transgenic mice does not have any effect on the disease course, even if it causes a large decrease in SOD1 enzymatic activity in cytosol. In G37R SOD1 transgenic mice the relative reduction of SOD1 activity is less in mitochondria than in cytosol [69]. This indicates that CCS independent pathways of SOD1 activation are more prominent in mitochondria than in cytosol. The enzymatic activity of G37R SOD1 in mitochondria might be partially due to mitochondrial protein sulfate isomerase that facilitates the formation disulfide bonds of its target proteins [47]. To elucidate further how CCS could influence mutant SOD1‐induced disease, transgenic mice expressing high level of WT human CCS were crossed with different SOD1 transgenic lines of mice harboring three kinds of human SOD1 mutations (β-barrel mutants G93A or G37R, truncation mutant L126Z, or metal binding region mutants G86R, H80G or H80G;G93A) or with a transgenic mice expressing WT human SOD1 (Table 1) [10,11,52]. Double transgenic mice expressing both CCS and G93A SOD1, or CCS and G37R SOD1 develop an accelerated neurological disease; they survive only about one month, whereas the parental G93A SOD1 and G37R SOD1 mice live for 8–9 months. Even in the end stage CCS/G93A SOD1 or CCS/G37R SOD1 mice do not exhibit any inclusionbased pathology. Instead, the disease is characterized by accelerated and augmented mitochondrial vacuolar pathology with selective COX deficiency [70]. Over-expression of CCS increases the levels of mutant SOD1 within the mitochondria of G93A SOD1 mice without altering total SOD1 levels [10]. CCS over-expression in G93A or G37R SOD1 mice also increases the amount of about 50 kDa SOD1 reactive species, which may be composed of misfolded, reduced SOD1 monomers [10,48]. In contrast to CCS/G93A SOD1 or CCS/G37R SOD1 mice, transgenic mice over-expressing both CCS and WT SOD1 have normal lifespan, do not manifest any abnormal neurological phenotype nor display mitochondrial vacuoles [10]. The reason might be that CCS can form heterodimers with WT SOD1 and isomerize C57–C146 disulfide bond within WT SOD1 more efficiently than within β-barrel SOD1 mutants, which have unstable regions specially in the metal binding area [46]. Overexpression of CCS increases the fraction of disulfide oxidized form of WT SOD1, but increases the fraction of reduced form in β-barrel mutants (Table 1). In the brain and spinal cord of WT SOD1 transgenic mice, CCS over‐expression results in a change in the SOD1 redox state, where virtually all of the WT SOD1 proteins are shifted to the oxidized form and only 1–4% remains reduced. In contrast, CCS over-expression in G93A SOD1 mice increases the proportion of the reduced form of G93A from 11% to 17–18% [11,22]. Thus, the interaction of CCS with these β-barrel SOD1 mutants might be partially non-productive in terms of insertion of copper and oxidation of the disulfide bond. Conversely, the formation of SOD1 C57–C146 disulfide bondis incomplete even in WT SOD1 in the presence of a specially generated mutant CCS, which cannot form CCS–SOD1 intermolecular disulfide bond, because the cysteine residues in CXC motif of domain 3 have been changed to serine [22]. The toxic effects of mutant SOD1 specially directed to mitochondria were studied by the generation of transgenic mice over-expressing SOD1 targeted to mitochondria, by using SOD1-mitofilin construct attached to a mouse prion promoter [71]. This mito-G93A SOD1 is anchored in inner membrane facing the IMS side and is therefore

exclusively localized in mitochondrial IMS. Mito-G93A SOD1 retains its enzymatic activity and leads to the same mitochondrial defects as regularly expressed in G93A SOD1 in transgenic mice, characterized by mitochondria cristae disorganization, swelling, and vacuolization. The mitochondria of mito-G93ASOD1 have impaired respiratory capacity, COX deficiency and defective calcium handling. Furthermore, mitoG93ASOD1 resulted in weight loss, motor dysfunction, motor neuron loss, muscle atrophy, in neural and muscle cells, but no muscle denervation [71]. Some of the pathological differences between G93ASOD1 and mito-G93ASOD1 are due to their expression levels in different tissues. G93A SOD1 is driven by a genomic promoter for human SOD1, which is ubiquitously expressed in almost all the tissues, whereas the expression of mito-G93A SOD1 is driven by a prion promoter, which is most active in the nervous system. G86R SOD1, H80G SOD1, H80G;G93A SOD1 and L126Z SOD1 exist almost 100% in the reduced form. CCS cannot normally interact with these SOD1 variants and does not have any effects on their redox state [11,52]. Therefore, these SOD1 mutants cannot take part in the oxidation–reduction reactions needed for their CCS mediated retention in IMS. CCS over expression has no impact on the pathology, disease course or survival of mice harboring G86R SOD1, H80G SOD1, H80G;G93A SOD1 or L126Z SOD1. Furthermore, CCS over expression does not affect the COX levels in these mutant SOD1 transgenic mice (Table 1) [11,52]. In mammalian cell cultures transfected both with CCS and with mutant SOD1, either β-barrel mutants or metal binding region mutants, CCS can prevent the formation of insoluble SOD1 aggregates, due to its chaperoning function [22,23]. This is in agreement with the findings that CCS/G93ASOD1 and CCS/G37R SOD1 transgenic mice do not display in their spinal cords SOD1 positive aggregates, characteristic to the end stage G93A and G37R SOD1 mice [10,11]. In animal models the accumulation of aggregates correlates with the neurodegeneration, being most visible in the end stages of the disease. CCS plays an important role in the localization of SOD1 in mitochondrial IMS. It is possible that CCS/ G93A SOD1 and CCS/G37R SOD1 mice succumb to the mitochondrial defects before they have accumulated detectable levels of SOD1 positive aggregates. It is also possible that in these mice mutant SOD1 forms soluble misfolded toxic conformations, which remained undetected. 7. The effects of SOD1 mutants on mitochondria SOD1 mutants interfere with various aspects of mitochondrial functions, including mitochondrial energy metabolism, transport, fission and fusion [72–76]. Mitochondrial accumulation of SOD1 mutants leads to defects in pores and channels, essential for the import and export of mitochondrial proteins, Ca++ and other ions [77–80]. Mutant SOD1 variants can incorporate into mitochondrial membranes altering their protein composition and/or damaging their structures [81–84]. SOD1 mutants also obstruct functions of several other mitochondrial proteins, including Bcl-2 where toxic BH3 domain is exposed [85], and an isoform of growth factor adapter Shc (p66Shc) which is normally involved in controlling mitochondrial redox state [86]. G93A SOD1 was shown to hinder the normal activity of cytochrome c, and lead to the production of toxic reactive oxygen species [47,87]. Structural properties of SOD1 mutants affect their sub-mitochondrial localization, possibly explaining the different consequences various SOD1 mutants have on mitochondrial pathology. G93A and G37R SOD1 localize mainly in IMS, but they associate also with the mitochondrial membranes in the brain and spinal cord of transgenic mice [31,72]. Both β-barrel mutant G93A SOD1 and metal-binding region mutant G85R SOD1 have been also found in the matrix [88]. A fraction of both β-barrel mutants, metal binding region mutants and truncation mutants are accumulating on the cytosolic side of the mitochondrial outer membrane in the spinal cords of transgenic mice, especially in the later stages of the disease [69,89]. Misfolded SOD1 mutants accumulate selectively into the outer membrane of the spinal

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cord mitochondria. This selectivity might be influenced by proteins expressed in the mitochondrial membranes as well as in the surrounding cytoplasm [89]. These proteins might not only be tissue specific but they might change during aging. This might partially explain why spinal cord motor neurons are particularly susceptible to the effects of ubiquitously expressed mutant SOD1, and why the disease manifests during later stages of life. In addition, neuronal cells, which are long lived, are vulnerable to misfolded SOD1 conformations, whereas rapidly dividing cells can dispose of them [32]. Attachment of misfolded SOD1 species to the mitochondrial membranes can hinder the transport of metabolites required for oxidative phosphorylation. The SOD1 aggregates might perturb the membrane permeability, cause leakiness of ions, and reduction of membrane potential obstructing the activity electron transport chain complexes [69,81]. They might also cause the generation of reactive oxygen or nitrogen species with damaging effects on respiratory chain complexes [90,91]. Localization of SOD1 in IMS is mostly dependent on its ability to undergo oxidation–reduction cycles and to productively cooperate with CCS. Therefore CCS can oxidatively fold and trap into IMS only WT SOD1 and β-barrel mutants, but not metal binding region or truncation mutants [11,39,52]. However, unstable, reduced forms of SOD1 mutants can be imported into IMS by CCS independent mechanism both in yeast and in mammalian cells [31,39]. The import of these SOD1 mutants is not physiologically regulated, and their accumulation in mitochondria is probably caused by their misfolding and aggregation [31]. Further evidence that the β-barrel and metal binding region SOD1 mutations have different effects on mitochondria, comes from recent studies of mutant SOD1 transgenic mice with reduced glutathione levels [92]. Knockout mice for the glutamatecysteineligase modifier subunit (GCLM-KO), which have a 70–80% reduction in total glutathione, were crossed with either G93A SOD1 mice, or with H46R/H48Q SOD1 mice. While GCLM-KO mice are viable and fertile, the life span of GCLM-KO/ G93A SOD1 mice is decreased 55% when compared to control mice. GCLM-KO/G93A SOD1 mice have increased oxidative stress, increased association of SOD1 with the mitochondria and aggravated mitochondrial pathology. GCLM-KO/G93A SOD1 mice have large reductions in COX-subunits levels. In contrast to the G93A SOD1 mice, reduced levels of glutathione do not decrease the lifespan of H46R/H48Q SOD1 mice. GCLM-KO/H46R/H48Q SOD1 mice display little or no mitochondrial pathology, and the levels of COX-subunits remain unchanged [92]. The mitochondrial respiratory chain defects in the central nervous system of familial ALS patients are difficult to assess because of long post-mortem intervals. Mitochondrial abnormalities and changes in mitochondria respiratory chain activities have been reported in the spinal cord and muscle biopsies of ALS patients. A patient with a heterozygous Q22R SOD1 mutation revealed COX deficiency in muscle fibers, indicating that low levels of SOD1 mutants are enough to contribute to mitochondrial toxicity in humans [93]. The activities of respiratory chain complexes, especially COX, are decreased in the brain and spinal cord of G93A SOD1 transgenic mice [72,94,95]. The defects are evident already at the onset of the disease indicating that they not only are the consequence of overall mitochondrial failure. Several different mechanisms might lead to respiratory chain defects. SOD1 mutants may form aggregates and cause a shift in the mitochondrial redox state making it more oxidizing and resulting in impairment of respiratory complexes [73]. Inner membranes damaged with mutant SOD1 structures could interfere with the assembly and/or function of electron transport chain complexes [96]. Mutant G93A SOD1 impairs the association of cytochrome c with mitochondrial inner membrane, which could contribute to COX defects [72]. The studies involving CCS over expression in the G93A or G37R SOD1 transgenic mice indicate to specific pathways contributing to SOD1 mitochondrial toxicity in these lines of mice. In these dual transgenic mice despite marked mitochondrial structural disruptions, deficiencies in electron transport chain complexes are limited to COX,

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while the levels of Complexes I, II, III and V are unaffected [70]. In contrast COX deficiency is not observed in mice expressing G86R SOD1, L126Z SOD1 or H80G;G93A SOD1 alone or in the context of CCS overexpression (Table 1) [11,52]. COX deficiency might be caused by pathological changes in redox and/or copper homeostasis in IMS. Cysteine rich, redox sensitive proteins involved in the import of proteins and metals to IMS and redox sensitive electron transport proteins might be especially affected by these changes. COX is the last complex in the electron transport chain, and it is also connected to disulfide relay system via cytochrome c [33]. COX is composed of several subunits and requires copper for enzymatic activity. The source of copper to COX is a pool of nonproteinaceous ligand-bound copper in the matrix [97]. The biogenesis of COX requires several accessory proteins containing cysteine motifs involved in transfer of copper to COX [98,99]. Copper is transferred by passing it from one copper binding protein to another, utilizing gradients of increasing copper-binding affinity [100]. It is probable that an abnormal redox environment, caused by excess of aberrantly folded mutant SOD1 and CCS in IMS, could lead to COX deficiency by interfering with COX subunits or accessory proteins, by altering their redox state and affecting their functions. In human patients, mutations in COX accessory proteins lead to a selective COX deficiency similar to that seen in CCS/G93A SOD1 and CCS/G37R SOD1 mice [101]. Inability of COX accessory proteins to take part in copper transport and other mitochondrial functions could ultimately have the same effects as mutations in those proteins.

8. Conclusion The basis for the differential pathology observed among the various mutant SOD1 transgenic mice lines could be explained by the biochemical differences in mutant SOD1 proteins, their stability and steady state levels, and their distribution in various cellular compartments. In transgenic mice over-expression of mutant SOD1 may aggravate and accelerate the disease, and it might also change some of the pathological characteristics of the disease. However, transgenic mouse models help to study the various molecular aspects responsible for the pathogenic changes in SOD1-related ALS. Mitochondrial dysfunction plays a critical role in the pathogenesis of mutant SOD1 mediated ALS. Mitochondria have shown to be a target in ALS pathogenesis and also contribute to the disease progression [74]. SOD1 mutants differ in copper or zinc binding, redox chemistry, interactions with CCS, disulfide bond formation, stability and propensity to misfold and aggregate. Only wild type SOD1 and β-barrel mutants can be trapped into the mitochondrial IMS by CCS mediated oxidative folding mechanism. Reduced SOD1 mutants and even aberrant reduced forms of wild type SOD1 can enter into the mitochondria by CCS independent pathways [39]. In mitochondria misfolded and aggregating SOD1 variants may lead to the production of high levels of superoxide and other toxic reactive oxygen species, overwhelming the mitochondrial oxidative defense system and ultimately leading to mitochondrial swelling [32,47]. The most characteristic pathological changes in SOD1-related ALS include formation of toxic fibrils and aggregates. Misfolded SOD1 conformations might interfere with subcellular structures, have abnormal copper and/or zinc binding properties, and contribute to oxidative stress [58]. The propensity of various SOD1 variants to unfold and form aberrant conformations correlates with their toxicity. The SOD1 positive multimeric fibrils and aggregates might have different conformations depending on the structure of the SOD1 variant [102]. Depending on structural properties, subcellular accumulations, CCS and other modifiers of various SOD1 mutations, the pathological consequences may be quite different [11,52,103]. Therefore, specific therapeutic interventions may need to be targeted for the particular type of SOD1 mutation.

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M. Son, J.L. Elliott / Journal of the Neurological Sciences 336 (2014) 1–7

Conflict of interest disclosure The authors do not have any conflict of interest to disclose. ARRIVE guidelines have been followed. Acknowledgments This work was supported by grants from the NINDS (R01 NS055315) and the Muscular Dystrophy Association (MDA) to Dr. Jeffrey L. Elliott. References [1] Dal Canto MC, Gurney ME. Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu,Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS). Brain Res 1995;676:25–40 [Pubmed: 7796176]. [2] Reaume AG, Elliott JL, Hoffman EK, Kowall NW, Ferrante RJ, Siwek DF, et al. Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat Genet 1996;13:43–7 [Pubmed: 8673102]. [3] Graffmo KS, Forsberg K, Bergh J, Birve A, Zetterstrom P, Andersen PM, et al. 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Mitochondrial defects in transgenic mice expressing Cu,Zn superoxide dismutase mutations: the role of copper chaperone for SOD1.

Several hypotheses have been proposed for the mechanisms underlying mutant Cu,Zn Superoxide Dismutase-related Amyotrophic Lateral Sclerosis. These inc...
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