A Proposed Mechanism for Neurodegeneration in Movement Disorders Characterized by Metal Dyshomeostasis and Oxidative Stress

Please cite this article in press as: Trist et al., A Proposed Mechanism for Neurodegeneration in Movement Disorders Characterized by Metal Dyshomeostasis and Oxidative Stress, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.004

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Perspective A Proposed Mechanism for Neurodegeneration in Movement Disorders Characterized by Metal Dyshomeostasis and Oxidative Stress Benjamin Guy Trist,1 Dominic James Hare,2,3 and Kay Lorraine Double1,* 1Discipline of Biomedical Science and Brain and Mind Centre, Sydney Medical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2050, Australia 2The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia 3Department of Pathology, The University of Melbourne, Parkville, VIC 3052, Australia *Correspondence: [email protected] https://doi.org/10.1016/j.chembiol.2018.05.004

Shared molecular pathologies between distinct neurodegenerative disorders offer unique opportunities to identify common mechanisms of neuron death, and apply lessons learned from one disease to another. Neurotoxic superoxide dismutase 1 (SOD1) proteinopathy in SOD1-associated familial amyotrophic lateral sclerosis (fALS) is recapitulated in idiopathic Parkinson disease (PD), suggesting that these two phenotypically distinct disorders share an etiological pathway, and tractable therapeutic target(s). Despite 25 years of research, the molecular determinants underlying SOD1 misfolding and toxicity in fALS remain poorly understood. The absence of SOD1 mutations in PD highlights mounting evidence that SOD1 mutations are not the sole cause of SOD1 protein misfolding occasioning oligomerization and toxicity, reinforcing the importance of non-genetic factors, including protein metallation and post-translational modification in determining SOD1 stability and function. We propose that these non-genetic factors underlie the misfolding and dysfunction of SOD1 and other proteins in both PD and fALS, constituting a shared and tractable pathway to neurodegeneration. Introduction Abnormal accumulation and dysfunction of cellular proteins is a pathological feature common to many chronic neurodegenerative disorders. In each disorder, the substantial loss of neurons within specific cell populations is traditionally associated with the misfolding of specific disease-associated proteins, which are believed to impart toxicity through either a loss or gain of function. More recent research, however, demonstrates a substantial overlap between the pathological proteomes of several neurodegenerative disorders (Budini et al., 2014; Clinton et al., 2010; Hebron et al., 2014; Helferich et al., 2016; Ishizawa et al., 2003; Masliah et al., 2001; Rosen et al., 2010; Zhang et al., 2015), with dysfunction and accumulation of proteins traditionally associated with only one condition now being observed in respective degenerating neuronal populations across multiple disorders. These observations suggest similar atypical biomolecular events promote common pathways to cell loss within vulnerable cell populations in multiple neurodegenerative disorders, with protein misfolding representing a central element unifying these toxic pathways. Identifying means of preventing the misfolding of specific proteins within these shared neurodegenerative pathways is therefore currently of intense interest, as these may assist development of new therapies directly targeting neurotoxic mechanisms across multiple disorders. Although the aggregation process of a number of these proteins has been well characterized in vitro, and many contributing biochemical influences have been identified, we still do not fully understand the molecular factors that underlie or modulate their accumulation in human disease. Dyshomeostasis of transition metals, particularly iron (Li and Reichmann, 2016), copper

(Manto, 2014), and zinc (Szewczyk, 2013), as well as the increased generation of reactive oxygen species (Kim et al., 2015), are recognized hallmarks of multiple neurodegenerative diseases that promote protein misfolding and associated neurotoxicity. Here we propose a shared pathway to neurodegeneration in Parkinson disease (PD) and superoxide dismutase 1 (SOD1)-linked familial amyotrophic lateral sclerosis (fALS), involving overlapping biometal dyshomeostasis, oxidative stress, and protein misfolding and dysfunction. A Shared Proteinopathy in PD and SOD1-fALS Rigidity and tremor in PD are the result of gradual dopaminergic neuron loss in the substantia nigra pars compacta (SNc), whereas progressive motor dysfunction in ALS is a consequence of the comparatively rapid degeneration of upper and lower motor neurons within the brainstem, motor cortices, and spinal cord. While shared genetic susceptibilities for PD and ALS have been reported for proteins involved in proteasomal degradation, regulation of membrane potential, protein trafficking, and angiogenesis (Hermosura and Garruto, 2007; Majoor-Krakauer et al., 1994; Payami et al., 2003; Quadri et al., 2011; van Es et al., 2011), identification of a shared, fundamental molecular pathway common to vulnerable neurons in both disorders has, until recently, remained elusive. A recent promising development comes from the discovery of the abnormal accumulation of the primary antioxidant enzyme, SOD1, in degenerating regions of the postmortem idiopathic PD brain. This wild-type SOD1 proteinopathy bears strong similarities to mutant SOD1 proteinopathy in degenerating brain and spinal cord of SOD1-associated fALS patients (Box 1) (Trist et al., 2017, 2018). Cell Chemical Biology 25, July 19, 2018 ª 2018 Elsevier Ltd. 1

Please cite this article in press as: Trist et al., A Proposed Mechanism for Neurodegeneration in Movement Disorders Characterized by Metal Dyshomeostasis and Oxidative Stress, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.004

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Perspective Box 1. Commonalities in SOD1 Proteinopathy in PD and SOD1-fALS d d d d d

Significant SOD1 deposition is specific to degenerating regions, where it is associated with neuronal loss. SOD1 aggregates possess a non-amyloid macrostructure, and contain SOD1, copper chaperone for SOD1, and ubiquitin proteins, but not pan or phosphorylated TAR DNA binding protein 43 or a-synuclein. SOD1 puncta, as well as spherical and amorphous inclusions, are identifiable within and surrounding neurons in vulnerable regions using conformation-specific antibodies that detect misfolded protein (B8H10, EDI clonalities). Soluble SOD1 protein has a reduced net charge, associated with the exposure of hydrophobic residues within the SOD1 dimer interface, an increased propensity for aggregation (Chiti et al., 2003), and a reduction in bound copper (Roudeau et al., 2015). Soluble SOD1 protein extracted from human tissues exhibits a reduction in specific activity.

Lessons Learnt from SOD1 Misfolding in Familial ALS Research from SOD1-associated fALS suggests that misfolded, metal-deficient variants of this enzyme underlie neuron loss (Box 2) (Hilton et al., 2015; Roberts et al., 2014), with toxicity likely stemming from both a loss of antioxidant activity and an apparent-yet-not-characterized neurotoxic gain of function (Saccon et al., 2013). The SOD1 homodimer has four metal-binding sites, incorporating two zinc(II) and two copper(II) ions via specific histidine and aspartic acid residues (Perry et al., 2010). While copper is solely responsible for catalyzing superoxide dismutation, both copper and zinc are important for thermodynamic stabilization of the fully formed protein, with copper shown to provide the greatest kinetic stability (Lynch and Colon, 2006). Several dozen identified mutations in the SOD1 gene produce metal-deficient SOD1, while other pathological ‘‘wild-type-like’’ mutations (e.g., G37R, G93A, and A4V) yield protein that retains wild-type metal-binding capacity (Hilton et al., 2015). The presence of substantial pools of metal-deficient SOD1 in fALS murine models that incorporate wild-type-like SOD1 mutations (Roberts et al., 2014; Williams et al., 2016) suggests that genetics are not the sole cause of SOD1 metal deficiency. Dyshomeostasis of the supply-demand balance for copper and zinc in motor neurons (Williams et al., 2016), and atypical post-translational modifications to SOD1, especially oxidative modifications (Martins and English, 2014; Petrov et al., 2016; Redler et al., 2011), also appear to be important modifiers of SOD1 structure and function. Re-evaluating the Role of Mutations in SOD1 Misfolding The finding that misfolded wild-type SOD1 in PD recapitulates conformations of mutant SOD1 in SOD1-fALS implies that pathological structural conformations can occur independent of mutations to SOD1, with the same net result of a dysfunctional, aggregated protein marked for proteasomal degradation. Destabilization of SOD1 by fALS-linked mutations, but also by non-genetic factors including de-metallation or atypical post-translational oxidative modifications, result in structural instability of the same micro-environments within the protein, especially the electrostatic loop, dimer interface, and zinc-binding regions (Figure 1) (Molnar et al., 2009; Sangwan et al., 2017; Sirangelo and Iannuzzi, 2017). It is important to note that the specific residue(s) altered by mutations or non-genetic factors are not necessarily within these specific micro-environments, mutation of non-metal-binding residues (G85R, D124V, D125H, and S134N) known to reduce SOD1 metal-binding capacity (Tiwari and Hayward, 2005). Structural instability within these regions exposes previously concealed epitopes that promote self-as2 Cell Chemical Biology 25, July 19, 2018

sembly into oligomers, which are shown to impart cytotoxicity irrelevant of SOD1 gene status (Sangwan et al., 2017; Schmitt and Agar, 2017). While the same regions of the protein are destabilized following any of these perturbations, it is important to consider the relative magnitudes of these effects with respect to the stability and toxicity of SOD1 protein. Oxidative modification of even single residues within the dimer interface yields a profound destabilization of the SOD1 dimer or monomer, with multiple modifications producing an effect that is equal to or greater than the most drastic of SOD1 mutations (Petrov et al., 2016). Adequately metallated wild-type and mutant SOD1 do not oligomerize under physiological conditions, but will readily do so upon the loss of bound metals (Banci et al., 2008; Banci et al., 2007), especially under conditions of oxidative stress (Furukawa and O’Halloran, 2005; Rakhit et al., 2002). Further, the copper content, rather than the total concentration of mutant SOD1, has been closely linked to disease severity and neuronal loss in multiple fALS murine models incorporating SOD1 mutations relevant to the human condition (Hilton et al., 2017; Roberts et al., 2014; Williams et al., 2016). It is important to note that the zinc content of SOD1 is also linked to protein stability and toxicity in vitro, however to date there is no direct evidence of zinc-deficient SOD1 in fALS patients or in animal models. Together these data indicate that, while mutations no doubt compromise the stability of SOD1, mutations alone are insufficient to trigger misfolding occasioning oligomerization and toxicity. The motor neuron-specific degeneration in SOD1-associated fALS provides further argument to this effect; SOD1 misfolding and toxicity is restricted to upper and lower motor neurons despite the ubiquitous expression of mutant SOD1 protein throughout the CNS. A model of SOD1 toxicity that places a greater emphasis on the destabilizing effects of non-genetic factors also has the potential to explain the late-onset of fALS despite the presence of a lifelong genetic risk, where age-related oxidative damage of SOD1 is proposed to be a key element in initial misfolding events in later life (Collier et al., 2017; Petrov et al., 2016). Although the focus has traditionally been on the effects of SOD1 mutations on protein stability and toxicity, these data argue that more close attention should now also be directed toward the singular or combined contributions of non-genetic factors within vulnerable neuronal populations to SOD1 misfolding, which are more predictive of the capacity for SOD1 oligomerization and toxicity. The destabilizing effects of SOD1 mutations should not be discounted, however, as they represent important modifying influences on the dynamics of SOD1 misfolding and oligomerization

Please cite this article in press as: Trist et al., A Proposed Mechanism for Neurodegeneration in Movement Disorders Characterized by Metal Dyshomeostasis and Oxidative Stress, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.004

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Perspective Box 2. Summary of Metal-Deficient SOD1 in SOD1-fALS d d d

d d

Mutations to the SOD1 gene account for 20% of fALS cases (Zou et al., 2017), and result in catalytic dysfunction and/or structural defects in the protein, including loss of metal-binding capacity and misfolding (Shaw and Valentine, 2007). Mutant SOD1 manifests a simultaneous and potentially synergistic loss and gain of function, reducing antioxidant activity within host neurons while generating neurotoxic protein assemblages (Saccon et al., 2013). Adequately metallated wild-type and mutant SOD1 do not oligomerize under physiological conditions, but will readily do so upon the loss of bound metals (Banci et al., 2008; Banci et al., 2007), especially under conditions of oxidative stress (Furukawa and O’Halloran, 2005; Rakhit et al., 2002). Metal status, rather than protein expression, is closely associated with disease severity and motor neuron survival (Roberts et al., 2014). Reduced copper binding to SOD1 severely diminishes antioxidant activity and alters protein conformation (Pratt et al., 2014), and is associated with shorter disease duration and increased motor neuron loss (Roberts et al., 2014; Williams et al., 2016).

in individuals in which they are present. Specific SOD1 mutations accentuate or moderate key structural perturbations to the protein conferred by non-genetic influences, such as mismetallation (Banci et al., 2008) and atypical post-translational modification (Petrov et al., 2016). The range of mutation genotypes therefore contributes to differential oligomerization propensities, resulting in variable sub-phenotypes within the broader symptoms of SOD1-fALS. In support of this, a model that incorporates the variable loss of stability and aggregation rates of individual fALS-linked SOD1 mutations accounts for greater than twothirds of the large variability in SOD1-fALS patient survival times (Schmitt and Agar, 2017). In addition, newly synthesized mutant proteins may be more susceptible in their immature states to aggregation in the unique biochemical environment of vulnerable neurons, due to the presence of cellular stressors like localized acidity or oxidative stress (Crow et al., 1997; Sirangelo and Iannuzzi, 2017). The recent identification of SOD1 misfolding and dysfunction in degenerating PD brain regions in the absence of SOD1 mutations (Trist et al., 2018) provides a timely and fortuitous human model for investigating the influence of non-genetic factors in SOD1 destabilization. Given the demonstrated primary importance of metallation status and post-translational modification in determining SOD1 thermodynamic stability, we believe the documented coincidence of copper deficiency (Davies et al., 2014) and excessive oxidative stress arising from both increased neuronal iron levels (Ward et al., 2014) and dopamine autoxidation (Burbulla et al., 2017) in vulnerable neuronal populations in PD has the capacity to precipitate SOD1 misfolding, dysfunction, and deposition in this disorder. Coincident Oxidative Stress and Metal Dyshomeostasis Underlie SOD1 Dysfunction in PD Dopaminergic neurons in the healthy SNc naturally sustain a substantial oxidative load, reflecting the metabolic cost of maintaining a complex axonal network and large soma, compounded by autoxidation of dopamine to reactive quinones and the subsequent formation of the neuromelanin biopolymer (Blesa et al., 2015). This is exacerbated by an age-related decrease in generalized antioxidant function (Venkateshappa et al., 2012) and concomitant increase in redox-active iron, which, when improperly chaperoned, further promotes both dopamine autoxidation and production of reactive oxygen species via Fenton/Haber-Weiss chemistry (Hare and Double,

2016). The high basal oxidative level in this unique neuronal population as we age likely predisposes vulnerability to additional, disease-associated impairment of antioxidant function and increased redox activity that has been well documented in this region in PD. Dismutation of cytosolic superoxide produced primarily by mitochondria (Brand et al., 2004), cytosolic oxidases, and cellular reductants (Cross and Jones, 1991) is the sole known function of SOD1, and is a primary mechanism of mitigating cytoplasmic oxidative stress in SNc dopaminergic neurons. Maturation of monomeric apo-SOD1 to the catalytically active holo-SOD1 dimer results from zinc(II) binding, followed by insertion of the catalytic copper(II) cofactor into the monomeric protein by copper chaperone for SOD1, intramolecular disulfide linking, and dimerization (Figure 2A) (Kawamata and Manfredi, 2010). SOD1 in human cells exists as a combination of these differentially metallated states (Bartnikas and Gitlin, 2003; Petrovic et al., 1996), and although the exact proportions within neurons are unknown, approximately 35% is believed to exist in the apo form. Specific to dopaminergic neurons in the PD SNc, increased redox activity stimulates compensatory expression of wild-type apo-SOD1 protein levels (Trist et al., 2017), in a similar manner to that observed in the degenerating fALS spinal cord (Milani et al., 2011). Maturation to active holo-SOD1 is, however, likely hindered by a neuronal copper deficiency specific to this brain region in PD (Davies et al., 2014; Genoud et al., 2017), reducing the antioxidant buffering capacity of SNc dopaminergic neurons by depleting a key line of defense against oxidative stress. Without adequate neuronal copper there is likely to be an accumulation of a relatively stable copper-deficient wild-type SOD1 intermediate (Figure 2B), similar to that described for mutant SOD1 in vulnerable regions in SOD1-fALS (Roberts et al., 2014). This intermediate possesses increased protein flexibility (Pratt et al., 2014), enabling oxidative modification of vulnerable free cysteine, arginine, and lysine residues within the normally inaccessible dimer interface (Lys3, Cys6, Lys9, Thr54, Cys111, and Arg115) (Petrov et al., 2016); conserved cysteine residues where the metal-stabilizing disulfide bridge is formed between Cys57 and Cys146 (Choi et al., 2005), or a number of histidine residues involved in proper metal coordination (Rakhit et al., 2002) (Figure 1). Oxidative modifications of wild-type SOD1 have been demonstrated to have a thermodynamically destabilizing effect that is at least as great as that of any SOD1 mutation (Petrov et al., 2016; Vassall et al., Cell Chemical Biology 25, July 19, 2018 3

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Perspective Figure 1. Structural Stability of SOD1 Protein Is Mediated by Specific Amino Acid Residues Crystal structure of the fully metallated dimeric SOD1 enzyme (A), depicted in ribbon (right hand monomer) and stick (left hand monomer) notation. The distribution of amino acid residues which are prone to oxidation (pink) (B), and which create the greatest destabilizing effect when oxidized, closely aligns with the dimer interface (red) and metal binding (orange and light blue) regions of SOD1 protein (A).

translational modification of SOD1 protein, especially oxidation, is also required.

2006). These modifications have especially been shown to stimulate oligomerization and aggregation of copper- or zinc-deficient forms of the protein, as well as apo-SOD1 (Chandran et al., 2010; Martins and English, 2014; Petrov et al., 2016; Rakhit et al., 2002). This pathway to deposition as insoluble aggregates is avoided in both the healthy brain and in PD brain regions that exhibit no or negligible neuronal loss until late-stage disease (Double et al., 2010), as adequate neuronal copper (Davies et al., 2014; Genoud et al., 2017) permits normal maturation of wildtype apo-SOD1 to holo-SOD1, and excess apo-SOD1 protein is rapidly discarded via the 20S proteasome. The early-disease-stage copper deficiency present in the PD brain may thus represent a key contributing factor underlying SOD1 misfolding in early PD. Quantification of soluble apo-, metal-deficient, and holo-SOD1, and of the metal content of aggregated SOD1, in human tissue is necessary to validate this shared pathway of SOD1 dysfunction in idiopathic PD and ALS. Identifying specific conformational changes elicited by atypical post4 Cell Chemical Biology 25, July 19, 2018

Oxidative Stress, Metal Dyshomeostasis, and Protein Misfolding: A Toxic Trio in PD We speculate that this mechanism, together with lessons learned from studying SOD1 mutations in SOD1-fALS, may explain other features of the parkinsonian neurodegenerative cascade. Mitochondrial dysfunction results from an interaction between oxidized wild-type SOD1 and the apoptosis regulator Bcl-2 (Pasinelli et al., 2004). Protein clearance machinery, such as ubiquitin, strongly associated with SOD1 aggregates in both SOD1-fALS and PD (Trist et al., 2017), are influenced by cellular copper levels, identified from the shared pathologies of rare copper metabolic disorders (Greenough et al., 2016), as well as inflammatory responses to neurodegeneration. In addition to promoting oligomerization of a-synuclein via nitrative stress (Soon et al., 2011), dysfunctional and misfolded SOD1 has also been shown to directly interact with oligomerized a-synuclein to potentiate the accumulation of both proteins (Helferich et al., 2015; Koch et al., 2016). Together, misfolded wild-type SOD1 (Bosco et al., 2010) and pathological a-synuclein (Volpicelli-Daley et al., 2014) also have the capacity to underlie the impairment of axonal transport observed in dopaminergic neurons in the PD SNc (De Vos and Hafezparast, 2017). Combined, these factors promote a self-perpetuating cycle of neurotoxicity in specific neuronal populations where metal dyshomeostasis, oxidative stress, and SOD1 dysfunction overlap. Although this article focuses on SOD1-mediated neurodegeneration in PD, there are other proteins whose structure and function are similarly altered by oxidative stress and copper dyshomeostasis, and which are known to be misfolded and dysfunctional in vulnerable PD brain regions. A reduction in copper metallation and atypical oxidative modification of ceruloplasmin both result in structural change, loss of function, and rapid degradation of the protein, which are characteristic of degenerating PD brain regions (Hellman et al., 2002; Olivieri et al.,

Please cite this article in press as: Trist et al., A Proposed Mechanism for Neurodegeneration in Movement Disorders Characterized by Metal Dyshomeostasis and Oxidative Stress, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.05.004

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Perspective 2011), although the exact residues and modifications involved are yet to be investigated. Ceruloplasmin dysfunction in PD is particularly intriguing, as the cuproprotein represents a nexus between iron and copper metabolism. Extracellular copper deficiency in PD impairs ceruloplasmin-mediated iron export from neurons by depleting available copper ions necessary for ferroxidase activity, which is essential for chaperoning iron(II) as it emerges from ferroportin onto vacant iron(III) binding sites on interstitial apo-transferrin (Ayton et al., 2013). Similarly, oxidation of Cys residues concealed within the two RNA binding motifs of TDP-43 results in loss of function and deposition of the protein into insoluble inclusion bodies (Chang et al., 2013), which have recently been reported in degenerating regions in the idiopathic PD brain (Trist et al., 2017). TDP-43 metal binding has not been thoroughly investigated; however, TDP-43 accumulation is prevented upon copper supplementation (Lovejoy and Guillemin, 2014), suggesting that a reduction in copper levels may potentiate TDP-43 accumulation via a direct or an indirect mechanism. Lastly, excessive oxidation of Cys and Met residues in the PDassociated DJ-1 protein has been linked to a loss of antioxidant function and proteopathic accumulation in PD, and elucidating the precise role of DJ-1 in health and disease has become a hot issue in PD research over the past decade (Wilson, 2011). A role for copper in DJ-1 structure and function was recently proposed following the identification of two copper-binding sites (Girotto et al., 2014), with oxidation-prone Cys residues central to copper coordination shown to be important in aggregation and apparent DJ-1 dysfunction. Further characterization of the role of copper in DJ-1 structure and function is needed to confirm whether copper deficiency may yield conceivable detrimental consequences, including an increased solvent accessibility of normally protected Cys residues, a loss of copperdependent functions, and potential structural consequences of reduced copper binding at the homodimer interface. The importance of an identified role for copper in DJ-1 activity cannot be overstated; considering that the most conclusive evidence of a biological role for DJ-1 involves chaperoning a-synuclein to prevent oligomerization (Shendelman et al., 2004); dysfunctional protein may thus represent a point where synucleinopathy, metal metabolism, and antioxidant activity intersect. Taken together, atypical SOD1 activity is unlikely to act within a vacuum, and we posit that proteins including ceruloplasmin, TDP-43, and DJ-1, all of which have been documented as misfolded in vulnerable regions of the PD brain, constitute mediators of neuronal loss with a possible cumulative effect in overlapping conditions of copper deficiency and increased oxidative stress. While copper is a demonstrated stabilizing factor for the proteins discussed above, it is also important to consider a possible influence of copper binding to the PD hallmark protein, a-synuclein. Both copper binding and oxidation of a-synuclein are reported to destabilize the protein in vitro, resulting in misfolding that subsequently disrupts its clearance, alters its cellular localization and promotes accumulation (Burai et al., 2015; Carboni and Lingor, 2015). Levels of intraneuronal copper are, however, either unchanged, or markedly reduced, in PD brain regions where substantial a-synuclein deposition is observed, including the SNc, locus coeruleus (Davies et al., 2014), and fusiform cortex (Genoud et al., 2017). These ex vivo data appear inconsistent with the hypothesis that an increased interaction with copper

stimulates a-synuclein misfolding and deposition in these brain regions in PD. Alternatively, decreased function of protective cuproproteins, such as SOD1, in the PD brain (Trist et al., 2017) may result in increased oxidation of a-synuclein and thus contribute to oligomerization in brain regions where oxidative stress becomes rampant. Despite substantial gaps in knowledge, it is evident that overlapping conditions of oxidative stress and copper deficiency adversely affect the structure and function of a range of cellular proteins, including SOD1, ceruloplasmin, TDP-43, and DJ-1. We propose that the resultant functional impairment and toxic gain of function of these proteins has the capacity to partially underlie pathological alterations to mitochondrial function, axonal transport, protein synthesis and degradation, nucleic acid processing and repair, and antioxidant defense observed in degenerating dopaminergic neurons. We strongly believe that future research should also be directed at determining the effect of these overlapping biochemical changes on the structure and function of other proteins implicated in PD pathogenesis, including Parkin, PINK1, LRRK2, and UCH-L1, which have currently not been investigated within this context. One Pathway Resulting in Divergent Phenotypes? Biochemical similarities between vulnerable neuronal populations in SOD1-associated fALS and PD suggest the above cascade has the potential to underlie the topographic pattern of neuronal loss in both disorders. Like nigral dopaminergic neurons, motor neurons are susceptible to oxidative stress during healthy aging due to a high metabolic output (Barber and Shaw, 2010), as well as age-related increases in reactive oxygen species. Further, while the endogenous copper status of human motor neurons in ALS has thus far not been reported, a supplydemand imbalance in SOD1 copper loading results in motor neuron toxicity in multiple animal models of SOD1-fALS (Williams et al., 2016). Finally, misfolded SOD1 in the PD brain identified using antibodies raised against fALS-associated variants (Trist et al., 2017; Trist et al., 2018) indicates shared misfolded conformations of wild-type protein and mutant SOD1, strengthening the growing body of research demonstrating that atypical wild-type SOD1 may recapitulate toxic conformations of mutant SOD1 (Bosco et al., 2010; Guareschi et al., 2012; Sangwan et al., 2017; Schmitt and Agar, 2017). Therefore it is plausible that the microchemical environment of stressed neurons may result in modification of multiple oxidation-prone amino acid residues within wild-type SOD1, resulting in destabilization and eventual oligomerization. It is also possible that other cuproproteins previously discussed share misfolded conformations in vulnerable regions of both disorders: ceruloplasmin (Conti et al., 2008; Manto, 2014), DJ-1 (Choi et al., 2006; Lev et al., 2009), and TDP-43 (Budini et al., 2014; Trist et al., 2017) have all been shown to exhibit misfolding or dysfunction in the ALS spinal cord, as well as the PD SNc. Similar disease-related biochemical changes within vulnerable neuronal populations in both disorders may therefore promote the evolution of a shared detrimental biomolecular cascade involving the misfolding and dysfunction of SOD1 and other cuproproteins. The manifestation of a shared neurodegenerative pathway as multiple distinct phenotypes can therefore be attributed to the degeneration of specific and distinct neuronal populations in each disease, which results in Cell Chemical Biology 25, July 19, 2018 5

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Perspective

Figure 2. Normal and Pathological Biosynthesis of the SOD1 Enzyme (A) At base levels of oxidative stress within a neuron, continuous transcription of the sod1 gene creates a cycling pool of monomeric, metal-free (apo-mSOD1) protein, with unused enzyme metabolized by the 20S proteasome. When oxidative stress increases as a result of increased metabolic activity, cellular redox sensors induce additional protein transcription, stimulate loading of zinc(II) ions by way of an as-yet unknown mechanism, and initiate copper(II) loading and dimerization mediated by copper chaperone for SOD1 (CCS). During this process, formation of an intramolecular disulfide bridge between Cys57 and Cys146 encapsulates the copper ion and stabilizes the monomer, which dimerizes through hydrophobic interactions between Cys6, Cys111, and loops IV and IIV. The solvent-inaccessible space between the monomers forms an active dimer with high stability, which carries out its biological function and returns superoxide levels to baseline. (B) In conditions of elevated oxidative stress and copper dyshomeostasis, as experienced by dopaminergic neurons in PD and motor neurons in ALS, the ability of SOD1 to dimerize and perform its antioxidant role is impaired. A systemic decrease in bioavailable copper limits the maturation of SOD1, resulting in the accumulation of copper-deficient zinc-containing protein, which is notably more stable than apo-SOD1. The lack of antioxidant activity of this intermediate species leaves increasing levels of free radicals and reactive oxygen species unchecked. Without copper, SOD1 protein also possesses increased flexibility, exposing regions of the protein that are normally buried and solvent inaccessible to the harsh oxidative environment of degenerating regions. Among others, the four oxidation-prone cysteine residues in each monomer are likely exposed in this conformation, with thiols in Cys6 and Cys111 particularly prone to oxidative (legend continued on next page)

6 Cell Chemical Biology 25, July 19, 2018

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Perspective differing disease onsets, symptoms, and durations, depending on the cell populations most affected. Future Outlook The majority of therapies aimed at modulating protein misfolding and aggregation in neurodegenerative disease have traditionally met with limited success, fueling the century-old debate about whether these events represent a causality or consequence of neurodegeneration. The recent shift toward recognition that end-stage protein deposits likely represent relatively benign species reflects an important progression in our understanding, as it directs focus toward earlier events underlying the misfolding of individual protein units prior to self-assembly. A number of strategies are now available that aim to reduce individual misfolded SOD1 units in the hope of ameliorating toxicity. Pyrimethamine and ISIS 333611 are two promising therapies that reduce wildtype or mutant SOD1 protein, eliminating the possibility of a neurotoxic SOD1 gain of function. While ISIS 333611 successfully cleared phase I clinical trials in 2012 (Miller et al., 2013) and is now in the process of entering into phase I/II trials in patients with ALS, pyrimethamine has recently successfully cleared phase I/II trials in SOD1-fALS patients, and is shown to successfully reduce SOD1 protein levels in patient CSF (Lange et al., 2017). Another compound, Arimoclomol, has progressed to phase II/III clinical trials in SOD1-fALS patients (ClinicalTrials. gov Identifier: NCT00706147), following confirmation of its safety, tolerability, and ability to cross the blood-brain barrier (Lanka et al., 2009). This compound amplifies heat-shock protein gene expression, with the aim of bolstering heat-shock proteindependent proteostasis pathways that play a role in regulating SOD1 misfolding and protein-protein interactions. While these therapies show promise in ameliorating the ALS phenotype, it must be noted that they all involve a reduction in, or removal of, SOD1 protein, which has been shown to result in, at best, a greater susceptibility to progressive cellular damage and deficits in response to injury or toxic stimuli (Saccon et al., 2013). While this appears more preferential to the aggressive ALS phenotype, it does highlight the physiological cost of SOD1 protein removal on the long-term resilience of neurons, especially in the context of aging and the appearance of other age-related biochemical changes. Based on these considerations we conclude that an ideal therapeutic strategy targeting misfolded SOD1 protein is one that avoids the reduction or removal of SOD1 protein, but instead stabilizes the protein and supports catalytic activity. Abundant data implicating metal-deficient SOD1 in SOD1fALS has led to the development of treatments targeting SOD1 metal status. Diacetylbis(N(4)-methylthiosemicarbazonato) copper(II), or CuII(atsm), is one such treatment which specifically delivers copper(II) to regions experiencing oxidative stress, hypoxia, and mitochondrial electron transport chain impairment (Donnelly et al., 2012; Hilton et al., 2017). Treatment with CuII(atsm) reduces motor neuron loss and increases survival in

multiple SOD1-fALS murine models (Hilton et al., 2017; Roberts et al., 2014; Williams et al., 2016), shown to be tightly linked to improved SOD1 copper loading, prompting human clinical trials in both SOD1-fALS and sALS (ClinicalTrials.gov Identifier: NCT02870634). The reduction in spinal cord TDP-43 accumulation in CuII(atsm)-treated mice further indicates that a restored stability and function of other copper-dependent proteins may also contribute to the efficacy of this compound in SOD1-fALS. Aside from the ability of this compound to slow neurodegeneration in these models, CuII(atsm) administration in the symptomatic disease phase was able to rescue the ALS phenotype, indicating that copper supplementation is able to halt the disease process. Upon administration to four separate animal models of PD, CuII(atsm) treatment similarly rescued neuronal loss and the parkinsonian phenotype, and prolonged survival time (Hung et al., 2012), prompting the initiation of a phase I clinical trial of CuII(atsm) in PD (ClinicalTrials.gov Identifier: NCT03204929). We propose that the efficacy of this treatment in PD models is similarly attributed to the improved stability and restored function of SOD1, and potentially of other copper-dependent proteins including ceruloplasmin, DJ-1, and TDP-43. Characterization of the structure and function of these proteins before and after CuII(atsm) treatment in these PD murine models is warranted, as well as developing means to identify preclinical PD patients who would most benefit from early intervention prior to neuron loss; it should not be ignored that within 1 year of clinical diagnosis up to 90% of dopaminergic neurons have been lost (Kordower et al., 2013), and attempted restoration of SOD1 activity in such cases would clearly be futile. These data suggest that normalization of neuronal copper levels could provide substantial neuroprotection in both PD and fALS, and that this may be linked, at least in part, to the stabilization and function of key disease-associated cuproproteins, including SOD1. ACKNOWLEDGMENTS This work was supported by funds from Parkinson’s NSW, Australia, and the University of Sydney (Discipline of Biomedical Science). D.J.H. is funded by the National Health and Medical Research Council (1122981) in partnership with Agilent Technologies, who provide materials and research support. AUTHOR CONTRIBUTIONS Conceptualization, B.G.T., D.J.H., and K.L.D.; Writing – Original Draft, B.G.T.; Writing – Review & Editing, B.G.T., D.J.H., and K.L.D.; Visualization, B.G.T. and D.J.H.; Supervision, D.J.H. and K.L.D.; Project Administration, D.J.H. and K.L.D.; Funding Acquisition, D.J.H. and K.L.D. REFERENCES Ayton, S., Lei, P., Duce, J.A., Wong, B.X., Sedjahtera, A., Adlard, P.A., Bush, A.I., and Finkelstein, D.I. (2013). Ceruloplasmin dysfunction and therapeutic potential for Parkinson disease. Ann. Neurol. 73, 554–559.

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