Benjamin G Trist

Sian Genoud

Stéphane Roudeau

Alexander Rookyard

Amr Abdeen

Veronica Cottam

Dominic J Hare

Melanie White

Jens Altvater

Jennifer A Fifita

Alison Hogan

Natalie Grima

Ian P Blair

Kai Kysenius

Peter J Crouch

Asuncion Carmona

Yann Rufin

Stéphane Claverol

Stijn Van Malderen

Gerald Falkenberg

David J Paterson

Bradley Smith

Claire Troakes

Caroline Vance

Christopher E Shaw

Safa Al- Sarraj

Stuart Cordwell

Glenda Halliday

Richard Ortega

Kay L Double
email: kay.double@sydney.edu.au

Altered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord

Keywords: superoxide dismutase-1, neurodegeneration, mislocalization, amyotrophic lateral sclerosis, post-translational modifications

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Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease characterized by the progressive loss of motor neurons in the cortex, brain stem and ventral horns of the spinal cord. Ten percent of ALS cases are hereditary (familial; fALS), while the remaining ~90% lack an overt familial history, referred to as sporadic (s)ALS. [START_REF] Mejzini | ALS Genetics, Mechanisms, and Therapeutics: Where Are We Now?[END_REF] The gene encoding superoxide dismutase 1 (SOD1) was the first dominantly-inherited genetic risk factor identified for ALS. [START_REF] Rosen | Mutations in Cu/Zn Superoxide-dismutase gene are associated with familial amyotrophic lateral sclerosis[END_REF] and over 200 SOD1 mutations have since been documented in fALS patients (http://alsod.iop.kcl.ac.uk). These mutations disrupt key structural or functional motifs within SOD1 protein, resulting in structurally-disordered mutant proteins that are strongly implicated in motor neuron death. [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF] A number of studies have also identified structurally-disordered wild-type SOD1 proteinopathy in the spinal cord of non-SOD1-linked fALS and sALS patients. [START_REF] Guareschi | An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1[END_REF][START_REF] Tokuda | Wild-type Cu/Znsuperoxide dismutase is misfolded in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Bosco | Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS[END_REF][START_REF] Rotunno | An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis[END_REF][START_REF] Maier | A human-derived antibody targets misfolded SOD1 and ameliorates motor symptoms in mouse models of amyotrophic lateral sclerosis[END_REF][START_REF] Pare | Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis[END_REF][START_REF] Forsberg | Misfolded SOD1 inclusions in patients with mutations in C9orf72 and other ALS/FTD-associated genes[END_REF][START_REF] Forsberg | Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients[END_REF] This finding, however, is not consistently reported, [START_REF] Cruz | Misfolded SOD1 is not a primary component of sporadic ALS[END_REF][START_REF] Kerman | Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form[END_REF][14] making it unclear whether wild-type SOD1 contributes to motor neuron death in ALS patients lacking SOD1 mutations.

Pathways hypothesized to underlie the self-assembly and toxicity of both wild-type and mutant SOD1 protein have been identified in silico and using in vitro systems, and in cellular and animal models of ALS. [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF] Evidence of these pathways in ALS patients in vivo or post-mortem, however, is scarce. This data is vital to ascertain whether SOD1 proteinopathy may indeed contribute to motor neuron death in familial and sporadic ALS patients through similar mechanisms to those identified in well-characterized disease models. Such knowledge may reveal productive treatment avenues, and improve the development and choice of preclinical models for therapeutic development, factors which are proposed to underlie recurrent failures to translate promising interventions into the clinic. [START_REF] Van Der Worp | Can animal models of disease reliably inform human studies?[END_REF] Data from transgenic mouse models, cultured cells and in vitro proteomic assays demonstrate SOD1 mutations disrupt copper and zinc binding, intramolecular disulfide bond formation and protein dimerization (recently reviewed) [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF] . These perturbations promote the accumulation of immature structurally-disordered SOD1 conformers, which elicit mitochondrial dysfunction, [START_REF] Pickles | Mitochondrial damage revealed by immunoselection for ALS-linked misfolded SOD1[END_REF] bioenergetic failure, [START_REF] Richardson | The effect of SOD1 mutation on cellular bioenergetic profile and viability in response to oxidative stress and influence of mutation-type[END_REF] reactive oxygen species accumulation [START_REF] Barber | Oxidative stress in ALS: key role in motor neuron injury and therapeutic target[END_REF] and ultimately neurodegeneration in preclinical models. Non-genetic factors can also provoke structural and/or functional alterations to wild-type SOD1 protein analogous to those associated with SOD1 mutations. These include post-translational modifications (PTM) to key amino acid residue side chains, as well as disruptions to molecular chaperones involved in SOD1 protein maturation. These alterations confer toxic properties to wild-type SOD1 mirroring those of some mutant proteins in vitro, [START_REF] Guareschi | An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1[END_REF] implying convergent molecular pathways resulting in SOD1 proteinopathy, irrelevant of SOD1 mutation status. [START_REF] Trist | A Proposed Mechanism for Neurodegeneration in Movement Disorders Characterized by Metal Dyshomeostasis and Oxidative Stress[END_REF] Despite this abundance of data from preclinical models, it is unclear whether abnormal SOD1 proteinopathy is unique to SOD1-fALS patients, or constitutes a shared feature amongst all forms of ALS.

In this study, we characterized SOD1 proteinopathy in post-mortem spinal cord tissues from familial and sporadic ALS patients, and profiled soluble and aggregated SOD1 protein biochemistry within these same tissues for evidence of non-genetic alterations to SOD1 that may underlie any proteinopathy. We show that structurally-disordered, immature SOD1 conformers becomes mislocalized from the ER-golgi network and nucleus to the cytoplasm in SOD1-linked (SOD1-)fALS, non-SOD1-linked (non-SOD1-)fALS and sALS spinal cord motor neurons, where they accumulate and often form large inclusions. These findings were associated with the demetallation and destabilization of enzymatically-active mature SOD1 protein, as well as alterations to SOD1 PTMs and molecular chaperones governing SOD1 maturation.

Heterogeneity in the presence of these alterations between ALS cases reinforces the likelihood that SOD1 proteinopathy arises from multiple biochemical pathways in ALS patients. Our data suggest some of these alterations may contribute to motor neuron death in ALS patients by promoting neurotoxic SOD1 proteinopathy, implying these pathways may constitute valid targets for therapeutic development.

Materials and methods

Human post-mortem tissues

Formalin-fixed and fresh frozen human post-mortem brain and spinal cord tissues from patients with SOD1-associated familial amyotrophic lateral sclerosis (SOD1-fALS; n = 3), non-SOD1associated fALS (non-SOD1-fALS; n = 4), sporadic ALS (sALS; n = 9) and age-matched controls (n = 10) were obtained from the MRC London Neurodegenerative Diseases Brain Bank biorepository of the NIH NeuroBioBank (Maryland, USA). Diagnoses of ALS were determined clinically using patient histories received from the donors' physicians. Pathological identification of motor neuron loss in the anterior horn of the spinal cord by brain bank neuropathologists confirmed clinical findings. All ALS cases were free of other neurological or neuropathological conditions. Genotyping confirmed SOD1 and C9ORF72 mutation status of fALS cases, as described below. Age-matched control cases were free of any clinically diagnosed neurological disorders and neuropathological abnormalities. Ethics approval was obtained from the University of Sydney Human Research Ethics Committee (approval number 2019/309). Demographic and clinical information for all cases are detailed in Supplementary Table 1. Diagnostic groups were matched for age and post-mortem interval (Kruskal-Wallis test, p > 0.05; Supplementary Table 2). A principal component analysis of all data collected in this study revealed neither of these factors significantly impacted variables of interest in the ventral spinal cord (Supplementary Figure 1, further details in statistical analyses). This analysis also found no impact of sex on measured variables, despite differences in the ratio of males to females between diagnostic groups (Supplementary Figure 2).

Fixed and fresh frozen tissues from the cervical and thoracic spinal cord represented regions of severe motor neuron degeneration in ALS, samples of occipital cortex represented a nondegenerating control region external to the spinal cord. Tissue availability varied between cases (Supplementary Table 1). Formalin-fixed tissues were embedded in paraffin and 7µm sections prepared for immunohistochemical analyses. Fresh frozen tissues were trimmed to remove most of the white matter. Fresh frozen spinal cord tissues were bisected into dorsal and ventral regions representing degenerating and non-degenerating spinal cord tissues in ALS respectively.

Paraffin-embedded formalin-fixed and fresh frozen tissue samples were randomly numbered by a secondary investigator (B.G.T. or S.G.) prior to experimentation to blind primary investigators (B.G.T., S.G., S.R., A.R., or J.A.F) to case diagnoses.

SOD1 and C9ORF72 genotyping

SOD1 and C9ORF72 genotyping was performed in ALS and control cases as previously described. [START_REF] Mccann | The genotype-phenotype landscape of familial amyotrophic lateral sclerosis in Australia[END_REF] DNA was extracted from fresh frozen human brain tissue from the occipital cortex A C C E P T E D M A N U S C R I P T using the DNeasy DNA extraction kit (Cat. #69506; Qiagen, Hilden, Germany), according to manufacturer's instructions. All five exons of SOD1, and at least 10bp of flanking sequence were sequenced using PCR amplification and Sanger sequencing. The repeat primed PCR method was used to amplify the C9orf72 hexanucleotide repeat sequence and was analysed by fragment analysis. [START_REF] Mccann | The genotype-phenotype landscape of familial amyotrophic lateral sclerosis in Australia[END_REF] All results were independently analysed by two team members. Positive samples were repeated twice to confirm results.

Immunohistochemistry

Fixed tissue sections used for 3,3′-Diaminobenzidine (DAB) staining and brightfield microscopy were deparaffinated and antigen retrieval performed in citrate buffer (pH 6; Fronine, Australia) at 95°C for 30 min. Sections were then washed with 50% ethanol, incubated with 3% H 2 O 2 (Fronine, Australia) in 50% ethanol to quench non-specific peroxidase activity, and blocked (0.5% casein, 1% bovine serum albumin in PBS) for 1.5 h at room temperature. Sections were then incubated overnight with appropriate primary antibodies diluted in blocking solution (Supplementary Table 3). Primary antibodies were detected using biotinylated IgG secondary antibodies (2 h, room temperature, 1:200 diluted in blocking solution) (Vector Laboratories, Burlingame, CA) followed by Vector Elite Kit tertiary antibody complex (2 h, room temperature, 1:100 diluted in PBS) (Vector Laboratories, Burlingame, CA), and visualised using a DAB (Sigma-Aldrich, St. Louis, MO, USA) solution containing cobalt and nickel ions, changing the chromogen colour from brown to black to enable distinction of DAB staining from pigmented neurons in the SNc. Sections were finally dehydrated and mounted with DPX, visualized and imaged using an Olympus VS 120 Slide Scanner at 40x magnification (Olympus-life science, Shinjuku, Tokyo, Japan), and images processed using OlyVIA (Version 3.1; Olympus-life science) or Fiji (National Institute of Health (NIH)) software.

A lack of consistency between immunostaining protocols investigating mammalian SOD1 misfolding may underlie variability in misfolded SOD1 detection within ALS post-mortem tissues and transgenic SOD1 mice. [START_REF] Pare | Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis[END_REF] Accordingly, our DAB immunostaining protocol for misfolded SOD1 protein adhered to guidelines proposed by Pare and colleagues [START_REF] Pare | Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis[END_REF] , except for performing Hematoxylin/Eosin staining on adjacent serial tissue sections, which could not be performed due to limited tissue availability. A no primary negative control (processed as above in the absence of primary antibodies) was also included for each diagnostic group to identify protocol-and tissue-related artefacts independent of primary antibody immunoreactivity.

Multiplexed immunofluorescence

Fixed tissue sections used for fluorescent staining and confocal microscopy were deparaffinated, antigen retrieved, quenched, blocked and incubated with appropriate primary antibodies (Supplementary Table 3) as above. Multiplexed fluorescent immunolabelling was then performed using OPAL fluorophores (Akoya Biosciences, Menlo Park, CA, USA) according to the manufacturer's instructions. Antibody stripping between OPAL fluorophore incubations was performed at 95°C for 40 min using AR6 commercial buffer (Akoya Biosciences, USA).

Following incubation with the final fluorophore sections were washed with PBS and coverslipped with 80% glycerol. Images were collected at 60x magnification using a Nikon C2+ Confocal Microscope System (Nikon, Minato City, Tokyo, Japan) and Nikon NIS-elements software (Version 5.20.02; Nikon, Japan), and were viewed and analysed using Fiji software (National Institute of Health, Bethesda, Maryland, USA).

As above, a no primary negative control was included for each diagnostic group to identify nonspecific fluorescent signals originating from autofluorescent tissue features (e.g. lipofuscin) or direct binding of secondary antibodies to tissues (Supplementary Figure 3). During acquisition of double-or triple-immunolabelled fluorescent images a sequential protocol was used where each fluorophore was excited and the corresponding channel was individually acquired, before moving onto the next fluorophore and channel, to prevent any spectral cross-contamination between channels. Negligible spectral overlap was identified between OPAL650, OPAL620 and OPAL520 fluorophores using this protocol (Supplementary Figure 3), indicating an absence of fluorophore cross-contamination across channels.

Neuronal quantification

Spinal cord motor neurons were quantified in DAB-immunostained serial 7µm fixed tissues sections, whereby cells immunopositive for neuron-specific class III β-tubulin (TUJ-1) in ventral horn grey matter were counted in every fifth tissue section for a total of 4 sections. Quantification was completed by a single investigator (BT) blinded to sample diagnosis. Grey matter area (mm 2 ) was recorded in Fiji software to enable conversion of neuron counts to neuronal densities (counts per mm 2 ). Due to the absence of neuronal cell bodies in dorsal horn grey matter of the spinal cord, the average area (mm 2 ) of grey matter in this region was used to approximate alterations to neuronal tract density. An index of neuronal loss was calculated for each investigated region of each case by normalizing neuronal density for each region/case to the mean neuronal density of the same investigated region for the healthy control group; Sample Index = 1 -(sample neuronal density / healthy control mean neuronal density for sample region).

Higher index values are associated with lower neuronal densities or greater neuron death. This index was used to explore correlations between neuron loss and quantified pathological features within investigated regions.

Quantification of pathological features of interest

Immunostaining for SOD1 and CCS was performed in ventral horn grey matter of the cervical and thoracic spinal cord as above, using appropriate primary antibodies (Supplementary Table 3). Pan and conformation-specific primary antibodies were employed for SOD1 immunostaining.

Pathologies of interest were SOD1 deposits, diffuse cytosolic SOD1, granular cytoplasmic SOD1, CCS deposits and diffuse cytosolic CCS. Counts of each pathology were expressed as the proportion of neurons exhibiting evidence of that pathology to account for differences in motor neuron number between cervical and thoracic spinal cord levels. Conformation-specific SOD1 antibodies yielded a 10-fold improvement in the identification of motor neuron inclusions compared with pan-SOD1 antibodies, supporting the strong involvement of disSOD1 conformers in SOD1 aggregation. Quantification of diffuse cytosolic disordered SOD1 staining was performed in sections stained with UβB conformation-specific SOD1 antibody and DAB. Using Fiji software, average DAB stain intensity was measured throughout the cross section of the neuron and was normalized to the average stain intensity within an area of similar size located immediately adjacent to the motor neuron of interest.

Fresh frozen tissue homogenization and fractionation

Fresh frozen tissue samples were homogenized in 5 volumes (µL) of homogenization buffer (20mM Tris-base, pH 7.6, 1:100 EDTA-free protease inhibitor (Sigma-Aldrich, USA), 1:10 EDTA-free phosphatase inhibitor (PhosSTOP; Roche, Basel, Switzerland)) per mg of tissue at 4°C using a hand-held tissue grinder with metal-free polypropylene probes (Kontes Pellet Pestle ® , Sigma-Aldrich, USA). Homogenates were aliquoted and stored at -80°C, or were centrifuged (16,000g, 30min, 4°C) and the supernatant and pellet collected, aliquoted and stored at -80°C. Protein concentrations within homogenates and supernatants were quantified using a Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, USA). Supernatant and pellet tissue fractions will subsequently be referred to as soluble and insoluble tissue extracts, respectively.

Measures were undertaken to prevent metal contamination of tissue extracts, including the use of plastic forceps for handling tissues, and the removal of trace metal impurities from buffers and glassware using Chelex resin (Bio-Rad) and a metal-chelating rinse solution (0.37% EDTA in 2.25mM K 3 PO 4 ; Sigma-Aldrich, USA), respectively. These measures were also incorporated into native isoelectric focussing experiments and SOD1 specific activity assays.

SOD1 specific activity

SOD1 antioxidant activity was quantified in soluble tissue extracts using a commercial SOD Assay Kit (Cat. #19160, Sigma-Aldrich, USA) and potassium cyanide (KCN), as previously described. [START_REF] Trist | Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain[END_REF] KCN is a potent inhibitor of SOD1 antioxidant activity. [START_REF] Reddy | Purification and characterization of Cu,Zn-superoxide dismutases from mungbean (Vigna-Radiata) seedlings[END_REF][START_REF] Iqbal | Use of cyanide and diethyldithiocarbamate in the assay of superoxide dismutases[END_REF] Duplicate samples containing 2ug protein were incubated with 15mM KCN or 20mM Tris buffer (pH 7.6; Sigma-Aldrich, USA) for 60min at room temperature, and SOD activity (U/mL) measured in both samples using the SOD Assay Kit, according to manufacturers' instructions. SOD1 activity (U/mL) was calculated by subtracting SOD activity measured in the KCN duplicate (non-SOD1 SOD activity) from the SOD activity measured in the Tris buffer duplicate (total SOD activity).

As validation of complete SOD1 inhibition in KCN duplicates, incubation of commercial SOD1 (0-200U/mL) with 15mM KCN for 60min at room temperature completely inhibited SOD1 activity up to ~50U/mL (Supplementary Figure 4), whereas the average total SOD activity in age-matched control samples was only 3.91U/mL in Tris buffer duplicates. The specific activity

Immunoblotting

10μg (SOD1) or 30μg (CCS, GFAP) total protein from soluble tissue extracts was incubated in loading buffer (100mM dithiothreitol, 3% SDS, 10% glycerol, 0.05% bromophenol blue, 62.5mM Tris-base, pH 7.4; Sigma-Aldrich, USA) at 95°C for 15min. Extracts were then loaded into Any kD Mini-PROTEAN TGX Precast Protein Gels (15 well, 15µL; Bio-Rad) and separated using the Bio-Rad Mini-PROTEAN Tetra Cell system (180V, 40min, 4°C). Separated proteins were transferred to Immun-blot PVDF membrane (0.2µm pore size; Bio-Rad) overnight at 9V and 4°C, before membranes were dried for 2h at room temperature and proteins stained with Sypro Ruby Protein Blot Stain (ThermoFisher Scientific, USA), according to the manufacturer's instructions. Sypro Ruby-stained membranes were imaged using a Chemi-Doc XRS imaging system (630BP30nm filter, Bio-Rad), and total protein in each sample quantified by densitometry using ImageLab software (Version 5.2, Bio-Rad). Membranes were then blocked for 2h with 5% skim milk diluted in PBS-T (PBS containing 0.1% Tween 20; Sigma-Aldrich, USA) and incubated overnight at 4°C with primary antibodies against SOD1, CCS and GFAP proteins (Supplementary Table 3) diluted in blocking solution. Primary antibodies were detected using appropriate horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) diluted in blocking solution (1:2,000) for 2h at room temperature. Protein signals were developed using ECL western blotting substrate (Bio-Rad), detected using a Chemi-Doc XRS imaging system (Bio-Rad), and quantified by densitometry using ImageLab software (Version 5.2, Bio-Rad), all according to manufacturers' instructions. Protein levels of SOD1, CCS and GFAP were normalized to total protein levels within the same sample to correct for variations in protein loading, and an internal standard (pooled from all tissue samples) was included on every gel to correct for variability between gels and immunoblot runs. SOD1 protein amount was confirmed to exhibit a linear correlation with SOD1 immunoblot densitometry (Supplementary Figure 5) from 0-1ug using a recombinant human SOD1 protein standard. This encompasses the protein loading amounts employed in our experiments, validing our SOD1 quantification method and accuracy. SOD1 and CCS reside within neurons and glia, [START_REF] Trist | Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain[END_REF][START_REF] Forsberg | Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis[END_REF] and CCS protein levels must take into account neuron loss and astrogliosis within degenerating regions in ALS, reported herein and elsewhere. [START_REF] Vargas | Astrogliosis in amyotrophic lateral sclerosis: role and therapeutic potential of astrocytes[END_REF] SOD1 and CCS protein levels were therefore normalized to a combined cellular index (CCI), which was generated by multiplying indices of neuronal loss (quantified by immunohistochemistry, Fig. 1i) and astrogliosis (quantified by GFAP immunoblot, Supplementary Figure 6).

Measurement of enzymatically-active SOD1 metallation

The metal content of enzymatically-active, dimeric SOD1 was quantified using size-exclusion chromatography (SEC) coupled with native isoelectric focussing (nIEF), synchrotron radiation X-ray fluorescence (SXRF) analyses and particle-induced X-ray emission spectroscopy (PIXE), according to our published method. [START_REF] Roudeau | Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow[END_REF] Briefly, spinal cord and brain tissue homogenates were subjected to SEC to purify mature SOD1 dimers from post-mortem tissues, and SOD1 identified within collected fractions using nano-liquid chromatography-tandem mass spectrometry at a high sequence coverage of 92%. SOD1-containing fractions were then applied along immobilized pH gradient (IPG) gels in duplicate and nIEF performed to further purify dimeric SOD1 according to its pI. SEC-nIEF collectively yielded a 99-fold enrichment of SOD1 protein in post-mortem tissue extracts. After the pI of enzymatically-active, dimeric SOD1 was identified within the first duplicate IPG gel using NBT staining and mass spectrometry, metal quantification was performed on the second duplicate IPG gel at the pI of active SOD1 using SXRF and PIXE. The SXRF analyses were performed in the microprobe hutch of the Hard X-ray Micro/Nano-Probe beamline P06 at the synchrotron PETRA III (DESY) in Hamburg (Germany). The analyses were carried out with an X-ray beam of 12 keV photon energy. The PIXE analyses were performed with 3 MeV proton beam (focussed down a spot size of 2 µm) produced at the AIFIRA facility of the CENBG (Gradignan, France) using a 3.5 MV singletron in-line particle accelerator (High Voltage Engineering Europe). For both techniques, triplicates were recorded for each sample and mean Cu/Zn ratios were calculated. Proteomic analyses using mass spectrometry identified minimal other metalloproteins within IPG gels at the pI of mature SOD1, [START_REF] Roudeau | Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow[END_REF] and their extremely low abundance compared with SOD1 (≤ 0.57%) in protein extracts suggests negligible metal contamination from these proteins. Neither SEC nor nIEF altered the pI or the copper:zinc ratio of a commercially-available human SOD1 control, [START_REF] Roudeau | Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow[END_REF] indicating preservation of endogenous metal binding stoichiometry in purified SOD1 protein.

Native isoelectric focussing (nIEF)

Soluble tissue extract aliquots (40μg total protein) were supplemented with DNase and RNase (Sigma-Aldrich, USA) to a final concentration of 10μg/mL and diluted to 140μL using trace metal-free 20mM Tris-base, pH 7.6. Duplicate immobilized pH gradient (IPG) gels (pH 4.5-5.5, 7 cm; Zoom ® IPG Strip, ThermoFisher Scientific, USA) were rehydrated passively using 140μL diluted soluble extract in an IPG re-swelling tray (Bio-Rad) for 2h at 10°C. The backing of the IPG gels was covered in mineral oil (Bio-Rad) to prevent drying during rehydration. Gels were then focussed in a Protean IEF Cell (Bio-Rad) for 12kVh at 10°C (50µA/gel; Supplementary Table 4). Immediately following nIEF SOD1 activity was detected in IPG gels using nitroblue tetrazolium, as described previously, [START_REF] Trist | Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain[END_REF] and the isoelectric point (pI) of SOD1 in each sample determined. One IPG strip rehydrated with commercially available human SOD1 (5μg, ≥2,500 units activity/mg protein; Sigma-Aldrich, USA) was included per sample run to control for variability between runs.

Tissue preparation for SOD1 aggregate metal quantification

The metal content of SOD1 aggregates in the ventral spinal cord of ALS cases was quantified in fresh frozen tissue sections using XFM at the Australian Synchrotron (Clayton, Victoria, Australia), according to our published method. [START_REF] Genoud | Simultaneous structural and elemental nanoimaging of human brain tissue[END_REF] Briefly, frozen spinal cord tissue blocks from 3 SOD1-fALS, 1 non-SOD1-fALS and 1 sALS case underwent serial cryosectioning (20µm) at -20°C. Immunohistochemical identification of SOD1 aggregates was performed on the first of two facing serial sections, using B8H10 conformation-specific SOD1 primary antibody coupled with DAB staining on a Superfrost™ microscope slide (ThermoFisher Scientific, USA), as described above. The adjacent section was reversed and mounted on a Si 3 N 4 window (5 & 5mm, 200nm film thickness, 200mm frame thickness; Australian National Fabrication Facility, QLD, Australia) with the matching tissue face exposed for synchrotron radiation analyses. We used an Olympus VS120 slide scanner (Olympus, Japan) to map the locations of SOD1 aggregates within

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immunolabelled sections using brightfield microscopy, and to map unlabelled facing sections mounted on Si 3 N 4 windows using differential interference contrast imaging. Images of facing sections were overlaid using OlyVIA 2.9 software (Olympus, Japan) and X, Y pixel coordinates of SOD1 aggregates identified in unlabelled sections from their location in DABimmunolabelled sections, often using distinct tissue features (blood vessels, dorsal or ventral horn boundaries) to triangulate aggregate locations in unlabelled sections. Pixel coordinates of aggregates were recorded and later transferred to motor stage positions at the beamline facility.

Australian Synchrotron XFM beamline operating parameters

The XFM beamline at the Australian Synchrotron 28 is a in-vacuum undulator hard x-ray microprobe with a Maia 384 detector, [START_REF] Ryan | Elemental X-ray imaging using the Maia detector array: The benefits and challenges of large solid-angle[END_REF] which allows the rapid imaging of large samples. A Kirkpatrick-Baez mirror microprobe was used to focus monochromatic 15.8keV X-rays to a 2µm spot. Samples were mounted on Perspex frame and scanned in fly-scan mode with 1µm step size and 5ms dwell per pixel. Total x-ray fluorescence emission spectra were collected by the Maia detector. Pixel coordinates from OlyVIA 2.9 were directly imported into the beamline using the integrated motor control system described previously. [START_REF] Howard | The XFM beamline at the Australian Synchrotron[END_REF]

Australian Synchrotron XFM data analysis

Elemental maps were produced by fitting the x-ray fluorescence (XRF) emission energies to the total XRF spectrum from each pixel. Model spectrum was produced with the assumption that dried tissues was 5µm thick. This quantitative deconvolution of elemental fluorescence was performed using GeoPIXE v7.2 which applies a first-order "matrix" correction to estimate the reabsorption of the elemental fluorescence by the tissue. [START_REF] Ryan | Nuclear microprobe-synchrotron synergy: Towards integrated quantitative real-time elemental imaging using PIXE and SXRF[END_REF] Images were exported as 32-bit tiffs for further analysis in Fiji software (National Institute of Health, USA) as previously described. [START_REF] Genoud | Simultaneous structural and elemental nanoimaging of human brain tissue[END_REF] Images were analysed as described previously. [START_REF] Genoud | Simultaneous structural and elemental nanoimaging of human brain tissue[END_REF] In brief, limit of detection (LOD) for each element was determined and each pixel corrected for background. A threshold of the upper 1% was also excluded from analysis. Aggregates were outlined by aligning brightfield microscopy photomicrographs with XFM images. Images were then normalised by pixel-wise division of Compton scatter emission to correct for variation in surface density and self-absorption effects,

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and aggregate measurements extracted for stacked XFM elemental maps. Extracted, corrected and normalised XFM data was imported into SPSS Statistics v26 (International Business Machines, Illinois, USA) with matching subject demographic data. Outliers were defined by SPSS as 'extreme values' ≥3 x the interquartile range (or 2 standard deviations) and excluded from analysis. A two-sided T-test was used to compare elemental levels in SOD1 aggregates with background tissue and active SOD1. A significant difference in Cu:Zn ratio from the expected 1:1 was inferred from a 95% confidence interval that did not span 1 (Wilcoxon signed rank test).

Immunoprecipitation

SOD1 protein was immunoprecipitated from post-mortem spinal cord tissue homogenates using Dynabeads M-280 Tosylactivated (Invitrogen, Carlsbad, CA, USA), according to manufacturer's instructions. Ten milligrams of Dynabeads M-280 Tosylactivated were conjugated to 100ug polyclonal SOD1 antibody (Enzo Life Sciences, Farmingdale, NY, USA; Supplementary Table 3) diluted to 40mg beads/mL in coupling buffer (0.1M boric acid, pH 9.5; 1.2M ammonium sulphate) overnight at 37°C. Dynabeads were blocked with 0.5% BSA in PBS (pH 7.4), washed with 0.1% BSA in PBS (pH 7.4), and incubated with ventral spinal cord tissue homogenates (200ug) diluted in PBS (pH 7.4) to 40mg beads/mL overnight at 4°C. Following PBS washes, immunoprecipitated proteins were eluted from Dynabeads using successive 10min incubations with 0.1M glycine (pH 3), eluants neutralized using an equivalent volume of ammonium bicarbonate (pH 8) and extracts dried under pressure using a vacuum concentrator. We observed minimal reductions in the amount of SOD1 protein captured over successive rounds of immunoprecipitation upon re-use of antibody-coupled Dynabeads (Supplementary Figure 7), and hence beads were re-used for 3 successive rounds of immunocapture. No measurable SOD1 protein was bound to antibody-coupled Dynabeads post elution (Supplementary Figure 7), negating the possibility of cross-contamination between samples assigned to successive rounds of immunocapture. No alterations to the antioxidant activity or metal content of a commerciallyavailable human SOD1 standard were observed following immunoprecipitation (Supplementary Figure 7), [START_REF] Kysenius | A versatile quantitative microdroplet elemental imaging method optimised for integration in biochemical workflows for low-volume samples[END_REF] indicating SOD1 protein was likely isolated from post-mortem tissues having retained its endogenous metal occupancy and protein conformation. SOD1 protein was also not

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identified in immunoprecipitates prepared using dynabeads that were not conjugated to our capture antibody (Supplementary Figure 7).

Sample preparation for mass spectrometry

Dried SOD1 immunoprecipitates were resuspended in 50 mM ammonium bicarbonate (pH 8) containing 6M urea, reduced with dithiothreitol (DTT; 10 mM final) for 30min at 56 °C, alkylated with iodoacetamide (IAA; 20 mM final) for 30min at room temperature in the dark, and finally quenched with a further 10 mM DTT for 30min at room temperature. Samples were diluted five-fold using 50 mM ammonium bicarbonate (pH 8) to reduce the concentration of urea to 1.2M and acetonitrile added (10% final), before in-solution digestion performed overnight at room temperature using 0.2µg sequencing-grade modified trypsin (Promega, Madison, WI, USA). Samples were then acidified using trifluoroacetic acid, desalted using Pierce C18 Tips (ThermoFisher Scientific, USA) according to manufacturer's instructions, and dried under pressure using a vacuum concentrator. Samples were resuspended in loading buffer (0.1% formic acid, 3% ACN) and transferred to HPLC vials immediately prior to mass spectrometry analyses.

Cysteine residues are highly susceptible to oxidation compared with other amino acids, [START_REF] Ahmad | Protein oxidation: an overview of metabolism of sulphur containing amino acid, cysteine[END_REF] therefore sample preparation for cysteine redox analysis utilized separate tissues prepared under a global reductive protein preparation strategy to limit the introduction of artifactual modifications during sample processing. [START_REF] Rookyard | A Global Profile of Reversible and Irreversible Cysteine Redox Post-Translational Modifications During Myocardial Ischemia/Reperfusion Injury and Antioxidant Intervention[END_REF] Samples were homogenized as described above, and homogenates incubated with N-ethylmaleimide (NEM; 20 mM final) for 10min at room temperature to alkylate free thiol groups. Excess NEM was quenched with DTT (25mM final) for 15min at room temperature, after which sample pH was adjusted above 7 using sodium hydroxide and samples reduced for 1h at room temperature. Homogenates were then incubated with MMTS (60 mM final) for 1.5h at 30 °C to quench DTT and alkylate nascent thiols, and proteins precipitated using chloroform/methanol precipitation. Proteins were resuspended in 50mM ammonium bicarbonate (pH 8) containing 6 M urea, diluted 5-fold using 50mM ammonium bicarbonate (pH 8), and protein concentration quantified using a BCA assay according to manufacturer's instructions. Acetonitrile was then added (10% final), and insolution digestion performed overnight at room temperature using sequencing-grade modified trypsin (1:50 trypsin:protein; Promega, Madison, WI, USA). Samples were then acidified using

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trifluoroacetic acid, dried under pressure using a vacuum concentrator, resuspended in loading buffer (0.1% formic acid, 3% ACN) and transferred to HPLC vials immediately prior to mass spectrometry analyses.

Mass spectrometry data acquisition

Label-free Fourier Transform Mass Spectrometry was employed to analyse immunoprecipitated protein extracts at Sydney Mass Spectrometry (Sydney, New South Wales, Australia). Analyses were performed using an UltiMate 3000 RSLCnano system (ThermoFisher Scientific, USA) coupled online via a Nanospray Ion Source (ThermoFisher Scientific, USA) to an Orbitrap Fusion Tribrid Mass Spectrometer (ThermoFisher Scientific, USA). Peptide digests were loaded onto an in-house packed ReproSil-Pur 120 C18-AQ analytical column (75µm id x 40 cm, 1.9µm particle size; Dr Maisch, Ammerbuch, Germany) regulated to 60°C using a PRSO-V2 Sonation column oven (Sonation, Baden-Wuerttemberg, Germany). A binary gradient of solvent A (0.1% formic acid in MilliQ water) to solvent B (0.1% formic acid in 80% ACN diluted with MilliQ water) was used for peptide elution at a separation flow rate of 300-450 nL/min over 90min (Supplementary Table 5). The mass spectrometer operated in positive ion mode at a 2.4 kV needle voltage. Data were acquired using Xcalibur software (Version 4.4.16.14, ThermoFisher Scientific, USA) in a data-dependent mode. MS scans (m/z 300-1650) were recorded at a resolution of R = 60 000 (@ m/z 200) and a standard automatic gain control target within 50msec. Dynamic exclusion was set to 9s with a mass tolerance of ±10ppm and top speed fragmentation in higher-energy collisional dissociation (HCD) mode was performed for the top 5 most abundant precursor ions. MS/MS scans with a target value of 200% were collected in the ion trap with a maximum fill time of 28 msec. Additionally, only +2 to +5 charged ions were selected for HCD fragmentation. Other settings were as follows: heated capillary temperature, 300°C; normalized HCD collision energy of 30% and an isolation width of 1.6 m/z. Monoisotopic precursor selection (MIPS) was set to Peptide and an intensity threshold was set to 2x10 [START_REF] Guareschi | An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1[END_REF] .

For cysteine redox analysis, liquid chromatography-mass spectrometry was conducted on a Q-Exactive HFX mass spectrometer (ThermoFisher scientific) coupled to an UltiMate 3000 RSLCnano UHPLC (ThermoFisher Scientific). Peptides were separated on a home-packed

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column (1.9 μm, 40 cm × 75 μm 120 Å pore size) of ReproSil Pur C 18 AQ beads (Dr. Maisch GmbH, AmmerbuchEntringen, Germany) regulated at 60 °C with a PRSO-V2 Sonation column oven. A binary gradient of Buffer A (0.1% formic acid) to Buffer B (90% ACN 0.1% formic acid) from 5% to 98% buffer B was used to elute peptide over 90min at a rate of 300 nL / min.

The MS was coupled to a Nanospray Ion Source (ThermoFisher Scientific) and was operated in positive ion data dependant acquisition mode with a needle voltage of 2.4 kV and ion transfer capillary temperature of 300 °C. Precursor ions were surveyed with an AGC target of 1e6, maximum fill time of 50ms and the orbitrap operating between 300-1650 m/z at a resolution of R = 60000 at 200m/z. The top 20 precursor ions with charges 2 + -4 + were taken for higher collisional energy dissociation with an isolation window of 1.4m/z, AGC target of 3e6, maximum ion fill time of 90ms and normalised collision energy of 27 (N)CE. This occurred with a dynamic exclusion window of 15s. Fragment ions were detected with a resolution of R = 15000 at 200 Sm/z. For PRM experiments the MS was operated in data independent acquisition mode consisting of a precursor ion scan followed by a loop of the scheduled inclusion list (Supplementary Table 6). Peptides were monitored with 16 min acquisition windows. The precursor ion scan occurred with a resolution of R = 30000 at 200m/z and fragment ion scans occurred with an isolation window of 0.7Da and a maximum fill time of 65 ms, otherwise MS parameters were as stated above (n=3 injection replicates per sample).

Mass spectrometry data analysis

Raw data generated from immunoprecipitated protein extracts was processed using MaxQuant software (Version 2.0.1.0, Max Planck Institute of Biochemistry, Planegg, Germany) under default settings. [START_REF] Cox | Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ[END_REF] Spectra were screened against Uniprot entry P00441 (SODC-HUMAN), corresponding to human SOD1, with peptides higher than 4600 Da or lower than 7 amino acids excluded from further analyses. Search parameters were as follows: mass accuracy of the MIPS precursor and peptide fragments was set to 10 ppm and 0.6 Da respectively. Only b-and y-ions were considered for mass calculation. Oxidation of methionines (+16 Da) and protein N-terminal acetylation (+42 Da) were considered as variable modifications for protein quantification, whereas carbamidomethylation of cysteines (+57 Da) as well as carbamylation (+43 Da) of lysines and residue N-termini were included as variable modifications for peptide identification

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and quantification. Given the presence of I113T and D101G mutations in SOD1-fALS samples, Xle->Thr and Asp->Gly were also included as variable modifications. PTMs of interest for comparison between ALS subgroups and controls were analysed in separate analysis batches, and included; 3-deoxyglucosone-derived hydroimidazolone (R; 144.0423), α-amino adipic acid (K; +14.9633), acetylation (K; +42.0106), allysine (K; -1.0316), carboxyethyllysine (K; +72.0211), carboxymethyllysine (K; 58.0055), deamidation (N/Q; +0.9840), glycation (K/R; +108.0211 with neutral loss of three water molecules), [START_REF] Keilhauer | HCD Fragmentation of Glycated Peptides[END_REF] methylglyoxal-derived hydroimidazolone (R; +54.0106)), nitration (W; +44.9976) oxidation (H/W; +15.9949), phosphorylation (S/T; +79.9663), succinylation (K; +100.0160), ubiquitination (K; 114.0429). A maximum of 5 modifications per peptide was allowed. Two missed trypsin cleavages were allowed. LFQ protein abundance was calculated using the MaxLFQ algorithm embedded in MaxQuant, requiring at least two ratio counts of unique peptides. Peptide validation was performed using the Andromeda algorithm and peptides were excluded if they possessed an andromeda score lower than 80 or a localization score lower than 0.8. The false discovery rate (FDR) for both peptides and proteins was adjusted to less than 1% using a target and decoy approach. The 'match between runs' option was enabled using a time window of 0.7 min. We achieved an average sequence coverage of 97.2% for SOD1 protein identified in sample immunoprecipitates. SOD1 protein was not identified in negative control immunoprecipitates prepared using dynabeads that were not conjugated to our capture antibody, suggesting negligible false discovery of SOD1 protein in ALS and control protein extracts. Modified peptide intensities were normalised to SOD1 protein LFQ intensities to account for protein and/or sample concentration variance. No differences in the relative levels of PTMs of interest were identified between immunoprecipitated and non-immunoprecipitated commercial SOD1 protein (Supplementary Table 7), implying our immunoprecipitation protocol did not significantly alter PTMs of interest. Strong correlations between the abundance of SOD1 protein in technical replicates, as well as the abundance of modified peptides in these replicates, highlights the reproducibility of our experimental workflow (Supplementary Figure 8).

Raw data generated from samples prepared for cysteine redox analysis was processed using Proteome Discoverer (ThermoFisher Scientific, version 2.4) and was searched against a Homo Sapiens Uniprot database (release 2020_05) with an in-house Mascot server (Matrix Science, London, UK) allowing for 2 missed trypsin cleavages and a minimum peptide length of 6

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residues. Cys-N-ethylmaleimide (+125.05 Da) was set as a fixed modification and Cysmethylthio (+45.99 Da), Met oxidation (+16.00 Da) and N/Q deamidation (+0.98 Da) as variable modifications. A precursor mass tolerance of 20 ppm and fragment mass tolerance of 0.2 Da was used with a strict FDR cut-off of 1% and relaxed FDR cut-off of 5%. The results from the DDA experiment were then used to build a spectral library in Skyline (version 20.02). Chromatogram integration was performed in Skyline and summed transition peak areas for each peptide were taken for further processing. Each modified Cys peptide was normalised to two non-modified SOD peptides to account for protein and/or sample concentration variance. Rather than averaging these values, which would weight the normalization more towards one of the two nonmodified peptides, normalised peak areas for both non-modified peptides were expressed as a ratio compared with the control diagnostic group, and were averaged to obtain final values.

Glutathione quantification

Glutathione concentrations were quantified in soluble extracts using a published Tietze enzymatic recycling assay, [START_REF] Ngamchuea | Rapid Method for the Quantification of Reduced and Oxidized Glutathione in Human Plasma and Saliva[END_REF] modified to be compatible with a microtitre plate spectrophotometer. Protein was precipitated using 5% 5-sulfosalicylic acid, samples promptly centrifuged (10,000g, 10min, 4°C), and supernatant collected and diluted five-fold using 20mM

Tris buffer (pH 8) to reduce 5-sulfosalicylic acid to 1% before assay commencement. All assay steps were undertaken within an Atmosbag glove bag (Cat. # Z530212, Sigma-Aldrich, USA) under anaerobic conditions (<1% O2) using ultra-high purity nitrogen gas (99.999%, BOC Healthcare), except for spectrophotometry measurements where plates were sealed with airtight adhesive strips and transported to the microplate plate reader. Adhesive strips did not alter sample absorption (Supplementary Figure 9). Anaerobic experimental conditions, together with storage of post-mortem tissues and extracts at -80°C prior to analysis, greatly reduces glutathione degradation, ensuring measurements are as close to endogenous glutathione levels as possible. [START_REF] Lin | Effects of Storage Time and Temperature on the Stability of Glutathione in Deproteinized Blood Sample[END_REF]

Inductively coupled plasma-mass spectrometry (ICP-MS)

Metal levels in soluble and insoluble tissue extracts were quantified using inductively coupled plasma-mass spectrometry (ICP-MS), according to our group's published methods. [START_REF] Genoud | Subcellular compartmentalisation of copper, iron, manganese, and zinc in the Parkinson's disease brain[END_REF] Soluble

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extracts were thawed on ice and diluted with 1% nitric acid (1:10 v/v; Suprapur grade, Merk Millipore) prior to analysis. Insoluble extracts were freeze-dried overnight, weighed, and digested overnight using concentrated nitric acid (50µL, 70%, Suprapur grade, Merk Millipore) at room temperature. Samples were then digested for a further 30min at 70°C, and incubated with concentrated hydrogen peroxide (30%, VWH International, PA, USA) for 60min at 70°C, and diluted with 1% nitric acid (1:10 v/v; Suprapur grade, Merk Millipore) prior to analysis.

Total metal levels in each sample were measured in triplicate using an Agilent Technologies 7700 ICP-MS system with a Teflon MiraMist concentric nebuliser and Scott-type double-pass spray chamber (Glass Expansion, VIC, Australia). Buffer controls containing 1% nitric acid were incorporated every 20 samples. Helium (3 mL/min) was used as a collision gas for removal of polyatomic interferences. Measured mass-to-charge (m/z) ratios were 44 (Ca), 55 (Mn), 56 (Fe), 63 (Cu), 66 (Zn), 78 (Se), 111 (Cd). External calibration was performed using multi-element standards (Sigma-Aldrich, USA) diluted in 1% HNO3 and yttrium (Y; m/z = 89; Accustandard, Connecticut, USA) was used as reference element via online introduction with a Teflon T-piece.

Measurements were background corrected to metal levels in buffer controls, adjusted for dilution factors and were standardized against original wet tissue weights. Samples below the instrument's limits of detection were excluded from analyses. select diagnostic groups for a given variable. Where appropriate, non-parametric data were transformed using X=Log(X) or X=SQRT(X) to meet parametric assumptions required for grouped analyses using Two-way ANOVA. Outliers were identified using the combined robust regression and outlier removal (ROUT) method with a maximum false discovery rate of 5%. Final values were reported as mean ± SEM. A p-value of less than 0.05 was accepted as the level of significance. Details of the statistical tests employed (test statistics, sample sizes, p values) for each variable of interest are included in the corresponding figure legend or results text section.

Statistical analyses

An n of 9 cases per diagnostic group was strongly powered to detect differences in SOD1 misfolding and conformational change (97.9-98.5% power, two-tailed t-test, α = 5%; SPSS software, IBM, Armonk, NY, USA) in a preliminary power analysis of pilot data describing the isoelectric point of wild-type SOD1 protein in the ventral spinal cord of a subset of sALS cases (n = 3) and age-matched controls (n = 3). Difficulties in sourcing and obtaining appropriate tissues from SOD1-fALS and non-SOD1-fALS cases limited the sample sizes of these diagnostic groups, however retrospective power analyses reveal data from both groups were still strongly powered to detect differences in many measured variables (SOD1 pI, % MNs exhibiting granular disSOD1, SOD1 protein levels, SOD1 spec act.) compared with age-matched controls due to the large effect sizes observed for these variables (77.2-100% power).

Principal component analysis (PCA)

PCA was performed, and corresponding plots constructed, using Rstudio 1.4.1717, R 4.1.0 (RStudio Team, RStudio: Integrated Development Environment for R, Boston, MA, USA) and associated packages FactoMinerR 2.4, 40 missMDA 1.18, 41 factoextra 1.0.7, [START_REF]Factoextra: Extract and Visualize the Results of Multivariate Data Analyses[END_REF] and tidyverse 1.3.1. [START_REF]Welcome to the Tidyverse[END_REF] The full reproducible code is available from the corresponding author upon reasonable request. Active variables (n=23) for the principal component analysis (PCA) are : % motor neurons containing granular SOD1 (MN_granular_disSOD1), SOD1 inclusions (MN_disSOD1), granular CCS (MN_granular_CCS) and diffuse cytoplasmic CCS (MN_diffuse_CCS), motor neuron density (neuronal_density), total (SOD1_act) and specific SOD1 activity (SOD1_spec_act), SOD1 (SOD1_level) and CCS protein levels (CCS_level), Cu:Zn atomic ratio within enzymatically-active soluble mature SOD1 (SOD1_CuZn_ratio), active SOD1 pI (pI), glutathione levels (GSH), and copper and zinc levels in whole tissues (Total_Cu, Total_Zn), as

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well as soluble and insoluble tissue extracts (Soluble_Cu, Soluble_Zn, Insoluble_Cu, Insoluble_Zn). SOD1 PTM data was also included, although these were grouped based on similarities in expected functional consequences; PTMoxoxidation His, Trp, Cys, nitration Trp; PTMchacetylation Lys, Phosphorylation Ser; PTMdeamdeamidation Asn; PTMage -CEL Lys; GlyGly_Lys91. Supplementary variables (case #, clinical diagnostic, age, sex, postmortem interval (PMI), site of onset) do not influence on the PCA analysis and allow for easier interpretation and visualisation of the results. The data were scaled before PCA and missing values were processed with the missMDA package. Our PCA revealed no significant differences in the relative levels of variables of interest between thoracic and cervical spinal cord levels (Supplementary Figure 10). Thoracic and cervical spinal cord values were therefore averaged for cases possessing data for both spinal cord levels, yielding one data point per case for grouped statistical analyses.

Results

Disordered SOD1 conformers are mislocalized and accumulate in spinal cord motor neurons of ALS cases.

Nascent, unfolded human SOD1 is structurally-disordered and lacks antioxidant activity. [START_REF] Banci | Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants[END_REF] Maturation of these immature species to enzymatically-active, mature SOD1 dimers is multifaceted, and hence SOD1 protein exists within healthy neurons as a complex mixture of immature, intermediate and mature conformers. [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF][START_REF] Culik | Effects of maturation on the conformational freeenergy landscape of SOD1[END_REF] The accumulation of structurally-disordered immature and intermediate conformers, hereon referred to as disordered (dis)SOD1, underlies the self-assembly and toxicity of mutant and wild-type SOD1 in vitro and in transgenic SOD1 mice. 46,47

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We employed immunohistochemistry to profile the subcellular distributions of mature and disSOD1 conformers in 7µm fixed post-mortem ventral spinal cord tissue sections collected from ALS patients and age-matched controls (Supplementary Table 1). Monoclonal and polyclonal pan-SOD1 antibodies recognizing full-length human SOD1 (Supplementary Table 3) identified diffuse cytosolic SOD1 immunoreactivity in 94% of spinal cord motor neurons in age-matched control cases (Fig. 1a), consistent with the primary subcellular localization of enzymatically-active mature SOD1. [START_REF] Pardo | Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons[END_REF] Faint SOD1-immunopositive cytoplasmic granules (2.5 -6µm diameter) were observed within 10-20% of control spinal cord motor neurons (Fig. 1a), which colocalized with cis-golgi (GM130; Fig. 1b) and endoplasmic reticulum (ER; calreticulin; Fig. 1b) markers, indicating partial localization of SOD1 to these compartments in control spinal cord motor neurons. Nuclear SOD1 immunostaining was also observed, consistent with a role for SOD1 as a nuclear transcription factor regulating antioxidant response genes. [START_REF] Tsang | Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance[END_REF] Owing to the broad amino acid sequence coverage of their binding epitopes, pan-SOD1 antibodies immunolabel multiple mature and immature SOD1 conformers, thus it is unclear which specific conformers exist in each of these compartments.

Conformation-specific SOD1 antibodies can selectively detect immature disSOD1 conformers in human post-mortem tissues. We probed adjacent control spinal cord sections with EDI, B8H10, and UβB conformation-specific antibodies (Supplementary Table 3), producing strong granular cytoplasmic SOD1 immunostaining within 66% of motor neurons, respectively, across all control cases (Fig. 1a). Colocalization of granular cytoplasmic SOD1 immunostaining with GM130 and calreticulin in sections probed with conformation-specific SOD1 antibodies (Fig. 1b) identified disSOD1 conformers within the ER-golgi network in control motor neurons, constituting a physiological population of unfolded SOD1 in this compartment. A greater intensity of nuclear SOD1 immunostaining is these sections compared with those probed with pan SOD1 antibodies (Fig. 1a) suggests immature conformers may mediate the transcriptional activity of SOD1, rather than the mature, enzymatically active protein. In contrast, low-intensity diffuse cytosolic immunoreactivity was present within only 34% of control spinal cord motor neurons in these tissue sections (Fig. 1a). This suggests disSOD1 conformers exist either transiently, or in significantly lower abundance, within the cytosol of control motor neurons under physiological conditions. Overall we show that mature SOD1 conformers likely constitute the dominant SOD1 species in the cytoplasm of healthy spinal cord motor neurons, whilst Using control motor neuron immunostaining as a baseline, we applied pan and conformationspecific immunostaining to ventral spinal cord tissues from SOD1-fALS, non-SOD1-fALS and sALS cases to evaluate mature and immature SOD1 deposition and subcellular localization. The proportion of motor neurons possessing granular SOD1 immunostaining was reduced by 64-70% in all ALS subgroups compared with controls (Fig. 1d,e), and was replaced by high-intensity diffuse cytosolic disSOD1 and/or disSOD1 protein inclusions in the perikarya and neurites of these neurons in all ALS cases (Fig. 1f, Table 1). Nuclear disSOD1 was also absent in ALS spinal cord motor neurons at end-stage disease (Fig. 1f). Given the broad combined epitope coverage of all antibodies employed, these data signify a subcellular re-distribution of disSOD1 conformers within vulnerable motor neurons in ALS; indeed, disordered SOD1 conformers were primarily no longer localized to the ER-golgi network in these patients (Fig. 1g). Reductions in spinal cord motor neuron density (Fig. 1h) were strongly correlated with lower proportions of granular disSOD1 motor neurons (Fig. 1i), but not higher proportions of motor neurons possessing disSOD1 inclusions (Fig. 1j,k). This is consistent with the involvement of smaller disSOD1 assemblages, rather than larger insoluble SOD1 deposits, in SOD1-mediated neuronal toxicity in ALS models. [START_REF] Gill | SOD1-positive aggregate accumulation in the CNS predicts slower disease progression and increased longevity in a mutant SOD1 mouse model of ALS[END_REF] Collectively, our findings indicate that immature disSOD1 conformers are mislocalized to the cytoplasm from the nucleus and ER-Golgi network in ALS spinal cord motor neurons by end-stage disease, where they accumulate and often form large inclusions. These alterations are a reliable feature of all forms of ALS, which may be associated with motor neuron degeneration.

Enzymatically-dysfunctional SOD1 conformers are more abundant in the ventral spinal cord of ALS cases.

Efficient superoxide detoxification by SOD1 is highly dependent upon correct protein folding and maturation. [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF] To assess whether the cytoplasmic mislocalization and accumulation of immature disSOD1 conformers impacts superoxide clearance by SOD1, we quantified SOD1 enzymatic activity in fresh frozen tissue from the ventral and dorsal spinal cord of our postmortem cohort. [START_REF] Trist | Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain[END_REF] Total SOD1 activity was unchanged in the ventral and dorsal spinal cord in all ALS subgroups compared with controls (Fig. 2a), indicating similar superoxide buffering capacities within these regions in ALS cases. These data are surprising considering oxidative stress is widely reported within the ALS spinal cord [START_REF] Barber | Oxidative stress in ALS: key role in motor neuron injury and therapeutic target[END_REF] an environment that would be be expected to result in an increased proportion of mature, enzymatically active SOD1 to increase superoxide clearance.

Therefore, to investigate whether SOD1 protein production and/or maturation are dysregulated in ALS, we quantified SOD1 protein levels using immunoblotting and calculated SOD1 enzymatic activity per unit of protein (specific activity). Ventral spinal cord SOD1 protein levels were moderately increased in all ALS subgroups compared with controls (2-2.9-fold; Fig. 2b), however SOD1 specific activity was reduced by 55-75% in this region of all ALS cases (Fig. 2c). These data are consistent with reductions in SOD1 activity measured in red blood cells, fibroblast and lymphoblast cell lines derived from SOD1-fALS patients. [START_REF] Saccon | Is SOD1 loss of function involved in amyotrophic lateral sclerosis?[END_REF] Levels of total SOD1 protein and specific activity were unchanged within the non-degenerating (Supplementary Figure 11) dorsal spinal cord (Fig. 2b,c) of all ALS cases, indicating these alterations are restricted to regions of neuron death. Lower SOD1 specific activity was strongly correlated with decreased motor neuron density and greater disSOD1 mislocalization to the cytoplasm in the ventral spinal cord of ALS cases (Fig. 2d,e), consistent with a reduced viability of SOD1deficient neurons under conditions of redox imbalance in Sod1 +/-mice. [START_REF] Barber | Oxidative stress in ALS: key role in motor neuron injury and therapeutic target[END_REF][START_REF] Saccon | Is SOD1 loss of function involved in amyotrophic lateral sclerosis?[END_REF] Overall, these data demonstrate enzymatically-dysfunctional SOD1 conformers selectively accumulate within vulnerable regions of the spinal cord in ALS (Fig. 2f).

Mature SOD1 destabilization may contribute to disordered SOD1 conformer accumulation in ALS spinal cord.

The accumulation of immature disSOD1 conformers in ALS motor neurons could result from disruptions to pathways governing SOD1 protein maturation, or alternatively from the destabilization of mature, enzymatically-active SOD1 dimers, hereon referred to as mature SOD1. Mature SOD1 contains one zinc and copper ion per monomer, which together underlie its exceptional structural stability and enzymatic activity. Immature SOD1, on the other hand, exhibits a reduction in the binding of its metal cofactors, which underlies it's greater structural disorder and reduced enzymatic activity. [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF] We evaluated mature SOD1 stability and functionality

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by quantifying metals bound to enzymatically-active SOD1 dimers (Fig. 3a). [START_REF] Roudeau | Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow[END_REF] Mature SOD1 purified from the ventral and dorsal spinal cord of control cases contained copper and zinc in an exact 1:1 ratio (Fig. 3b), [START_REF] Roudeau | Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow[END_REF] consistent with the theoretical copper:zinc ratio bound to a mature SOD1 dimer. By contrast, the copper:zinc ratio of mature SOD1 was significantly elevated in the ventral spinal cord of 42% of ALS cases (5-of-12; 1 Cu : 0.6 Zn) compared with controls (Fig. 3b), including 2 SOD1-fALS (I113T and D101G SOD1 mutants), 1 non-SOD1-fALS (C9ORF hexanucleotide repeat expansion mutant) and 2 sALS cases. Mature SOD1 metallation was unchanged in the non-degenerating occipital cortex of affected cases (Supplementary Figure 12), suggesting perturbed SOD1 zinc binding results from biochemical factors unique to the ventral spinal cord. Our data provide the first evidence of alterations to the metallation of mature mutant and wild-type SOD1 in a proportion of familial and sporadic ALS patients, which may contribute to SOD1 pathology in these patients.

Alterations to SOD1 protein isoelectric point (pI) reflect conformational changes [START_REF] Pergande | Isoelectric Point Separations of Peptides and Proteins[END_REF] that can alter both its aggregation propensity and the electrostatic guidance of anionic superoxide towards the active site. Mature SOD1 pI was significantly elevated in the ventral spinal cord in all ALS subgroups, and in the dorsal spinal cord of SOD1-fALS cases, compared with controls (Fig. 3c,d), suggestive of conformational changes to mature SOD1 in these regions. Whilst elevated pIs in both spinal cord sub-regions of SOD1-fALS cases suggests SOD1 mutations may contribute to these alterations in SOD1-fALS, only one of the two mutant proteins examined in this study exhibits a reduced surface charge compared with wild-type SOD1, [START_REF] Prudencio | Variation in aggregation propensities among ALS-associated variants of SOD1: correlation to human disease[END_REF] implicating alternative unknown factors. Likewise, the selectivity of this increase to the ventral spinal cord in other ALS cases indicates non-genetic factors unique to this region underlie such changes in these cases. Subcellular mislocalization of SOD1 to the cytoplasm within spinal cord motor neurons, for example, may contribute to the reduction in mature SOD1 surface charge in this region Fig. 3e). Mature SOD1 pI was also increased in the ventral spinal cord of ALS cases exhibiting SOD1 zinc deficiency (n = 5), compared with control (n = 10) and remaining ALS cases (n = 11; Fig. 3f). Whilst conformational change in mature SOD1 was not associated with reduced enzymatic activity (data not shown; Spearman's r = -0.296, p = 0.13), it was correlated with greater motor neuron loss and deposition of SOD1 in the ventral spinal cord of ALS cases (Fig. 3g,h), consistent with data demonstrating reduced surface charge of SOD1 dimers promotes neurotoxic SOD1 aggregation from a native-like dimeric state in vitro. [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF] Overall, these Zinc-deficient mature SOD1 is unlikely to undergo self-assembly from a native-like dimeric state in ALS cases.

To ascertain whether zinc-deficient mature SOD1 may indeed aggregate directly from a disordered dimer-like state in vivo, we assessed whether the metal content of ventral spinal cord disSOD1 deposits reflects the elevated copper:zinc ratio of soluble mature SOD1 in this region in select ALS patients. Copper and zinc levels were quantified within 45 individual SOD1 aggregates, ranging from 2.5-30µm in diameter, and 25 areas of surrounding grey matter tissue of a similar size, in frozen unstained ventral spinal cord sections from 3 SOD1-fALS and 1 sALS case (Fig. 4a,b). Compton inelastic scattering was also measured simultaneously as an index of tissue matter density, revealing increased density within SOD1 aggregates compared with surrounding tissues (Fig. 4c). Normalization of metal levels to Compton scatter values ensured that varying tissue matter densities between aggregates, as well as between aggregates and surrounding tissues, did not confound interpretation of metal data. We identified a 70% increase in zinc (Fig. 4d), and a 12% reduction in copper (Fig. 4e), within SOD1 aggregates compared with surrounding grey matter tissues, constituting a significant decrease in the copper:zinc ratio within SOD1 aggregates compared with surrounding tissues (Fig. 4f) and the copper:zinc ratio of soluble mature SOD1 measured in these same cases (Fig. 4g). We speculate that these data indicate zinc-deficient, copper-containing mature SOD1 may not form aggregates from a nativelike dimeric state in vivo. Instead, we propose that zinc-deficiency promotes mature SOD1 dissolution into alternative metal-deficient disSOD1 conformers, which subsequently aggregate, as reported elsewhere. [START_REF] Roberts | Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis[END_REF] We recognize that this may also involve the recruitment of additional zinc-binding proteins, including CCS, [START_REF] Trist | Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain[END_REF] which contribute to the reduced copper:zinc ratio within SOD1 aggregates. The post-translational modification of key amino acid residue side chains within SOD1 protein regulates its structure (Fig. 5a,b), maturation, metallation, subcellular localization and enzymatic function. [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF][START_REF] Banks | Mechanisms of SOD1 regulation by post-translational modifications[END_REF] Recognizing that disSOD1 proteinopathy in ALS patients may result from altered SOD1 PTMs, we constructed post-translational fingerprints of SOD1 protein immunoprecipitated in its native state from post-mortem ventral and dorsal spinal cord of ALS patients and controls, identifying 42 individual PTMs to 29 residues (19%) of SOD1 protein across all diagnostic groups (Fig. 5c, Supplementary Tables 8,9).

Oxidation of SOD1 His48 and His63 was significantly increased (2-fold) in the ventral spinal cord of ALS cases possessing Zn-deficient soluble SOD1, compared with controls and remaining ALS cases (Supplementary Tables 8,9). This finding is consistent with a role for oxidation of metal-coordinating His residues in inducing SOD1 mismetallation in vitro [START_REF] Mulligan | Early steps in oxidation-induced SOD1 misfolding: implications for non-amyloid protein aggregation in familial ALS[END_REF] (Fig. 5d).

Significant increases in Trp32 oxidation (1.7-fold) and nitration (3.4-fold) were also identified within SOD1 protein isolated from the ventral spinal cord of three SOD1-fALS and five sALS cases, respectively, compared with controls (Supplementary Tables 8,9). Considering the role of this residue in redox-induced aggregation and propagation of disSOD1 in cellular and animal models of ALS [START_REF] Crown | Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection[END_REF] (Fig. 5e), these modifications may contribute to the development of SOD1 proteinopathy in affected cases.

Whilst Lys succinylation was unchanged between ALS cases and controls (Supplementary Table 8), Lys3 acetylation and Ser98 phosphorylation were significantly reduced in six and four sALS cases, respectively, compared with controls (Supplementary Tables 8,9). Although reduced Lys3 acetylation may increase the susceptibility of Lys3 to destabilizing PTMs, we did not detect any increase in oxidation of this residue between diagnostic groups (Supplementary Table 8). This reduction may, however, underlie the elevated active SOD1 pI in these cases (Fig. 3), which is proposed to promote SOD1 aggregation and intercellular propagation in vitro. [START_REF] Abdolvahabi | Arresting amyloid with coulomb's law: acetylation of ALS-linked SOD1 by aspirin impedes aggregation[END_REF] Functionally, neither alteration is likely to underlie reduced SOD1 specific activity within the ventral spinal cord of sALS patients given their respective locations within SOD1's dimer interface and β-barrel (Fig. 5f). Reduced Ser98 phosphorylation may, however, impede SOD1- [START_REF] Tsang | Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance[END_REF] Deamidation of Gln15, Asn26, Asn53 and Asn131 was significantly increased in mutant SOD1 protein isolated from the ventral spinal cord of all SOD1-fALS cases compared with controls (Supplementary Tables 8,9). Comparatively mild (2-fold) increases in Asn26 deamidation may destabilize local hydrogen bonding networks in loop II, with similar increases in Gln15 deamidation capable of disrupting hydrogen bonding across the dimer interface to diminish dimer stability (Fig. 5g). Excessive (>10-fold) deamidation of Asn53 within the disulfide loop GDNT motif, and Asn131 within the electrostatic loop, on the other hand, can significantly impact coupling of SOD1 to copper chaperone for SOD1 (CCS) and greatly reduce the stability of mature SOD1 dimers [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF][START_REF] Shi | Deamidation of asparagine to aspartate destabilizes Cu, Zn superoxide dismutase, accelerates fibrillization, and mirrors ALS-linked mutations[END_REF] (Fig. 5g). This may promote SOD1 aggregation in these patients.

Whilst no sites of glycation were detected within SOD1 protein isolated from ALS cases and controls, levels of the advanced glycation end-product carboxyethyllysine were elevated at electrostatic loop residues Lys122 and Lys128 in mutant SOD1 protein isolated from ventral spinal cord of all SOD1-fALS cases compared with controls (Supplementary Tables 8,9). In vitro data suggest charge alterations resulting from these PTMs may disrupt superoxide diffusion towards the active site [START_REF] Fujii | Oxidative stress caused by glycation of Cu,Zn-superoxide dismutase and its effects on intracellular components[END_REF] and alter stabilizing bonds between the electrostatic and disulfide loops, promoting structural disorder and aggregation [START_REF] Kato | New consensus research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 (SOD1) gene mutations: inclusions containing SOD1 in neurons and astrocytes[END_REF] (Fig. 5h).

Tryptic digestion of ubiquitin-conjugated proteins results in Gly-Gly dipeptide remnants on conjugated lysine residues, which serve as indicators of protein ubiquitination. [START_REF] Peng | A proteomics approach to understanding protein ubiquitination[END_REF] Consistent with immunohistochemical analyses of SOD1 aggregates previously reported by our group, [START_REF] Trist | Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain[END_REF] we identified a significant 2.5-fold increase in ubiquitination of mutant SOD1 at Lys91 in all SOD1-fALS cases compared with controls (Supplementary Tables 8,9), indicative of efforts by the ubiquitin-proteosome system to clear mutant disSOD1 from the ventral spinal cord of these cases. It is unclear why similar elevations in ubiquitination were not observed in non-SOD1-fALS or sALS cases exhibiting wild-type disSOD1 deposition, which suggests wild-type disSOD1 does not elicit the same proteostatic response from the UPS as mutant SOD1.

Analysis of free thiol groups in whole protein extracts prepared specially for cysteine redox analysis [START_REF] Rookyard | A Global Profile of Reversible and Irreversible Cysteine Redox Post-Translational Modifications During Myocardial Ischemia/Reperfusion Injury and Antioxidant Intervention[END_REF] 8,9). Whilst the identity of these modifications remains unclear, increases in any candidate modificationsulfenic acid, S-nitrosylation, S-glutathionylation, S-palmitoylation, disulfide bondingnegatively impacts the subcellular localization, structure and/or aggregation propensity of SOD1 protein in vitro. [START_REF] Valle | Cysteine Modifications in the Pathogenesis of ALS[END_REF][START_REF] Redler | Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers[END_REF][START_REF] Toichi | Disulfide Scrambling Describes the Oligomer Formation of Superoxide Dismutase (SOD1) Proteins in the Familial Form of Amyotrophic Lateral Sclerosis[END_REF] Indeed, all four cases exhibiting elevated reversible cysteine modifications possessed significantly higher SOD1 aggregate densities compared with other ALS cases examined (Supplementary Table 9), consistent with a role for redox-induced aggregation of SOD1 in these cases. [START_REF] Rakhit | Oxidation-induced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis[END_REF] It is unclear whether these modifications are upstream or downstream of redox imbalance documented in this region, [START_REF] Barber | Oxidative stress in ALS: key role in motor neuron injury and therapeutic target[END_REF] however we speculate that they likely form a cycle culminating in oxidative stress and a deteriorating antioxidant capacity within ALS spinal cord motor neurons. Whilst we did not identify irreversible Cys PTMs in these cases (sulfinic/sulfonic acid), the greater magnitude of corresponding reductions in unmodified Cys57 (9.3-fold) and Cys146 (8.8-fold) residues (Fig. 5j, Supplementary Table 8) compared with reversible PTMs (2-to-4-fold) in these cases suggests they may be present but are undetectable in complex whole protein extracts.

In summary, we identified significant differences in levels of 15 PTMs to 14 SOD1 protein residues (9%) across 69% of ALS cases compared with controls (Fig. 5k, Supplementary Table 8,9). Importantly, only one of these alterations was also present in the dorsal spinal cord of affected cases (Supplementary Table 8), suggesting that the unique biochemical environment of the diseased ventral spinal cord may underlie alterations to SOD1 PTMs in these cases. Further examinations into these unknown pre-disposing factors for each altered PTM are warranted.

Disruptions to pathways governing SOD1 protein maturation may contribute to SOD1 pathology in ALS cases.

In addition to direct alteration of SOD1 protein biochemistry, the impairment of key molecular chaperones facilitating SOD1 protein maturation promotes disSOD1 proteinopathy in vitro. [START_REF] Luchinat | A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants[END_REF][START_REF] Banci | Atomic-resolution monitoring of protein maturation in live human cells by NMR[END_REF] To evaluate whether disrupted CCS may contribute to disSOD1 pathology in our ALS cases we profiled the subcellular distribution of CCS protein within spinal cord motor neurons and quantified CCS protein levels. Monoclonal and polyclonal antibodies recognizing full-length

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human CCS (Supplementary Table 3) identified CCS protein within cytoplasmic granules of a similar size and shape to those described for immature disSOD1 in approximately 37% of control spinal cord motor neurons (Fig. 6a), consistent with preferential binding of CCS to immature SOD1 to promote metal insertion and maturation to mature SOD1. [START_REF] Luchinat | A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants[END_REF][START_REF] Banci | Atomic-resolution monitoring of protein maturation in live human cells by NMR[END_REF] Low-intensity diffuse cytosolic CCS immunoreactivity was virtually absent within control spinal cord motor neurons (Fig. 6a) Supplementary Table 10).

The proportion of motor neurons possessing granular CCS immunostaining was significantly reduced in all ALS subgroups compared with controls (Fig. 6a,b), indicating alterations to the subcellular localization of CCS within these neurons. The proportions of motor neurons lacking granular CCS and disSOD1 immunostaining were strongly correlated (Fig. 6c), suggesting CCS either contributes, or responds, to disSOD1 mislocalization to the cytoplasm. Diffuse CCS cytoplasmic staining was more intense and more prevalent in motor neurons of ALS cases compared with controls (Fig. 6a,d), and was associated with strong diffuse disSOD1 pathology (Supplementary Figure 13). CCS protein levels were significantly increased in the ventral, but not dorsal, spinal cord of ALS cases compared with controls (Fig. 6e), and CCS and SOD1 protein levels were strongly correlated in the ventral spinal cord (Fig. 6f). CCS-immunopositive protein inclusions of varying morphologies were also observed in the perikarya and neuritic processes of 67-88% of SOD1-fALS motor neurons, as well as occasionally (<10%) in motor neurons of two non-SOD1-fALS and three sALS cases (Fig. 6a, Supplementary Table 10).

Glutathione in its reduced redox state (GSH) can also facilitate SOD1 copper loading and disulfide bond formation. [START_REF] Carroll | Mechanisms for activating Cu-and Zncontaining superoxide dismutase in the absence of the CCS Cu chaperone[END_REF] We quantified a 2.5-to-14-fold reduction in GSH concentrations between all ALS subgroups compared with controls (Fig. 6g). Considering reductions in GSH were correlated with reductions in SOD1 specific activity (Fig. 6h), we speculate that alterations to CCS-independent SOD1 maturation pathways may contribute to immature disSOD1 accumulation in ALS patients by attenuating SOD1 copper loading.

Aside from disruptions to molecular chaperones governing SOD1 maturation, reduced copper or zinc bioavailability may contribute to SOD1 mismetallation in ALS cases (Fig. 3,4). We identified a 47-71% increase in whole tissue zinc levels in the ventral and dorsal spinal cord of all ALS subgroups except the SOD1-fALS dorsal spinal cord (Fig. 6i), suggesting mature SOD1 zinc-deficiency in select ALS cases is not likely to arise from a bulk reduction in zinc 11). To assess whether the compartmentalization of copper or zinc may also be altered in these patients, we partitioned whole tissue extracts into cytosolic and interstitial components (soluble fraction), and those encased within membranes, cellular organelles and other compartments (insoluble fraction). [START_REF] Genoud | Subcellular compartmentalisation of copper, iron, manganese, and zinc in the Parkinson's disease brain[END_REF] Whilst zinc levels remained significantly elevated in both tissue fractions (Fig. 6i), copper was reduced 28-49% selectively in the soluble ventral spinal cord tissue fraction of all ALS subgroups compared with controls (Fig. 6i). Our data suggest zinc is elevated globally in all subcellular compartments of both spinal cord subregions, whilst copper is reduced selectively within the cytosol of ALS spinal cord motor neurons and glia, and the surrounding interstitium. Strong correlations between these biometal alterations and CCS protein levels reinforce a regulatory role for these metals in CCS expression [START_REF] Bertinato | Copper deficiency induces the upregulation of the copper chaperone for Cu/Zn superoxide dismutase in weanling male rats[END_REF] (Fig. 6j,k), whilst additional correlations with higher proportions of motor neurons lacking granular disSOD1 or CCS immunostaining (Fig. 6l,m) suggests immature disSOD1 and CCS mislocalization to the cytoplasm are associated with biometal dysregulation in the ventral spinal cord. Copper redistribution within this region was also associated with reduced GSH (Fig. 6n), consistent with a role for GSH in cellular copper handling, [START_REF] Banci | Affinity gradients drive copper to cellular destinations[END_REF] which may impact SOD1 specific activity in ALS patients by reducing the availability of SOD1's catalytic copper cofactor (Fig. 6o).

Collectively we provide the first post-mortem tissue-derived evidence that alterations to cellular biometals and molecular chaperones facilitating SOD1 protein maturation may contribute to SOD1 mismetallation, aggregation, enzymatic dysfunction and subcellular redistribution in the ventral spinal cord of ALS patients.

Alterations to SOD1 biochemistry clearly demarcate ALS cases from controls

Alterations to SOD1 biochemistry, metallochaperones, and tissue biometal levels, were identified in all ALS cases compared with controls, indicating such factors may constitute biomarkers for ALS. By applying a principal component analysis (PCA) to 23 parameters examined in this study we were able to accurately distinguish ALS cases from controls (Fig 7a).

The first three dimensions of this analysis accounted for the majority (64.6%) of variation between cases, with the clear separation of ALS cases from controls largely a result of dimension 1. This suggests that variables which contributed most to this dimension should be investigated Importantly, we did not observe a clear separation of SOD1-fALS, non-SOD1-fALS and sALS cases across the three primary PCA dimensions (Fig 7a). This is consistent with mounting data demonstrating similarities in the biochemistry of aberrant wild-type and mutant SOD1 proteinopathy in familial and sporadic forms of ALS. [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF] When considered as a single cohort however, we note substantial dispersion of ALS cases across dimensions 2 and 3 of our analysis. This is indicative of substantial heterogeneity in alterations to SOD1 biochemistry between individual ALS cases, in particular whole tissue copper levels, multiple SOD1 PTMs, SOD1 activity and soluble SOD1 metallation (Fig 7c,d). It is possible that such dispersion of ALS cases may arise from differences in the site of disease onset (limb vs bulbar; Fig 7e), or from differences between ALS patient subgroups (SOD1-fALS vs sALS).

Despite substantial heterogeneity in alterations to SOD1 biochemistry between ALS patients, our data suggest SOD1 biochemistry and other variables measured in this study may be able to distinguish ALS cases from controls, warranting further studies examining these factors in larger patient cohorts post-mortem and in the clinic.

Discussion

Despite several decades of intensive research into ALS-associated disease mechanisms in cellular and animal models of SOD1 proteinopathy, this is the first study to construct distinct profiles of the subcellular localization, relative abundance and deposition of mature and immature SOD1 conformers in spinal cord motor neurons of familial and sporadic ALS cases, and age-matched controls. We demonstrate that while large SOD1 protein inclusions are not reliable hallmarks of disease in spinal cord motor neurons of non-SOD1-linked fALS and sALS, the mislocalization of immature, structurally-disordered SOD1 from the nucleus and ER-Golgi network to the cytoplasm are characteristic of this vulnerable region in all forms of ALS. The presence of soluble or insoluble disSOD1 in non-SOD1-fALS and sALS cases is currently controversial. [START_REF] Guareschi | An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1[END_REF][START_REF] Tokuda | Wild-type Cu/Znsuperoxide dismutase is misfolded in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Bosco | Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS[END_REF][START_REF] Rotunno | An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis[END_REF][START_REF] Maier | A human-derived antibody targets misfolded SOD1 and ameliorates motor symptoms in mouse models of amyotrophic lateral sclerosis[END_REF][START_REF] Pare | Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis[END_REF][START_REF] Forsberg | Misfolded SOD1 inclusions in patients with mutations in C9orf72 and other ALS/FTD-associated genes[END_REF][START_REF] Forsberg | Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients[END_REF][START_REF] Cruz | Misfolded SOD1 is not a primary component of sporadic ALS[END_REF][START_REF] Forsberg | Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis[END_REF] We and others [START_REF] Pare | Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis[END_REF] posit this largely derives from methodological differences between studies. Accordingly, our disSOD1 immunostaining protocol adhered to logical guidelines recently proposed by Pare and colleagues for the use of conformation-specific SOD1 antibodies, [START_REF] Pare | Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis[END_REF] which aim to minimize false-positives whilst maximizing tissue antigenicity to avoid false-negatives.

Accompanying our profiles of mature and immature SOD1 abundance, subcellular localization and deposition, we provide the first evidence that immature disSOD1 accumulation in the cytoplasm of ALS spinal cord motor neurons likely arises from both the destabilization of mature SOD1 and from abnormalities in SOD1 maturation in this region of ALS patients. Our novel SEC-nIEF workflow is the first to ensure metallation analyses focus selectively on enzymatically-active mature SOD1, [START_REF] Roudeau | Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow[END_REF] identifying reduced zinc binding and conformational change within mature SOD1 dimers; a finding which has yet to be reported in any transgenic ALS model. We are also the first to conduct such analyses on SOD1 protein isolated in it's native state from human tissues, a factor which we propose is likely to underlie discrepancies between our results and those previously reported from human SOD1-overexpressing transgenic animal and cellular models of ALS. Whilst such over-expression is able to generate a robust ALS phenotype in these models, even wild-type human SOD1 is frequently isolated from these systems exhibiting reduced metallation, [START_REF] Lelie | Copper and zinc metallation status of copper-zinc superoxide dismutase from amyotrophic lateral sclerosis transgenic mice[END_REF][START_REF] Hayward | Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis[END_REF] questioning whether changes identified accurately resemble those characterizing human ALS patients. Indeed, Lelie and colleagues [START_REF] Lelie | Copper and zinc metallation status of copper-zinc superoxide dismutase from amyotrophic lateral sclerosis transgenic mice[END_REF] found copper and zinc binding by endogenous mouse SOD1 was much closer to the expected 1:1 ratio compared with wild-type (Cu:Zn -~1:3) and mutant (Cu:Zn -~1:2.2) human SOD1 isolated from the spinal cord of transgenic mice of the same strain. These discrepancies are proposed to arise from the over-saturation of endogenous metallochaperones driving SOD1 metallation, or from dissimilarities between endogenous and transgenic human SOD1 proteins, warranting further investigation.

We also provide preliminary evidence of disruptions to SOD1's molecular chaperones, CCS and GSH, as well as atypical post-translational modification of structural motifs (e.g. GDNT) governing the maturation of SOD1 in ALS post-mortem tissues. Whilst a previous study by Watanabe and colleagues [START_REF] Watanabe | Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues[END_REF] only identified CCS-immunopositive inclusions in lumbar spinal cord motor neurons of one-of-seventeen sALS cases, and neither of two SOD1-fALS cases, we highlight substantial differences in methodology between this and our study; spinal cord subregions examined, antibodies and dilutions employed and number of spinal cord sections [START_REF] Watanabe | Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues[END_REF] This has already been demonstrated to some extent in vitro, [START_REF] Boyd | Mutations in Superoxide Dismutase 1 (Sod1) Linked to Familial Amyotrophic Lateral Sclerosis Can Disrupt High-Affinity Zinc-Binding Promoted by the Copper Chaperone for Sod1 (Ccs)[END_REF] whereby CCS is reported to possess a reduced efficacy in promoting zinc binding to mutant disSOD1 compared with wild-type SOD1. [START_REF] Boyd | Mutations in Superoxide Dismutase 1 (Sod1) Linked to Familial Amyotrophic Lateral Sclerosis Can Disrupt High-Affinity Zinc-Binding Promoted by the Copper Chaperone for Sod1 (Ccs)[END_REF] We speculate that this suggests structural disorder in immature mutant or wild-type proteins could subsequently result in under-metallation of mature SOD1 or SOD1 aggregate precursors, warranting further investigation. CCS also influences the subcellular compartmentalization of disSOD1 within organelles such as mitochondria by modulating SOD1 disulfide status. [START_REF] Kawamata | Import, Maturation, and Function of SOD1 and Its Copper Chaperone CCS in the Mitochondrial Intermembrane Space[END_REF] This suggests CCS mislocalization may precede and drive that of disSOD1 in ALS spinal cord motor neurons, although alterations to CCS biochemistry have not been thoroughly investigated in ALS post-mortem cases to date. Further profiling of chaperone-SOD1 interactions under physiological and pathological conditions is required to confirm the potential roles of CCS and GSH in the development of atypical SOD1 proteinopathy in ALS.

Many of the altered SOD1 PTMs in this study promote disSOD1 accumulation, [START_REF] Tsang | Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance[END_REF]76 selfassembly and mislocalization in vitro, [START_REF] Trist | Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic[END_REF][START_REF] Banks | Mechanisms of SOD1 regulation by post-translational modifications[END_REF] and are thus also likely to contribute to the development of disSOD1 proteinopathy in the vulnerable ventral spinal cord of ALS patients. Indeed, immature disSOD1 protein is thought to exist on the precipice of aggregation, with even small disturbances in charge or hydrophobicity capable of surpassing small energy barriers between folding states to promote self-assembly. [START_REF] Mojumdar | Partially native intermediates mediate misfolding of SOD1 in single-molecule folding trajectories[END_REF] Additionally, the absence of all but one of these alterations in the dorsal spinal cord of our ALS cases suggests SOD1 proteinopathy is promoted selectively within the ventral spinal cord by endogenous biochemical factors within this region.

Importantly, whilst the majority of biochemical changes to SOD1 identified in this study are specific to the ventral spinal cord, this does not necessarily equate to an aetiological contribution of all of these factors in motor neuron degeneration. Using our post-mortem data alone, it is difficult to establish whether alterations to SOD1 biochemistry precede, or follow, motor neuron death in ALS patients. It is indeed possible that some of the observed changes are attributable to cell death pathways activated by alternative aetiological factors, rather than any important SOD1-dependent mechanism. This is certainly the case for factors we report herein for the first

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time or which are seldom investigated elsewhere to date -disSOD1 and CCS subcellular mislocalization, and alterations to SOD1 phosphorylation, deamidation, acetylation. Whilst reported structural and/or functional consequences of such changes for SOD1 may often imply associations with neuronal damage, [START_REF] Tsang | Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance[END_REF][START_REF] Shi | Deamidation of asparagine to aspartate destabilizes Cu, Zn superoxide dismutase, accelerates fibrillization, and mirrors ALS-linked mutations[END_REF][START_REF] Kawamata | Import, Maturation, and Function of SOD1 and Its Copper Chaperone CCS in the Mitochondrial Intermembrane Space[END_REF] direct examination of the cytotoxicity of these perturbations is required to examine any causal relationships. For other alterations, however, there is ample data from cellular and animal models of ALS to suggest an aetiological link with ALS; hence the inclusion of these variables for investigation in this study. Soluble disSOD1 conformers are widely linked to the protein's toxicity in cellular and animal models of ALS, 46,[START_REF] Gill | SOD1-positive aggregate accumulation in the CNS predicts slower disease progression and increased longevity in a mutant SOD1 mouse model of ALS[END_REF][START_REF] Brotherton | Cellular toxicity of mutant SOD1 protein is linked to an easily soluble, non-aggregated form in vitro[END_REF] especially those that are metal-deficient [START_REF] Roberts | Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis[END_REF][START_REF] Hilton | Metal-deficient SOD1 in amyotrophic lateral sclerosis[END_REF] or are atypically oxidized [START_REF] Ezzi | Wild-type superoxide dismutase acquires binding and toxic properties of ALS-linked mutant forms through oxidation[END_REF] or glycated. [START_REF] Sirangelo | Glycation in Demetalated Superoxide Dismutase 1 Prevents Amyloid Aggregation and Produces Cytotoxic Ages Adducts[END_REF] Taken together, we posit that our data implicate mechanisms of SOD1-dependent toxicity in the death of spinal cord motor neurons in ALS patients.

Interestingly, substantial heterogeneity in the post-translational profile of SOD1 protein between ALS cases (Supplementary Table 9) suggests molecular mechanisms resulting in SOD1 proteinopathy and toxicity are variable between individual ALS patients, which may contribute to variability in disease progression in affected individuals. A similar concept was proposed by Wang and colleagues, [START_REF] Wang | Protein aggregation and protein instability govern familial amyotrophic lateral sclerosis patient survival[END_REF] who reported biochemical differences between mutant SOD1 proteins accounted for 69% of variability in SOD1-fALS patient survival times. Heterogeneity in additional disease-linked mechanisms between our ALS cases may combine with disSOD1 proteinopathy to influence rates of disease progression, including TDP-43 mislocalization and deposition, reported in a related study. The presence of multiple PTM alterations to SOD1 in 63% of cases examined (Supplementary Table 9) also indicates several pathways resulting in SOD1 self-assembly may be present in many patients. Mechanistically, we speculate that structural disorder induced by SOD1 mutations, or early-stage alterations to SOD1 PTMs, may promote subsequent alterations by increasing the solvent accessibility of new PTM sites. This may explain the greater number of alterations to mutant SOD1 in our SOD1-fALS cases compared with wild-type SOD1 in other ALS cases, although this may differ for other mutations not examined herein. Considering the similar disSOD1 conformations identified between cases using conformation-specific disSOD1 antibodies, we posit that these distinct pathways often culminate in common structural endpoints, reinforcing existing studies demonstrating similar disordered motifs within mutant and wild-type SOD1 proteins in ALS patients. Heterogeneity in ALS-linked pathologies has no doubt contributed to the limited success of past clinical trials in this disorder, suggesting multimodal or personalized treatment approaches may be required to meaningfully attenuate disease progression. For example, we reason that the cysteine-reactive compound Ebselen is unlikely to improve SOD1 folding and maturation in patients exhibiting redox modification of SOD1's cysteine residues, [START_REF] Capper | The cysteine-reactive small molecule ebselen facilitates effective SOD1 maturation[END_REF] and cases exhibiting increased oxidation of key proteins such as SOD1 are likely to respond better to antioxidant therapies. Similar categorization of SOD1-fALS patients into metal-binding-region and wildtype-like SOD1 mutants has already been proposed to identify those most likely to respond to copper supplementation using diacetylbis(4-methylthiosemicarbazonato)copper II (Cu II (atsm)), given the negligible protection reported in cultured cells expressing metal-binding-region SOD1 mutants compared with wild-type-like mutants. 86 A notable caveat of this proposal is the current lack of clinical diagnostic assays capable of differentiating these patient subgroups, which will be key to the successful implementation of such personalized approaches. Whilst our study provides preliminary evidence that heterogenous SOD1 biochemistry between ALS patients may enable such stratification of these patients into subgroups that are more amenable to specific therapeutic approaches, difficulties in sourcing greater numbers of familial (SOD1, C9ORF) ALS cases prevents any concrete conclusions being drawn. Further studies examining these factors in larger patient cohorts are warranted.

Overall, we provide post-mortem evidence of pathways of SOD1 neurotoxicity in the spinal cord of ALS patients, which have previously only been characterized in cellular and animal models of ALS. Altered SOD1 biochemistry clearly differentiates all forms of ALS from controls, indicating SOD1 likely constitutes a valuable therapeutic target in all patient groups, irrelevant of SOD1 gene status. Residues are labelled in wild-type SOD1 using three letter amino acid codes and the side chains (d-h) of altered residues are highlighted in red. Details of specific ALS cases exhibiting alterations to each PTM are presented in Supplementary Table 9. Reversible PTMs to Cys57 and Cys146 (i) were significantly increased in SOD1 protein isolated from three SOD1-fALS cases and one sALS case (cluster), compared with controls and remaining ALS cases (One-way ANOVA: Cys 57, p < 0.0001, F = 36.72; Cys146, p < 0.0001, F = 120.2; Dunnet's multiple comparisons post-hoc tests: p < 0.0001 when comparing both Cys57 and Cys146 to controls and remaining ALS cases). Corresponding significant reductions in unmodified Cys57 and Cys146 (j) residues were also identified in these cases compared with controls and remaining ALS cases (Kruskal-Wallis H Test with Dunn's multiple comparisons post-hoc tests for both residues; Cys 57 -cluster vs control: p = 0.03; Cys146 -cluster vs control: p = 0.04). Complete details of statistical analyses presented in i and j are presented in Supplementary Table 8. Data in i and j represent mean±SEM. *p<0.05, ****p<0.0001. k. Distribution of all residues whose posttranslational modification is significantly altered in ALS cases.

(

  King's College, London, UK) and the University of Maryland Brain and Tissue Bank, a

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  of SOD1 (U/mL/SOD1 protein) was determined by normalising SOD1 activity to SOD1 protein levels measured by immunoblotting.

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  Statistical analyses were performed using IBM SPSS Statistics (Version 27, IBM, Armonk, New York, United States) and GraphPad prism (Version 7.02, Graphpad, San Diego, CA, USA). Parametric tests or descriptive statistics with parametric assumptions (standard two-way and oneway ANOVA, Pearson's r and t-test) were used for variables meeting the associated assumptions, with data normality assessed using either the D'Agostino-Pearson (omnibus K2) normality test or the Shapiro-Wilk (Royston) normality test. Two-and one-way ANOVAs were paired with Sidak's and Dunnet's multiple comparisons post-hoc tests, respectively, to assess pair-wise comparisons between select diagnostic groups for a given variable. Non-parametric tests or statistics (Kruskal-Wallis test, Spearman's r and Mann-Whitney U-test) were used for variables where the observed data did not fit the assumptions of parametric tests, with Kruskal-Wallis tests paired with Dunn's multiple comparisons post-hoc tests to assess pair-wise comparisons between

  are present in relatively greater amounts in the nucleus and ER-Golgi network of these neurons.

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  results indicate that conformational change and mismetallation of mature SOD1 may contribute to the accumulation of dysfunctional, immature SOD1 conformers in the ventral spinal cord of ALS patients.

  are altered specifically in the vulnerable ventral spinal cord of ALS cases.

  mediated transcription of protective antioxidant genes by impairing SOD1 nuclear translocation.

  revealed 2-and 4-fold increases in reversible PTMs to Cys57 and Cys146, respectively, in mutant SOD1 protein isolated from the ventral spinal cord of all SOD1-fALS cases, and in wild-type SOD1 protein isolated from the ventral spinal cord of one sALS case, compared with controls and remaining ALS cases (Fig.5i, Supplementary Tables

  levels remained unchanged in these tissues (Fig.6h; Supplementary Table

  further as potential biomarkers of ALS; disSOD1 and CCS subcellular localization, disSOD1 deposition, active SOD1 surface charge (pI) and tissue zinc levels (Fig7b).

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  analyzed per patient. Perhaps most importantly for SOD1-fALS cases, we analyzed tissues from patients carrying I113T and D101G SOD1 mutations, which likely impact SOD1-CCS coupling and metal transfer differently compared with A4V and unknown SOD1 mutants examined byWatanabe et al.

4

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AAAA

  Liu HN, Sanelli T, Horne P, et al. Lack of evidence of monomer/misfolded superoxide dismutase-1 in sporadic amyotrophic lateral sclerosis. Ann Neurol. Jul 2009;66(1):75-80. doi:10.1002/ana.21704 Aquilina JA, Yerbury JJ. Susceptibility of Mutant SOD1 to Form a Destabilized Monomer Predicts Cellular Aggregation and Toxicity but Not In vitro Aggregation Propensity. Front Neurosci. 2016;10:499. doi:10.3389/fnins.2016.00499 Ronchi D, Magri F, Comi GP, Corti S. SOD1 misplacing and mitochondrial dysfunction in amyotrophic lateral sclerosis pathogenesis. Front Cell Neurosci. 2015;9:336. doi:10.3389/fncel.2015.00336 Farrawell NE, Yerbury MR, Plotkin SS, McAlary L, Yerbury JJ. CuATSM Protects Against the In Vitro Cytotoxicity of Wild-Type-Like Copper-Zinc Superoxide Dismutase Mutants but not Mutants That Disrupt Metal Binding. ACS Chem Neurosci. Mar 20 2019;10(3):1555-1564. doi:10.1021/acschemneuro.8b00527

Figure 1 .

 1 Figure 1. DisSOD1 accumulation, aggregation and mislocalization in the vulnerable ventral spinal cord of ALS cases. a, Pan-SOD1 antibodies, as well as conformation-specific antibodies raised against disordered mutant SOD1 (B8H10), disordered SOD1 with an exposed dimer interface (EDI) and disordered SOD1 with an unfolded β-barrel (UBB) were employed to profile the distribution of mature SOD1 and disSOD1 conformers within control (n = 10) ventral spinal cord tissues. These SOD1 antibodies (Supplementary Table3) detected granular cytoplasmic staining (arrowheads) and diffuse cytosolic staining (arrows) within control motor neurons. b, Visualization of granular SOD1 immunostaining within control spinal cord motor neurons using the UBB conformation-specific SOD1 antibody and DAB chromogen. Granular SOD1 immunostaining strongly colocalized with the ER-golgi markers Calreticulin and GM130 in control motor neurons. DAPI was also employed to visualize cell nuclei. c, No immunostaining was observed in spinal cord tissue sections processed as above in the absence of primary antibodies. d, Motor neurons possessing granular SOD1 immunostaining were less abundant in all ALS subgroups compared with controls (One-way ANOVA: p < 0.0001, F = 55.82; Dunnet's multiple comparisons post-hoc tests: p < 0.0001 for all comparisons). e, Granule numbers within surviving ALS motor neurons exhibiting granular SOD1 immunostaining were equivalent to those within control motor neurons (Kruskal-Wallis H test, p = 0.073, H = 6.975). f, In addition to granular and diffuse cytosolic immunostaining, conformation-specific antibodies labelled a variety of disSOD1 inclusions in ventral spinal cord motor neurons of SOD1-fALS (n = 3), non-SOD1-fALS (n = 4) and sALS (n = 9) cases; punctate inclusions (dotted arrowheads), globular inclusions (single downwards arrowheads) and fibrillar skein-like inclusions (double downwards arrowheads). Case numbers (Supplementary Table1) are listed in the top left corner of each panel of a and f. g, Disordered SOD1 conformers immunolabelled by the UBB SOD1 antibody were not colocalized with the ER-golgi markers Calreticulin and GM130 in motor neurons of ALS cases. DAPI was also employed to visualize cell nuclei. Scale bars in a, b, c, f and g represent 25µm. h, Significant reductions in motor neuron density were observed in all ALS subgroups compared with controls (One-way ANOVA: p < 0.0001, F = 30.65; Dunnet's multiple comparisons post-hoc tests: p < 0.0001 for all comparisons). i, The proportion of granular spinal

Figure 2 .

 2 Figure 2. Alterations to SOD1 protein levels and SOD1 specific activity, but not total SOD1 activity, in the vulnerable ventral spinal cord of all ALS cases. a, Total SOD1 antioxidant activity was unchanged in the ventral and dorsal spinal cord of ALS cases compared with controls (Two-way ANOVA; F (3, 36) = 1.888, p = 0.1491). b, SOD1 protein levels were significantly increased in the VSpC (Two-way ANOVA with Sidak's multiple comparisons posthoc test; SOD1-fALS: p = 0.0035; non-SOD1-fALS: p = 0.03; sALS: p = 0.0065), but not DSpC (SOD1-fALS: p = 0.677; non-SOD1-fALS: p = 0.974; sALS: p = 0.999), of all ALS subgroups compared with controls. Representative SOD1 immunoblots (SOD1 2 antibody, Supplementary Table 3) and Sypro Ruby blot staining for total protein in the VSpC are shown. c, SOD1 specific activity (antioxidant activity per unit of SOD1 protein) was significantly decreased in the VSpC (Two-way ANOVA with Sidak's multiple comparisons post-hoc test; SOD1-fALS: p = 0.0488; non-SOD1-fALS: p = 0.0011; sALS: p = 0.0002), but not DSpC (Two-way ANOVA with Sidak's multiple comparisons post-hoc test; SOD1-fALS: p = 0.495; non-SOD1-fALS: p = 0.661; sALS: p = 0.724), of all ALS subgroups compared with controls. Data in a-c represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001. d,e Reductions in SOD1 specific activity were strongly correlated with higher indices of neuron loss (d) and greater proportions of motor neurons lacking granular staining with UBB conformation-specific SOD1 antibody (e).Spearman's r coefficient, p values and the number of XY pairs analysed (n) are stated within panels d and e. A correlation is strong if Spearman's r = 0.5 or higher.

Figure 3 .

 3 Figure 3. Mature SOD1 metallation and surface charge are altered in the vulnerable ventral spinal cord of ALS cases. a. Experimental workflow for measuring mature SOD1 metal

Figure 4 .

 4 Figure 4. Cu:Zn ratios within individual SOD1 aggregates are distinct from purified soluble mature SOD1. a. Experimental workflow for identifying and analysing SOD1 aggregate metal content in post-mortem spinal cord tissues from ALS patients. Following serial sectioning of frozen post-mortem spinal cord tissues onto alternating superfrost slides and Si 3 N 4 synchrotron grids (1), SOD1 aggregates were immunostained on superfrost slides using B8H10 conformation-specific SOD1 antibody (2) and their locations mapped as regions of interest (ROI) using an Olympus Slide Scanner (3). Tissue maps containing ROIs were superimposed over differential interference contrast images of facing, serial, unstained frozen tissue sections mounted on Si 3 N 4 grids (4). Synchrotron radiation was directed at ROIs within unstained tissues mounted on Si 3 N 4 grids using the X-ray fluorescence microscopy (XFM) beamline of the Australian Synchrotron (5), and X-ray fluorescence emission spectra and Compton scatter spectra collected by the Maia detector (6). b,c. Compton scatter and XFM images of representative SOD1 aggregates in the ventral spinal cord of 3 SOD1-fALS and 1 sALS case (b). Scale bars represent 10µm, regions of interest are marked by white dashed circles. Case numbers and SOD1 mutations are listed in the top left hand corner of each panel. Higher Compton scatter values, represented by darker pixels, signify greater tissue matter densities in SOD1 aggregates compared with surrounding ventral horn grey matter tissue (Surr. Tissue; c; Unpaired t-test; p < 0.0001, t = 4.731, df = 66). d-g, Zinc was increased by 70% (d; Unpaired t-test; p < 0.0001, t = 9.339, df = 67), and copper reduced by 12% (e; Unpaired t-test; p < 0.0001, t = 5.012, df = 62), within SOD1 aggregates compared with surrounding grey matter tissues, resulting in a significant decrease in the atomic ratio of copper:zinc within SOD1 aggregates compared with surrounding tissues (f; Mann-Whitney U test; p < 0.0001, U = 8.5) and the copper:zinc ratio of soluble mature SOD1 measured in these cases (g; Mann-Whitney U test; p < 0.0001, U = 0). Data in c-g represent mean±SEM. ****p<0.0001.

Figure 5 .

 5 Figure 5. SOD1 protein structure and locations of PTMs which are significantly altered in the ventral spinal cord of fALS and sALS cases. a,b. Mature SOD1 is dimeric, with each monomer comprising an eight-stranded β-barrel (grey) with one bound Cu (orange) and Zn (cyan) ion. The electrostatic loop (blue) contains charged and polar residues important for guiding anionic superoxide towards Cu in the active site. Three histidine residues and one aspartic acid residue (cyan) within the metal-binding loop (green) facilitate Zn coordination, whilst four histidine residues (orange) mediate Cu coordination. The disulfide loop (yellow) is a

Figure 6 .

 6 Figure 6. Alterations to the subcellular localization and levels of CCS protein, as well as in the levels of reduced glutathione, copper and zinc, in the vulnerable ventral spinal cord of ALS cases. a, Pan-CCS antibodies were employed to profile the distribution of CCS within ventral spinal cord motor neurons from SOD1-fALS (n = 3), non-SOD1-fALS (n = 4) and sALS (n = 9) cases, and controls (n = 10). CCS antibodies (Supplementary Table3) detected granular cytoplasmic staining (arrowheads) and diffuse cytosolic staining (arrows) within ALS and control motor neurons. In addition to granular and diffuse cytosolic immunostaining, CCS antibodies labelled motor neuron inclusions of punctate (dotted arrowheads), globular (single downwards arrowheads) and fibrillar skein-like morphologies (double downwards arrowheads).

1 Figure 6 2 1 Figure 7 2

 1617 Figure 6

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Acknowledgements

Tissues were received from the University of Maryland Brain and Tissue Bank, a biorepository of the NIH NeuroBioBank, as well as from the Sydney Brain Bank, which is supported by Neuroscience Research Australia and the University of New South Wales, and from The London Neurodegenerative Diseases Brain Bank, which receives funding from the Medical Research

Funding

This study was supported by ForeFront, a large collaborative research group dedicated to the study of neurodegenerative diseases and funded by the National Health and Medical Research Council of Australia Program Grant (1132524), Dementia Research Team Grant (1095127) and CogSleep Centre of Research Excellence (1152945). B.G.T., and K.L.D. are funded by the National Health and Medical Research Council of Australia (1181864), Parkinson's NSW, The University of Sydney, MND Research Australia and The Michael J Fox Foundation for Parkinson's Research in partnership with the Shake It Up Australia Foundation. J.A.F is funded by MND Research Australia (Bill Gole Fellowship). P.J.C and K.K were supported by funds from the The Motor Neurone Disease Research Institute of Australia (Betty Laidlaw MND Research Project; Jenny Barr Smith MND Research Project), FightMND (Translational Research Grant), and the University of Melbourne. C.V. is funded by Motor Neurone Disease Association UK. G.H. is an NHMRC Senior Leadership Fellow (1176607).

Data availability

The data and materials that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

All authors report no conflicts of interest.

Supplementary material

Supplementary material is available at Brain online. Large dots with coloured ellipses represent the centroid for that category or grouping and its 95% confidence interval. a, PCA plot of SOD1-fALS (green), non-SOD1-fALS (orange), sALS (red) and control (blue) cases across dimension 1 (x axis), dimension 2 (z axis) and dimension 3 (y axis). b-d, The % contribution of the top 10 most influential variables comprising dimensions 1 -3. The mean % contribution of all variables to each dimension is indicated by a red dotted line. e, PCA plot of ALS cases grouped according to site of onset; bulbar (blue) or limb (red).

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