Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow

Studies of the metal content of metalloproteins in tissues from the human central nervous system (CNS) can be compromised by preparative techniques which alter levels of, or interactions between, metals and the protein of interest within a complex mixture. We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and either proton or synchrotron X-ray fluorescence within electrophoresis gels to analyze the endogenous metal content of copper-zinc superoxide dismutase (SOD1) purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis and Parkinson’s disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. Validating our novel approach, we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein versus confounding metalloproteins. We analyzed tissues from nine healthy individuals and five CNS regions (occipital cortex, substantia nigra, locus coeruleus, dorsal spinal cord, and ventral spinal cord). We found that Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28, a ratio very close to the expected value of 1. Our methodological workflow can be applied to the study of endogenous native SOD1 in a pathological context and adapted to a range of metalloproteins from human tissues and other sources

Co-deposition of SOD1, TDP-43 and p62 proteinopathies in ALS:evidence for multifaceted pathways underlying neurodegeneration

Multiple neurotoxic proteinopathies co-exist within vulnerable neuronal populations in all major neurodegenerative diseases. Interactions between these pathologies may modulate disease progression, suggesting they may constitute targets for disease-modifying treatments aiming to slow or halt neurodegeneration. Pairwise interactions between superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43) and ubiquitin-binding protein 62/sequestosome 1 (p62) proteinopathies have been reported in multiple transgenic cellular and animal models of amyotrophic lateral sclerosis (ALS), however corresponding examination of these relationships in patient tissues is lacking. Further, the coalescence of all three proteinopathies has not been studied in vitro or in vivo to date. These data are essential to guide therapeutic development and enhance the translation of relevant therapies into the clinic. Our group recently profiled SOD1 proteinopathy in post-mortem spinal cord tissues from familial and sporadic ALS cases, demonstrating an abundance of structurally-disordered (dis)SOD1 conformers which become mislocalized within these vulnerable neurons compared with those of aged controls. To explore any relationships between this, and other, ALS-linked proteinopathies, we profiled TDP-43 and p62 within spinal cord motor neurons of the same post-mortem tissue cohort using multiplexed immunofluorescence and immunohistochemistry. We identified distinct patterns of SOD1, TDP43 and p62 co-deposition and subcellular mislocalization between motor neurons of familial and sporadic ALS cases, which we primarily attribute to SOD1 gene status. Our data demonstrate co-deposition of p62 with mutant and wild-type disSOD1 and phosphorylated TDP-43 in familial and sporadic ALS spinal cord motor neurons, consistent with attempts by p62 to mitigate SOD1 and TDP-43 deposition. Wild-type SOD1 and TDP-43 co-deposition was also frequently observed in ALS cases lacking SOD1 mutations. Finally, alterations to the subcellular localization of the three proteins were tightly correlated, suggesting close relationships between the regulatory mechanisms governing the subcellular compartmentalization of these proteins. Our study is the first to profile spatial relationships between SOD1, TDP-43 and p62 pathologies in post-mortem spinal cord motor neurons of ALS patients, previously only studied in vitro. Our findings suggest interactions between these three key ALS-linked proteins are likely to modulate the formation of their respective proteinopathies, and perhaps the rate of motor neuron degeneration, in ALS patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40478-022-01421-9

Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: A Methodological Workflow

We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and high sensitivity X-ray-based metal detection within electrophoresis gels to analyze the metal content of single proteins purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. An important metalloprotein in the human central nervous system is copper-zinc superoxide dismutase (SOD1), an antioxidant enzyme linked to the aetiology of both amyotrophic lateral sclerosis and Parkinson’s disease. Abnormal SOD1 metallation is suspected to play a role in the pathogenic aggregation of SOD1 in both disorders, although data describing SOD1 metal occupancy in human tissues has not previously been reported. Validating our novel approach we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein vs confounding metalloproteins. We found Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28 in human central nervous system tissues from healthy individuals, a ratio close to the expected value of 1. Our methodological workflow can be adapted to study a range of metalloproteins from human tissues and other sources.<br /

Coupling SEC and IEF to SXRF for metallation analysis of SOD1 enzyme isolated from the human central nervous system

Copper-zinc superoxide dismutase enzyme (SOD1) is one of the 16,227 expressed proteins in the central nervous system (CNS). Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis (ALS) and Parkinson’s disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. The analysis of metals in metalloproteins under native conditions remains a difficult exercise, especially when the protein is present at physiological levels in a complex tissue. In this work, we developed a methodological workflow that allows synchrotron X-ray fluorescence (SXRF) analysis of metalloenzymes isolated from small amounts of tissue. This protocol allowed us to characterize the metallation of endogenous SOD1 in the human CNS from only 20 mg of post-mortem tissue. In a first 2D-chromatography step, a soluble protein extract is prepared from post-mortem CNS tissues and then separated according to molecular weight using Size Exclusion Chromatography (SEC), and then according to isoelectric point by native isoelectric focusing (IEF) in gel. The processed samples were analyzed by SXRF in the microprobe hutch of the Hard X-ray Micro/Nano-Probe beamline P06 at PETRA III (DESY) in Hamburg (Germany). In total, we analyzed post-mortem CNS tissue from 30 individuals (17 without neurological disease and 13 ALS cases). The Cu/Zn atomic ratio in active SOD1 from control cases was 1.12 ± 0.28 (mean±sd) and did not differ between CNS regions or individuals. To our knowledge, this is the first report of a ratio value close to the theoretical value of 1 in active SOD1 isolated from human tissue. In addition to its excellent detection limit (∼0.1 µg/g), SXRF also offers excellent repeatability (Cu/Zn ratio in SOD1 standard=0.93 +/-0.01, n=9). Furthermore, our protocol preserves both the metallation and the activity of SOD1, and reduces proteome complexity by 97.8%. 2D chromatography enriches SOD1 99-fold compared to IEF alone, and also improves the accuracy and precision of the measurements (mean Cu/Zn=1.12 vs 2.39, sd = 0.28 vs 1.48). Using this SXRF-based method we revealed that SOD1 metallation is altered in ALS. This method is applicable to other metalloproteins when only small amounts of tissue are available and excellent sensitivity and reproducibility are required

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

International audienceAberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce.We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration.We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases.Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development

Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson’s disease brain

Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and oxidative stress. Emerging data regarding overlapping proteinopathy in traditionally distinct neurodegenerative diseases suggest that disease-modifying treatments targeting these pathological features may exhibit efficacy across multiple disorders. Here, we describe proteinopathy distinct from classic synucleinopathy, predominantly comprised of the anti-oxidant enzyme superoxide dismutase-1 (SOD1), in the Parkinson’s disease brain. Significant expression of this pathology closely reflected the regional pattern of neuronal loss. The protein composition and non-amyloid macrostructure of these novel aggregates closely resembles that of neurotoxic SOD1 deposits in SOD1-associated familial amyotrophic lateral sclerosis (fALS). Consistent with the hypothesis that deposition of protein aggregates in neurodegenerative disorders reflects upstream dysfunction, we demonstrated that SOD1 in the Parkinson’s disease brain exhibits evidence of misfolding and metal deficiency, similar to that seen in mutant SOD1 in fALS. Our data suggest common mechanisms of toxic SOD1 aggregation in both disorders and a potential role for SOD1 dysfunction in neuronal loss in the Parkinson’s disease brain. This shared restricted proteinopathy highlights the potential translation of therapeutic approaches targeting SOD1 toxicity, already in clinical trials for ALS, into disease-modifying treatments for Parkinson’s disease