Unveiling the double-edged sword: SOD1 trimers possess tissue-selective toxicity and bind septin-7 in motor neuron-like cells

Abstract Misfolded soluble trimeric species of superoxide dismutase 1 (SOD1) are associated with increased death in neuron-like cell models and greater disease severity in amyotrophic lateral sclerosis (ALS) patients compared to insoluble protein aggregates. The mechanism by which structurally independent SOD1 trimers cause cellular toxicity is unknown but may be a driver of disease pathology. Here, we uncovered the SOD1 trimer interactome – a map of potential tissue-selective protein binding partners in the brain, spinal cord, and skeletal muscle. We identified binding partners and key pathways associated with SOD1 trimers, comparing them to those of wild-type SOD1 dimers. We found that trimers may affect normal cellular functions such as dendritic spine morphogenesis and synaptic function in the central nervous system and cellular metabolism in skeletal muscle. We also identified key pathways using transcriptomic data from motor neuron-like cells (NSC-34s) expressing SOD1 trimers. We discovered differential gene expression in cells that express SOD1 trimers with selective enrichment of genes responsible for protein localization to membranes and a global upregulation of cellular senescence pathways. We performed detailed computational and biochemical characterization of protein binding for septin-7, an SOD1 trimer binding partner. We found that septin-7 preferentially binds SOD1 trimers and co-localizes in neuron-like cells. We explore a double-edged sword theory regarding the toxicity of SOD1 trimers. These trimers are implicated in causing dysfunction not only in the central nervous system but also in muscle tissues. Our investigation highlights key protein factors and pathways within each system, revealing a plausible intersection of genetic and pathophysiological mechanisms in ALS through interactions involving SOD1 trimers. Summary In amyotrophic lateral sclerosis (ALS), misfolded soluble species of superoxide dismutase 1 (SOD1) are associated with disease severity and, specifically, trimeric forms of SOD1 are toxic in neuron-like cells compared to insoluble aggregates. The role of toxic SOD1 trimers in cells is unknown. Using molecular engineering and pull-down experiments, we found that SOD1 trimers have tissue-selective protein interactions that affect pathways such as dendritic spine morphogenesis and synaptic function in the nerves, energy, and amino acid metabolism in skeletal muscle. We investigated the SOD1 trimer transcriptome to reveal a global upregulation of genes associated with cellular senescence compared to SOD1 dimers. We further validated septin-7, a shared brain and spinal cord protein binding hit, using integrative computational and biochemical approaches, and confirmed that septin-7 binds SOD1 trimers and not native dimers. Taken together, we show evidence that SOD1 trimers play a central role in the convergence of ALS pathophysiology. Graphical abstract