Induction of wild-type SOD1 misfolding, aggregation and its cell-to-cell propagation

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration and loss of motor neurons that appears to spread through the neuroaxis in a spatiotemporally restricted manner. Misfolded Cu/Zn superoxide dismutase (SOD1) has been detected in all ALS patients, despite SOD1 mutations accounting for only 2% of total cases, while the presence of inclusions containing pathological TAR-DNA binding protein-43 (TDP-43) represent a hallmark of all non-SOD1/FUS familial ALS. We previously reported that TDP-43 and FUS can trigger misfolding of human wild-type SOD1 (HuWtSOD1) in living cells, however the mechanisms and consequences are unknown. Here, we used immunocytochemistry, immunoprecipitation and cell viability studies to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. Furthermore, we developed several chimeric SOD1-GFP proteins that we validated to aggregate in the presence of pathological SOD1 or TDP-43 seed. We used this assay, along with immunofluorescence, live-cell microscopy and flow cytometry studies, to show that intermolecular conversion of SOD1 by pathological TDP-43 is mediated by tryptophan residues in both proteins. Furthermore, we used the reporter proteins to show that human spinal cord extracts prepared from familial, but not sporadic, ALS patients can trigger SOD1 aggregation in cultured cells. Finally, we used this system to show that small molecules, akin to 5-fluorouridine, can block this intermolecular kindling of SOD1 aggregation, and demonstrated that our assay can be used as a high-throughput tool for screening drugs against induced SOD1 aggregation. Altogether, our studies indicate that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of tryptophan-dependent propagated SOD1 misfolding. Furthermore, it is key to recognize that elucidation of the pathogenic role of a simple structural motif in ALS may provide a framework for understanding other neurodegenerative diseases in which propagated protein misfolding is shown to occur.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat