SARS-CoV-2 Nucleocapsid Protein forms biomolecular condensates and interacts with ALS-associated RNA binding proteins in vitro

As we have entered the endemic phase of COVID-19, there is a need to assess the long-term effects of SARS-CoV-2 infection on the trajectory of neurodegenerative disorders. Herein, we investigate whether the SARS-CoV-2 RNA-binding nucleocapsid protein (NCP; N-protein) may influence molecular processes relevant to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We detected N-protein immunoreactivity in ALS spinal cord motor neurons, including cytoplasmic N-protein-positive puncta that colocalized with cytoplasmic TAR DNA-binding protein 43 (TDP-43). In vitro, optogenetically induced N-protein biomolecular condensates (BMCs) showed reversible, liquid-like behavior and recovered after photobleaching. Using this model, we found that these condensates colocalized with TDP-43 and the RNA-binding proteins G3BP1/2, PABPC1 and TIA1. Condensate formation was associated with TDP-43 cytoplasmic mislocalization, altered TARDBP expression, and altered exon 3 splicing of POLDIP3. The N-terminal portion of N-protein influenced BMC formation, whereas truncated C-terminal proteoforms did not form detectable BMCs or show detectable TDP-43 colocalization in this assay. Extending these observations to a human cell model, we show that optogenetically induced N-protein BMCs colocalize with TDP-43 in human iPSC-derived motor neurons. Collectively, these findings support further investigation into potential molecular links between SARS-CoV-2 exposure and the trajectory of neurodegenerative disease.