Altered Nucleolar Phase Separation by NPM1 in ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is a lethal and incurable neurodegenerative disorder commonly associated with repeat expansion in the C9orf72 gene; termed C9-ALS. A major pathological feature of this is the accumulation of arginine-rich (R-rich) dipeptide repeat (DPR) polypeptides in neurons. R-rich DPRs interact with low complexity domains in proteins, accumulate in membraneless organelles (MLOs), alter the material properties of MLOs, and induce cell death. The molecular mechanisms underlying pathogenesis, though, remain unknown. R-rich DPRs infiltrate nucleoli, co-localize with nucleophosmin (NPM1), and alter NPM1 phase separation in vitro. NPM1 is crucial to the maintenance of nucleolar liquid-like properties through its ability to phase separate with proteins and nucleic acids. Elucidating the effects of DPR interactions with NPM1 on the liquid-like properties and overall architecture of nucleoli that ultimately lead to nucleolar dysfunction and cell death is critical to understanding C9-ALS. Here we employ poly(PR) as an archetypal R-rich DPR to (1) identify the interactions mediating phase separation with NPM1; (2) elucidate the mechanisms causing the DPR-dependent dissolution of in vitro NPM1/DPR droplets; and (3) confirm hypotheses stimulated by our biophysical results regarding the mechanisms of DPR-mediated nucleolar disruption in cells. Results from multiple, complementary biochemical and biophysical techniques show that (1) NPM1/DPR interactions are mediated by acidic tracts within the intrinsically disorder region of NPM1, (2) R-rich DPRs dissolve NPM1-containing droplets in vitro by sequestering NPM1 into large saturated complexes, and (3) exogenous poly(PR) induces NPM1 release from nucleoli, disrupting nucleolar organization and function. These results support the hypothesis that R-rich DPRs, mediate their toxic effects in part through saturation/sequestration of NPM1, perturbing NPM1 mediated phase separation in nucleoli, disrupting nucleolar function, and inducing cell death.