Insights into Nucleocytoplasmic Transport Decline in FUS-mediated ALS and Nanobody Biologics for SOD1-mediated ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. The pathogenic mechanisms driving ALS are unclear, and no effective treatment is available. Herein, focusing on two ALS-linked proteins: Fused in Sarcoma (FUS) and Superoxide dismutase 1 (SOD1), I investigated the molecular bases of ALS pathophysiology and propose potential biologics for preclinical testing.

Altered nucleocytoplasmic transport is an emerging, yet poorly understood disease pathway in FUS-mediated ALS and neurodegeneration. To understand the mechanism(s) underlying FUS-induced nucleocytoplasmic transport decline, I performed biochemical analyses of an interaction between FUS and the nucleoporin Nup62 that was recently uncovered by our lab. Phase separation of FUS and Phe/Gly-rich nucleoporins including Nup62 is implicated in their respective cellular functions. Using recombinant proteins, I found that FUS and Nup62 alter the phase separation properties of each other in a manner that is modulated by RNA. Our results implicate that the cytoplasmic versus nuclear environment influences FUS/Nup62 interaction, thereby supporting the notion that nucleocytoplasmic transport dysregulation is driven by ALS-mutation-induced mislocalization of FUS within the cytoplasm. Further, I found that the C-terminal domain of Nup62 plays an important role in the FUS/Nup62 interaction. Since this domain mediates Nup62 localization to the nuclear envelope, our results support a model whereby an interaction with cytoplasmic mutant FUS impairs Nup62 homeostasis in nucleocytoplasmic transport.

As the first known genetic cause of ALS, SOD1 pathobiology has been studied longer than that of FUS. Notably, ALS-linked mutations induce toxic, misfolded SOD1 conformations which are potentially targetable for therapy. Herein, I characterized anti-SOD1 nanobodies that are capable of modulating the cellular properties of SOD1. Notably, a beneficial effect of anti-SOD1 nanobody expression was observed in human ALS-SOD1 neurons. Thus, this study supports the therapeutic potential of nanobody-based biologics for ALS-SOD1.