A Deletion of the Nuclear Localization Signal Domain in the Fus Protein Induces Stable Post-stress Cytoplasmic Inclusions in SH-SY5Y Cells

Abstract

Mutations in Fused-in-Sarcoma (FUS) gene involving the nuclear localization signal (NLS) domain lead to juvenile-onset Amyotrophic Lateral Sclerosis (ALS). The mutant protein mislocalizes to the cytoplasm, incorporating it into Stress Granules (SG). Whether SGs are the first step to the formation of stable FUS-containing aggregates is still unclear. In this work, we used acute and chronic stress paradigms to study the SG dynamics in a human SH-SY5Y neuroblastoma cell line carrying a deletion of the NLS domain of the FUS protein (homozygous: ΔNLS–/–; heterozygous: ΔNLS+/–). Wild-type (WT) cells served as controls. We evaluated the subcellular localization of the mutant protein through immunoblot and immunofluorescence, in basal conditions and after acute stress and chronic stress with sodium arsenite (NaAsO2). Cells were monitored for up to 24 h after rescue. FUS was expressed in both nucleus and cytoplasm in the ΔNLS+/– cells, whereas it was primarily cytoplasmic in the ΔNLS–/–. Acute NaAsO2 exposure induced SGs: at rescue,>90% of ΔNLS cells showed abundant FUS-containing if compared to less than 5% of the WT cells. The proportion of FUS-positive SGs remained 15–20% at 24 h in mutant cells. Cycloheximide did not abolish the long-lasting SGs in mutant cells. Chronic exposure to NaAsO2 did not induce significant SGs formation. A wealth of research has demonstrated that ALS-associated FUS mutations at the C-terminus facilitate the incorporation of the mutant protein into SGs. We have shown here that mutant FUS-containing SGs tend to fail to dissolve after stress, facilitating a liquid-to-solid phase transition. The FUS-containing inclusions seen in the dying motor neurons might therefore directly derive from SGs. This might represent an attractive target for future innovative therapies.

Role of the Ubiquitin System in Stress Granule Metabolism

“…The SG-associated protein FUS (fused in sarcoma) is exported from the nucleus to the cytoplasm under stress conditions and associates with SGs. Several disease-linked mutations in FUS cause its cytoplasmic mislocalization and increase its association with SGs [ 87 , 99 , 100 , 101 ]. At SGs, mutant FUS may then aggregate into cross-beta fibril structures, thereby promoting a liquid-to-solid phase transition of SGs [ 31 , 94 , 100 ].…”

“…Several disease-linked mutations in FUS cause its cytoplasmic mislocalization and increase its association with SGs [ 87 , 99 , 100 , 101 ]. At SGs, mutant FUS may then aggregate into cross-beta fibril structures, thereby promoting a liquid-to-solid phase transition of SGs [ 31 , 94 , 100 ].…”

Emerging Roles of RNA-Binding Proteins in Neurodevelopment

“…( D ) List of RBP domains associated with disease and dysfunction. Common diseases and abnormalities are included [ 249 , 250 , 251 , 252 , 253 , 254 ].…”

IFNγ protects motor neurons from oxidative stress via enhanced global protein synthesis in FUS‐associated amyotrophic lateral sclerosis

“…While some mutations affect the N‐terminal region, arginine‐glycine–glycine box (RGG) and RNA recognition motif (RRM) region, most missense mutations affect the nuclear localization signal (NLS) domain in the C‐terminus of the protein. These mutations result in cytoplasmic mislocalization and nuclear clearance of FUS and yield an aggressive disease phenotype [ 9 , 10 ]. The most common FUS mutation affects arginine 521 (R to H, C, or G) [ 11 ].…”