Recruitment into stress granules prevents irreversible aggregation of FUS protein mislocalized to the cytoplasm

Abstract

Fused in sarcoma (FUS) belongs to the group of RNA-binding proteins implicated as underlying factors in amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. Multiple FUS gene mutations have been linked to hereditary forms, and aggregation of FUS protein is believed to play an important role in pathogenesis of these diseases. In cultured cells, FUS variants with disease-associated amino acid substitutions or short deletions affecting nuclear localization signal (NLS) and causing cytoplasmic mislocalization can be sequestered into stress granules (SGs). We demonstrated that disruption of motifs responsible for RNA recognition and binding not only prevents SG recruitment, but also dramatically increases the protein propensity to aggregate in the cell cytoplasm with formation of juxtanuclear structures displaying typical features of aggresomes. Functional RNA-binding domains from TAR DNA-binding protein of 43 kDa (TDP-43) fused to highly aggregation-prone C-terminally truncated FUS protein restored the ability to enter SGs and prevented aggregation of the chimeric protein. Truncated FUS was also able to trap endogenous FUS molecules in the cytoplasmic aggregates. Our data indicate that RNA binding and recruitment to SGs protect cytoplasmic FUS from aggregation, and loss of this protection may trigger its pathological aggregation in vivo.

Keywords: FUSopathy; RNA-binding proteins; amyotrophic lateral sclerosis; cytoplasmic RNP complexes; protein aggregation; proteinopathy.

Figure 1. Characterization of mutant FUS proteins transiently expressed in neuroblastoma SH-SY5Y cells. (A) Scheme of FUS domain organization and GFP-fusion constructs. (B) All GFP-labeled FUS variants utilized in the study are expressed at a comparable level in neuroblastoma cells as revealed with anti-GFP antibody. Total protein extracts for western blot analysis were prepared from cells lysed 24 h after transfection with corresponding expression plasmid. C is an unrelated cytosolic GFP-fusion protein used as a control. The size of proteins in kDa is shown. (C) Diffuse distribution of cytoplasmically localized FUS variants FUS R522G, FUS 1–513, and FUS ΔRRMcyt is detected in transfected cells at early stages of the ectopic protein accumulation (4–12 h post-transfection) or in cells with relatively low levels of its expression. In contrast, in most cells expressing FUS 1–359, small aggregates are formed very early after transfection. Scale bar: 20 µm. (D) Intracellular distribution of GFP-FUS fusion proteins and morphology of aggregates formed in the cytoplasm 36 h post-transfection. See also Videos S1 and S2. (E) Twenty-four hours after transfection, the majority of cells expressing FUS 1–359 possess aggregates of some form, and more than half of them display large juxtanuclear structures. (F) Juxtanuclear aggregates formed by FUS 1–359 protein are negative for endosomal/lysosmal compartment markers LAMP-1 and LAMP-2. Scale bars: (D), 15 µm; (C and F), 20 µm.

Figure 2. Aggregates formed by truncated FUS 1–359 in SH-SY5Y cells display typical characteristics of aggresomes. (A) Large juxtanuclear structures formed by FUS 1–359 are clustered around the centrosome, visualized with centrosome marker gamma-tubulin (arrowheads). (B) Anti-vimentin staining of cells expressing FUS 1–359 reveals a characteristic “vimentin cage” around large juxtanuclear structures. (C) Formation of juxtanuclear aggregates by FUS 1–359 (arrow) is prevented by nocodazole-induced disruption of microtubules. Scale bar: 15 µm for all panels.

Figure 3. Truncated FUS 1–359 is not recruited into stress granules in naïve SH-SY5Y cells. Representative confocal images of cells transfected with plasmids expressing various GFP-tagged FUS variants and stained with antibodies that detect stress granules. Foci spontaneously formed by FUS R522G or FUS 1–513 in a fraction of transfected cells are positive for TIAR (A) or G3BP1 (B), markers for stress granules, whereas neither large juxtanuclear, nor small dispersed aggregates formed by FUS 1–359 contain these proteins. (C) Emetine treatment disperses foci formed by FUS R522G, but has no effect on FUS 1–359 aggregates. Scale bars: 15 µm for all panels.

Figure 4. Truncated FUS 1–359 is not recruited into stress granules upon oxidative insult. (A) Cells transfected with GFP-tagged FUS expressing plasmids were treated with 0.5 mM sodium arsenite for 1 h and stained with anti-TIAR antibody. Foci formed by FUS R522G and FUS 1–513 are positive for TIAR, while FUS 1–359 aggregates (arrows) do not co-localize with TIAR-positive stress granules (arrowheads). (B) Cycloheximide treatment blocks formation of stress granules including those containing FUS 1–513, but does not affect FUS 1–359 aggregates. Scale bar: 15 µm for all panels.

Figure 5. Aggregation of truncated FUS 1–359 in cultured cells can be prevented by fusion with functional RNA-binding domains of TDP-43. (A) Scheme of FUS-TDP-43 chimeric proteins. (B) Western blot analysis of FUS 1–359 and FUS-TDP-43 fusions using anti-GFP antibody demonstrates their equal levels upon expression in neuroblastoma SH-SY5Y cells. The size of proteins in kDa is shown. (C) Intracellular distribution of GFP-tagged chimeric proteins in transfected SH-SY5Y cells reveals granule-like aggregates with diffuse distribution of FUS-TDP-43 with intact RRMs and formation of aggresomes when RNA-binding capacity of RRM1 is abolished by 3 amino acid substitutions, L106D, V108D, L111D (3D mutant). Scale bar: 15 µm for both panels. (D and E) FUS-TDP-43 with intact RRMs (D) but not FUS-TDP-43 bearing substitutions in RRM1 disrupting RNA binding (E) is able to enter stress granules induced by arsenite treatment. Scale bar: 15 µm for all panels.

Figure 6. Truncated FUS 1–359 protein is able to sequester both normal and mutated FUS variants in aggregates. Cells were co-transfected with shown GFP-tagged FUS variants, and stained with antibody against C terminus of FUS. Cytoplasmic variants FUS R522G and FUS 1–513 are recruited into morphologically distinct aggregates formed by FUS 1–359 (arrowheads). In some, relatively rare cells FUS 1–359 recruits into these aggregates even a fraction of co-expressed wild-type FUS or endogenous wild-type FUS retained in the cytoplasm (lower panel). Scale bar: 10 µm for all panels.

Figure 7. A mechanism of pathological aggregation of FUS protein in the cell cytoplasm. A proposed sequence of internal and external events triggering the development of FUSopathy. FUS-containing stress granules are shown in yellow, and FUS aggregates in green.