Intranuclear aggregation of mutant FUS/TLS as a molecular pathomechanism of amyotrophic lateral sclerosis

Abstract

Dominant mutations in FUS/TLS cause a familial form of amyotrophic lateral sclerosis (fALS), where abnormal accumulation of mutant FUS proteins in cytoplasm has been observed as a major pathological change. Many of pathogenic mutations have been shown to deteriorate the nuclear localization signal in FUS and thereby facilitate cytoplasmic mislocalization of mutant proteins. Several other mutations, however, exhibit no effects on the nuclear localization of FUS in cultured cells, and their roles in the pathomechanism of fALS remain obscure. Here, we show that a pathogenic mutation, G156E, significantly increases the propensities for aggregation of FUS in vitro and in vivo. Spontaneous in vitro formation of amyloid-like fibrillar aggregates was observed in mutant but not wild-type FUS, and notably, those fibrils functioned as efficient seeds to trigger the aggregation of wild-type protein. In addition, the G156E mutation did not disturb the nuclear localization of FUS but facilitated the formation of intranuclear inclusions in rat hippocampal neurons with significant cytotoxicity. We thus propose that intranuclear aggregation of FUS triggered by a subset of pathogenic mutations is an alternative pathomechanism of FUS-related fALS diseases.

Keywords: Amyloid; Amyotrophic Lateral Sclerosis (Lou Gehrig's Disease); Neurodegenerative Diseases; Prions; Protein Aggregation.

FIGURE 1.

Domain organization of FUS. FUS is composed of an N-terminal region rich in Ser, Tyr, Gly, and Gln (SYGQ), three regions rich in the RGG sequence (RGG1, RGG2, and RGG3), an RNA recognition motif (RRM), and a zinc finger motif (ZnF). Mutations in FUS causing fALS are also indicated; RGG1 and a C-terminal region of RGG3 are hot spots for pathogenic mutations.

FIGURE 2.

Purified GST-FUS proteins associate with E. coli endogenous RNAs. A, purification of GST-FUS with GSH-Sepharose resins is examined by SDS-PAGE. E. coli cells overexpressing GST-FUS were lysed and then fractionated by centrifugation into soluble supernatant (sup) and insoluble pellets (pel). The supernatant was treated with GSH-Sepharose resins and separated into the fraction unbound and bound to the resins (unbound and bound, respectively). A band corresponding to GST-FUS is indicated by an arrow. B and C, examination on the purification of GST-FUS proteins with UV-visible absorption spectroscopy. GSH-Sepharose resins binding GST-FUS proteins were washed with a buffer containing 0.1 m NaCl (dotted curve) or 1 m NaCl (solid curve), and then the bound GST-FUS proteins were eluted from the resins and examined with UV-visible spectrophotometer (B). The spectrum of the solution washed out with a buffer containing 1 m NaCl was also shown in C. D, E. coli endogenous nucleic acids bound to GST-FUS were dissociated by washing GSH-Sepharose resins with a buffer containing 1 m NaCl (see B and C). After treatment with either RNase or DNase, the washes were analyzed with urea-PAGE.

FIGURE 3.

Purification and aggregation of GST-FUS-His proteins. A, E. coli cells overexpressing GST-FUS-His were lysed and then fractionated by centrifugation into soluble supernatant (sup) and insoluble pellets (pel). GST-FUS-His in the supernatant fraction was purified with GSH-Sepharose resins (GST) and then with Ni²⁺-affinity chromatography (GST/His). Samples were analyzed by SDS-PAGE using a 10% polyacrylamide gel stained with Coomassie Brilliant Blue. A band corresponding to GST-FUS-His protein was indicated by an arrow. B, Western blotting analysis of purified GST-FUS and GST-FUS-His proteins was performed by using anti-GST antibody. Full-length GST-FUS and GST-FUS-His are indicated by arrows. A GST-FUS sample was found to contain several forms of GST-FUS in which FUS was truncated, whereas most of such truncated proteins were successfully removed in a GST-FUS-His sample. C, UV-visible spectrometric analysis of 10 μg GST-FUS-His quantified by a Bradford assay was performed after purification with GSH-Sepharose resins (dotted curve) and then further with Ni²⁺-affinity chromatography (solid curve). D, 5 μm GST-FUS-His in a TN-trehalose buffer with 250 mm imidazole at pH 8.0 (filled circles) or 6.8 (open circles) was incubated at room temperature, and the solution turbidity was monitored by measuring the absorption at 350 nm. Inset, after incubation for 2 h, sample solutions (T) were fractionated into soluble supernatant (S) and insoluble pellets (I) by ultracentrifugation and analyzed by SDS-PAGE using 10% polyacrylamide gel. GST-FUS-His was found to form insoluble aggregates at pH 8.0 but not at pH 6.8.

FIGURE 4.

G156E mutation facilitates the formation of insoluble aggregates of GST-FUS-His. A, 5 μm GST-FUS-His in a TN-trehalose buffer with 250 mm imidazole, pH 6.8, was incubated at room temperature, and the solution turbidity was monitored by measuring the absorption at 350 nm: wild-type (open circles), G156E (filled circles), G225V (open triangles), M254V (filled triangles), and P525L (open square). B, after incubation for 2 h, the samples were collected, fractionated into soluble supernatant (Sol) and insoluble pellets (Insol) by ultracentrifugation and then analyzed by SDS-PAGE using 10% polyacrylamide gel. C, insoluble pellets collected from the GST-FUS^G156E-His sample incubated for 2 h were examined by an electron microscope after negative staining. A bar at the lower left represents 200 nm. D, after incubation for 2 h, 5 μm GST-FUS-His in TN-trehalose buffer with 250 mm imidazole, pH 6.8, was mixed with 25 μm thioflavin T and then examined by fluorescence spectrometry: GST-FUS^WT-His (broken curve) and GST-FUS^G156E-His (solid curve).

FIGURE 5.

Intracellular localization of wild-type and mutant FUS in SH-SY5Y cells. SH-SY5Y cells were first transfected with HA-FUS with the indicated mutations: A, WT; B, G156E; C, G225V; D, R522G; E, P525L. After differentiation and incubation overnight, the cells were fixed, stained with anti-HA-fluorescein antibody (green), and observed using a confocal microscope. Merged images with nuclei counterstained by DAPI (blue) were also shown in the lower panels. Intranuclear foci observed in cells expressing HA-FUS^G156E were indicated with arrows.

FIGURE 6.

FUS^G156E forms intranuclear inclusions in rat hippocampal primary neurons. A–E, rat hippocampal primary neurons at DIV 7 were transfected with a plasmid for expression of HA-FUS^WT (A), HA-FUS^G156E (B–D), or HA-FUS^P525L (E) and incubated for 2 days at 37 °C. Cells were fixed, stained with anti-HA (red) and anti-MAP2 (green) antibodies, and observed using a confocal microscope. Nuclei were counterstained with Hoechst 33342. In each panel, the image immunostained with anti-HA antibody (magnification: ×63) and the merged images (magnification: ×40) are shown at the left and right, respectively. F, mean intensity of immunofluorescence observed by staining with anti-MAP2 antibody was measured in a cell body with Image J software and found to significantly decrease when HA-FUS with pathogenic mutations were expressed in cells. Measurements were performed in 15, 12, 18, and 6 cells for NT (non-transfected cells), and cells expressing HA-FUS^WT, HA-FUS^G156E, and HA-FUS^P525L, respectively.

FIGURE 7.

A seeded aggregation of wild-type FUS protein with aggregates of FUS^G156Ein vitro and in vivo. A, 0.5 μm (monomer-based concentration) GST-FUS^G156E-His aggregates were added to 5 μm GST-FUS^WT-His in a TN-trehalose buffer with 250 mm imidazole, pH 6.8 (10% seeding), and then the solution turbidity was monitored by measuring the absorbance at 350 nm (filled circles). Without addition of seeds (GST-FUS^G156E-His aggregates), GST-FUS^WT-His did not form aggregates (open circles). B, after incubation for 20 min in A, the GST-FUS^WT-His samples with and without GST-FUS^G156E-His seeds were fractionated into soluble supernatant (Sol) and insoluble pellets (Insol) by ultracentrifugation and then analyzed by SDS-PAGE using 10% polyacrylamide gel. C–E, rat hippocampal primary neurons at DIV 7 were transfected with a plasmid for expression of myc-FUS^WT together with a plasmid for expression of HA-FUS^WT (C) or HA-FUS^G156E (D and E) and incubated for 2 days at 37 °C. Cells were fixed, stained with anti-HA (red) and anti-myc (green) antibodies, and observed using a confocal microscope. Nuclei were counterstained with Hoechst 33342.

FIGURE 8.

Effects of pathogenic mutations on the intracellular behavior of FUS. FUS protein translated at the cytoplasm (upper right) is transported to the nucleus by function of its C-terminal PY-NLS and then regulates various processes such as translation and splicing (upper left). Impairment of PY-NLS by mutations in the C-terminal region (e.g. P525L) has been shown to significantly reduce the nuclear fractions of FUS. Even with intact PY-NLS, we have found here that the G156E mutation increases aggregation propensities of FUS in the nucleus (lower left). Once aggregates of mutant FUS form, seeding reactions further facilitate the aggregation of FUS including wild-type counterparts, resulting in loss of physiological functions of FUS and possibly exerting cytotoxicity. The amino acid sequence of the FUS SYGQ domain is also shown, and the concentration of Gln, Asn, and Tyr residues is evident near Gly¹⁵⁶ (underlined).