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ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

Murakami T, Qamar S, Lin JQ, Schierle GS, Rees E, Miyashita A, Costa AR, Dodd RB, Chan FT, Michel CH, Kronenberg-Versteeg D, Li Y, Yang SP, Wakutani Y, Meadows W, Ferry RR, Dong L, Tartaglia GG, Favrin G, Lin WL, Dickson DW, Zhen M, Ron D, Schmitt-Ulms G, Fraser PE, Shneider NA, Holt C, Vendruscolo M, Kaminski CF, St George-Hyslop P - Neuron (2015)

Bottom Line: One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation.Nuclear FUS granules may be similarly affected.Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Tanz Centre for Research in Neurodegenerative Diseases, and Departments of Medicine, Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 3H2, Canada.

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FUS Constructs, Cellular Localization, and Neurotoxicity of Mutant FUS(A) FUS constructs and mutations. Full-length FUS constructs include wild-type FUS(WT) and ALS/FTD mutations (R522G, R524S, P525L, and R495X), or FUS (FUS501), which mimics several ALS/FTD truncation mutants. LC motif at residues 2–214 was deleted in FUS(WT)ΔLC, FUS(P525L)ΔLC, and FUS501ΔLC. FUS(LC)-only constructs include wild-type FUS(LC), pathogenic S96del or G156E mutants, or clinically benign P21H and N63S sequences. LC, low-complexity motif; G-Rich, glycine-rich region; RRM, RNA recognition motif; RGG-rich, arginine-glycine-glycine-rich region.(B) FUS(WT) is located in neuronal nuclei; mutant FUS accumulates as 8 M urea-soluble assemblies in neuronal cytoplasm. Western blot of high-salt buffer extracted whole-worm lysates (top), 8 M urea extracted lysates (middle), and α-tubulin (bottom).(C) FUS LC domain is necessary and sufficient for FUS assembly and neurotoxicity. Deletion of LC domain from FUS(P525L) and FUS 501 reduces formation of 8 M urea-soluble FUS (left panel), improves motor function (middle panel), and lifespan in mutant FUS lines (right panel), but has no impact on FUS(WT) animals. N.S., not significant. ∗∗p < 0.01; ∗∗∗p < 0.001.(D) FUS LC domain is necessary and sufficient for FUS assembly and neurotoxicity. Overexpression of wild-type FUS(LC) alone is associated with increased 8 M urea-soluble assemblies (left panel), impaired motor function (middle panel), and lifespan (right panel). The ALS/FTD FUS(LC)-mutations (S96del and G156E) further augment neurotoxicity. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Error bars are SEM.
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fig1: FUS Constructs, Cellular Localization, and Neurotoxicity of Mutant FUS(A) FUS constructs and mutations. Full-length FUS constructs include wild-type FUS(WT) and ALS/FTD mutations (R522G, R524S, P525L, and R495X), or FUS (FUS501), which mimics several ALS/FTD truncation mutants. LC motif at residues 2–214 was deleted in FUS(WT)ΔLC, FUS(P525L)ΔLC, and FUS501ΔLC. FUS(LC)-only constructs include wild-type FUS(LC), pathogenic S96del or G156E mutants, or clinically benign P21H and N63S sequences. LC, low-complexity motif; G-Rich, glycine-rich region; RRM, RNA recognition motif; RGG-rich, arginine-glycine-glycine-rich region.(B) FUS(WT) is located in neuronal nuclei; mutant FUS accumulates as 8 M urea-soluble assemblies in neuronal cytoplasm. Western blot of high-salt buffer extracted whole-worm lysates (top), 8 M urea extracted lysates (middle), and α-tubulin (bottom).(C) FUS LC domain is necessary and sufficient for FUS assembly and neurotoxicity. Deletion of LC domain from FUS(P525L) and FUS 501 reduces formation of 8 M urea-soluble FUS (left panel), improves motor function (middle panel), and lifespan in mutant FUS lines (right panel), but has no impact on FUS(WT) animals. N.S., not significant. ∗∗p < 0.01; ∗∗∗p < 0.001.(D) FUS LC domain is necessary and sufficient for FUS assembly and neurotoxicity. Overexpression of wild-type FUS(LC) alone is associated with increased 8 M urea-soluble assemblies (left panel), impaired motor function (middle panel), and lifespan (right panel). The ALS/FTD FUS(LC)-mutations (S96del and G156E) further augment neurotoxicity. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Error bars are SEM.

Mentions: To rigorously test the idea that mutations in FUS alter its assembly into RNP granules, we first explored the biology of FUS in a C. elegans model of FUS proteinopathy (Murakami et al., 2012). In this model, equivalent levels of wild-type or mutant human FUS protein are expressed under the neuron-specific Prgef-1 promoter with a GFP tag, which does not alter the activity or location of FUS (Murakami et al., 2012). We focused on animals (n ≥ 3 lines per mutant) expressing wild-type FUS (FUS(WT)) or one of five ALS/FTD mutants: FUS (R495X), FUS(R522G), FUS(R524S), FUS(P525L), and FUS501(which models several ALS/FTD-associated C-terminal truncating mutations) (Figure 1A). These mutations span a wide range of predicted aggregation propensities as calculated using the Zyggregator method (Tartaglia and Vendruscolo, 2008): FUS(R495X): Zagg = 0.44; FUS(R522G): Zagg = 0.09; FUS(R524S): Zagg = 0.05; FUS(P525L): Zagg = 0.12; FUS501: Zagg = 0.52 (Figure S4). The aggregation propensities of these mutants correlate with their phenotypic severities in C. elegans and in humans, ranging from mild (R522G) to severe (FUS (P525L), FUS (R495X), and FUS501) (Murakami et al., 2012) (Figure S5).


ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

Murakami T, Qamar S, Lin JQ, Schierle GS, Rees E, Miyashita A, Costa AR, Dodd RB, Chan FT, Michel CH, Kronenberg-Versteeg D, Li Y, Yang SP, Wakutani Y, Meadows W, Ferry RR, Dong L, Tartaglia GG, Favrin G, Lin WL, Dickson DW, Zhen M, Ron D, Schmitt-Ulms G, Fraser PE, Shneider NA, Holt C, Vendruscolo M, Kaminski CF, St George-Hyslop P - Neuron (2015)

FUS Constructs, Cellular Localization, and Neurotoxicity of Mutant FUS(A) FUS constructs and mutations. Full-length FUS constructs include wild-type FUS(WT) and ALS/FTD mutations (R522G, R524S, P525L, and R495X), or FUS (FUS501), which mimics several ALS/FTD truncation mutants. LC motif at residues 2–214 was deleted in FUS(WT)ΔLC, FUS(P525L)ΔLC, and FUS501ΔLC. FUS(LC)-only constructs include wild-type FUS(LC), pathogenic S96del or G156E mutants, or clinically benign P21H and N63S sequences. LC, low-complexity motif; G-Rich, glycine-rich region; RRM, RNA recognition motif; RGG-rich, arginine-glycine-glycine-rich region.(B) FUS(WT) is located in neuronal nuclei; mutant FUS accumulates as 8 M urea-soluble assemblies in neuronal cytoplasm. Western blot of high-salt buffer extracted whole-worm lysates (top), 8 M urea extracted lysates (middle), and α-tubulin (bottom).(C) FUS LC domain is necessary and sufficient for FUS assembly and neurotoxicity. Deletion of LC domain from FUS(P525L) and FUS 501 reduces formation of 8 M urea-soluble FUS (left panel), improves motor function (middle panel), and lifespan in mutant FUS lines (right panel), but has no impact on FUS(WT) animals. N.S., not significant. ∗∗p < 0.01; ∗∗∗p < 0.001.(D) FUS LC domain is necessary and sufficient for FUS assembly and neurotoxicity. Overexpression of wild-type FUS(LC) alone is associated with increased 8 M urea-soluble assemblies (left panel), impaired motor function (middle panel), and lifespan (right panel). The ALS/FTD FUS(LC)-mutations (S96del and G156E) further augment neurotoxicity. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Error bars are SEM.
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fig1: FUS Constructs, Cellular Localization, and Neurotoxicity of Mutant FUS(A) FUS constructs and mutations. Full-length FUS constructs include wild-type FUS(WT) and ALS/FTD mutations (R522G, R524S, P525L, and R495X), or FUS (FUS501), which mimics several ALS/FTD truncation mutants. LC motif at residues 2–214 was deleted in FUS(WT)ΔLC, FUS(P525L)ΔLC, and FUS501ΔLC. FUS(LC)-only constructs include wild-type FUS(LC), pathogenic S96del or G156E mutants, or clinically benign P21H and N63S sequences. LC, low-complexity motif; G-Rich, glycine-rich region; RRM, RNA recognition motif; RGG-rich, arginine-glycine-glycine-rich region.(B) FUS(WT) is located in neuronal nuclei; mutant FUS accumulates as 8 M urea-soluble assemblies in neuronal cytoplasm. Western blot of high-salt buffer extracted whole-worm lysates (top), 8 M urea extracted lysates (middle), and α-tubulin (bottom).(C) FUS LC domain is necessary and sufficient for FUS assembly and neurotoxicity. Deletion of LC domain from FUS(P525L) and FUS 501 reduces formation of 8 M urea-soluble FUS (left panel), improves motor function (middle panel), and lifespan in mutant FUS lines (right panel), but has no impact on FUS(WT) animals. N.S., not significant. ∗∗p < 0.01; ∗∗∗p < 0.001.(D) FUS LC domain is necessary and sufficient for FUS assembly and neurotoxicity. Overexpression of wild-type FUS(LC) alone is associated with increased 8 M urea-soluble assemblies (left panel), impaired motor function (middle panel), and lifespan (right panel). The ALS/FTD FUS(LC)-mutations (S96del and G156E) further augment neurotoxicity. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Error bars are SEM.
Mentions: To rigorously test the idea that mutations in FUS alter its assembly into RNP granules, we first explored the biology of FUS in a C. elegans model of FUS proteinopathy (Murakami et al., 2012). In this model, equivalent levels of wild-type or mutant human FUS protein are expressed under the neuron-specific Prgef-1 promoter with a GFP tag, which does not alter the activity or location of FUS (Murakami et al., 2012). We focused on animals (n ≥ 3 lines per mutant) expressing wild-type FUS (FUS(WT)) or one of five ALS/FTD mutants: FUS (R495X), FUS(R522G), FUS(R524S), FUS(P525L), and FUS501(which models several ALS/FTD-associated C-terminal truncating mutations) (Figure 1A). These mutations span a wide range of predicted aggregation propensities as calculated using the Zyggregator method (Tartaglia and Vendruscolo, 2008): FUS(R495X): Zagg = 0.44; FUS(R522G): Zagg = 0.09; FUS(R524S): Zagg = 0.05; FUS(P525L): Zagg = 0.12; FUS501: Zagg = 0.52 (Figure S4). The aggregation propensities of these mutants correlate with their phenotypic severities in C. elegans and in humans, ranging from mild (R522G) to severe (FUS (P525L), FUS (R495X), and FUS501) (Murakami et al., 2012) (Figure S5).

Bottom Line: One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation.Nuclear FUS granules may be similarly affected.Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Tanz Centre for Research in Neurodegenerative Diseases, and Departments of Medicine, Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 3H2, Canada.

Show MeSH
Related in: MedlinePlus