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Fused in sarcoma (FUS) protein lacking nuclear localization signal (NLS) and major RNA binding motifs triggers proteinopathy and severe motor phenotype in transgenic mice.

Shelkovnikova TA, Peters OM, Deykin AV, Connor-Robson N, Robinson H, Ustyugov AA, Bachurin SO, Ermolkevich TG, Goldman IL, Sadchikova ER, Kovrazhkina EA, Skvortsova VI, Ling SC, Da Cruz S, Parone PA, Buchman VL, Ninkina NN - J. Biol. Chem. (2013)

Bottom Line: Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases.To address this question, we designed a variant of FUS, FUS 1-359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding.These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5-4.5 months and death of affected animals within several days of onset.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom.

ABSTRACT
Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1-359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1-359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5-4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1-359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.

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Proteinopathy in the nervous system of FUS-TG mice.A–H, paraffin sections were stained with an antibody (ab) recognizing an N-terminal epitope in both mouse and human FUS (A–D, A′–D′) or antibody 1480 specific to human protein (E–H). Multiple intracellular FUS-positive inclusions were revealed in the cytoplasm (arrowheads), nucleus (inset), and axons (arrows) of spinal cord (A′) and brainstem (B′) neurons of symptomatic FUS-TG but not of non-transgenic littermate (A and B, respectively) mice. Similar inclusions were detected in the brainstem (F) and spinal cord (G) of transgenic mice using human FUS-specific antibody. Truncated FUS accumulates in axons (white arrows) and forms spheroids (black arrows) in brainstem tracts of symptomatic FUS-TG (C′) but not of non-transgenic littermate (C) mice. In cortical neurons of symptomatic FUS-TG mice (D′), FUS accumulates and forms inclusions in cell bodies (black arrows) and neurites (white arrows), whereas in non-transgenic littermates, endogenous FUS is confined to cell nuclei (D). E, staining of a spinal cord section from a non-transgenic animal shows that human FUS-specific antibody 1480 does not react with mouse FUS protein. H, FUS-positive inclusions (arrowheads) are occasionally detected in neurons of 9-month-old transgenic mice of low-expressing line 6. Scale bars: A–F, 50 μm; G, 30 μm; H, 15 μm.
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Figure 2: Proteinopathy in the nervous system of FUS-TG mice.A–H, paraffin sections were stained with an antibody (ab) recognizing an N-terminal epitope in both mouse and human FUS (A–D, A′–D′) or antibody 1480 specific to human protein (E–H). Multiple intracellular FUS-positive inclusions were revealed in the cytoplasm (arrowheads), nucleus (inset), and axons (arrows) of spinal cord (A′) and brainstem (B′) neurons of symptomatic FUS-TG but not of non-transgenic littermate (A and B, respectively) mice. Similar inclusions were detected in the brainstem (F) and spinal cord (G) of transgenic mice using human FUS-specific antibody. Truncated FUS accumulates in axons (white arrows) and forms spheroids (black arrows) in brainstem tracts of symptomatic FUS-TG (C′) but not of non-transgenic littermate (C) mice. In cortical neurons of symptomatic FUS-TG mice (D′), FUS accumulates and forms inclusions in cell bodies (black arrows) and neurites (white arrows), whereas in non-transgenic littermates, endogenous FUS is confined to cell nuclei (D). E, staining of a spinal cord section from a non-transgenic animal shows that human FUS-specific antibody 1480 does not react with mouse FUS protein. H, FUS-positive inclusions (arrowheads) are occasionally detected in neurons of 9-month-old transgenic mice of low-expressing line 6. Scale bars: A–F, 50 μm; G, 30 μm; H, 15 μm.

Mentions: To confirm a high aggregation propensity of the engineered FUS 1–359 protein (Fig. 1A) in the cytoplasm of eukaryotic cells, we expressed it as a GFP fusion in the human neuroblastoma SH-SY5Y cell line. Indeed, in contrast to FUS lacking epitopes 2 and 3 of NLS (18) but retaining all other domains (FUS 1–513), which was diffusely distributed or occasionally formed small cytoplasmic granules even 48 h after transfection, FUS 1–359 rapidly aggregated and formed large perinuclear inclusions (Fig. 1B, arrows) as early as 12 h after transfection despite the same levels of expression for both proteins (Fig. 1C). Therefore, we proceeded to generation and characterization of transgenic mice expressing human FUS 1–359 protein in their nervous system under control of the Thy-1 promoter (Fig. 1D). Transgenic lines FUS 1–359 TG 6 and FUS 1–359 TG 19 were established from two independent founders. In the central nervous system of hemizygous FUS 1–359 TG 19 mice, the 55-kDa truncated human protein was expressed at a lower level than the 70-kDa endogenous mouse FUS protein (Fig. 1E), and these animals developed a severe neurological phenotype early in their life. Animals of FUS 1–359 TG line 6 expressed substantially less human protein in their nervous system when compared with line 19 (Fig. 1F), and only in aging homozygous line 6 mice were FUS-positive aggregates observed in a small proportion of lower motor neurons (see Fig. 2H). Therefore, we focused our studies on characterizing pathology in hemizygous FUS 1–359 TG 19 mice (herein “FUS-TG”). These mice were indistinguishable from their wild type littermates until the age of 2.5–4.5 months, at which point transgenic animals abruptly developed severe motor dysfunction. A typical pattern at the onset of clinical signs included gait impairment caused by asymmetrical pareses and eventual complete paralyzes of limbs (Fig. 1, G and H, and supplemental Video S1). The disease rapidly progressed and spread to other groups of muscles, and typically affected animals became emaciated and dehydrated and the lost righting reflex and the ability to move freely within several days of onset. Because transformation of a visibly healthy animal into one exhibiting severe pathological phenotypes starts any time within an ∼2-month interval and always occurs extremely quickly, it was not possible to obtain statistically sound data for any parameters of the disease progression for a cohort of transgenic mice. However, it was possible to predict with absolute confidence that after an individual animal develops the first signs of unstable gait or/and displays slightly compromised performance in the inverted grid test, it will reach the terminal stage of the disease within a maximum of 2 weeks. The lifespan of FUS-TG mice did not exceed 5 months (Fig. 1I).


Fused in sarcoma (FUS) protein lacking nuclear localization signal (NLS) and major RNA binding motifs triggers proteinopathy and severe motor phenotype in transgenic mice.

Shelkovnikova TA, Peters OM, Deykin AV, Connor-Robson N, Robinson H, Ustyugov AA, Bachurin SO, Ermolkevich TG, Goldman IL, Sadchikova ER, Kovrazhkina EA, Skvortsova VI, Ling SC, Da Cruz S, Parone PA, Buchman VL, Ninkina NN - J. Biol. Chem. (2013)

Proteinopathy in the nervous system of FUS-TG mice.A–H, paraffin sections were stained with an antibody (ab) recognizing an N-terminal epitope in both mouse and human FUS (A–D, A′–D′) or antibody 1480 specific to human protein (E–H). Multiple intracellular FUS-positive inclusions were revealed in the cytoplasm (arrowheads), nucleus (inset), and axons (arrows) of spinal cord (A′) and brainstem (B′) neurons of symptomatic FUS-TG but not of non-transgenic littermate (A and B, respectively) mice. Similar inclusions were detected in the brainstem (F) and spinal cord (G) of transgenic mice using human FUS-specific antibody. Truncated FUS accumulates in axons (white arrows) and forms spheroids (black arrows) in brainstem tracts of symptomatic FUS-TG (C′) but not of non-transgenic littermate (C) mice. In cortical neurons of symptomatic FUS-TG mice (D′), FUS accumulates and forms inclusions in cell bodies (black arrows) and neurites (white arrows), whereas in non-transgenic littermates, endogenous FUS is confined to cell nuclei (D). E, staining of a spinal cord section from a non-transgenic animal shows that human FUS-specific antibody 1480 does not react with mouse FUS protein. H, FUS-positive inclusions (arrowheads) are occasionally detected in neurons of 9-month-old transgenic mice of low-expressing line 6. Scale bars: A–F, 50 μm; G, 30 μm; H, 15 μm.
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Figure 2: Proteinopathy in the nervous system of FUS-TG mice.A–H, paraffin sections were stained with an antibody (ab) recognizing an N-terminal epitope in both mouse and human FUS (A–D, A′–D′) or antibody 1480 specific to human protein (E–H). Multiple intracellular FUS-positive inclusions were revealed in the cytoplasm (arrowheads), nucleus (inset), and axons (arrows) of spinal cord (A′) and brainstem (B′) neurons of symptomatic FUS-TG but not of non-transgenic littermate (A and B, respectively) mice. Similar inclusions were detected in the brainstem (F) and spinal cord (G) of transgenic mice using human FUS-specific antibody. Truncated FUS accumulates in axons (white arrows) and forms spheroids (black arrows) in brainstem tracts of symptomatic FUS-TG (C′) but not of non-transgenic littermate (C) mice. In cortical neurons of symptomatic FUS-TG mice (D′), FUS accumulates and forms inclusions in cell bodies (black arrows) and neurites (white arrows), whereas in non-transgenic littermates, endogenous FUS is confined to cell nuclei (D). E, staining of a spinal cord section from a non-transgenic animal shows that human FUS-specific antibody 1480 does not react with mouse FUS protein. H, FUS-positive inclusions (arrowheads) are occasionally detected in neurons of 9-month-old transgenic mice of low-expressing line 6. Scale bars: A–F, 50 μm; G, 30 μm; H, 15 μm.
Mentions: To confirm a high aggregation propensity of the engineered FUS 1–359 protein (Fig. 1A) in the cytoplasm of eukaryotic cells, we expressed it as a GFP fusion in the human neuroblastoma SH-SY5Y cell line. Indeed, in contrast to FUS lacking epitopes 2 and 3 of NLS (18) but retaining all other domains (FUS 1–513), which was diffusely distributed or occasionally formed small cytoplasmic granules even 48 h after transfection, FUS 1–359 rapidly aggregated and formed large perinuclear inclusions (Fig. 1B, arrows) as early as 12 h after transfection despite the same levels of expression for both proteins (Fig. 1C). Therefore, we proceeded to generation and characterization of transgenic mice expressing human FUS 1–359 protein in their nervous system under control of the Thy-1 promoter (Fig. 1D). Transgenic lines FUS 1–359 TG 6 and FUS 1–359 TG 19 were established from two independent founders. In the central nervous system of hemizygous FUS 1–359 TG 19 mice, the 55-kDa truncated human protein was expressed at a lower level than the 70-kDa endogenous mouse FUS protein (Fig. 1E), and these animals developed a severe neurological phenotype early in their life. Animals of FUS 1–359 TG line 6 expressed substantially less human protein in their nervous system when compared with line 19 (Fig. 1F), and only in aging homozygous line 6 mice were FUS-positive aggregates observed in a small proportion of lower motor neurons (see Fig. 2H). Therefore, we focused our studies on characterizing pathology in hemizygous FUS 1–359 TG 19 mice (herein “FUS-TG”). These mice were indistinguishable from their wild type littermates until the age of 2.5–4.5 months, at which point transgenic animals abruptly developed severe motor dysfunction. A typical pattern at the onset of clinical signs included gait impairment caused by asymmetrical pareses and eventual complete paralyzes of limbs (Fig. 1, G and H, and supplemental Video S1). The disease rapidly progressed and spread to other groups of muscles, and typically affected animals became emaciated and dehydrated and the lost righting reflex and the ability to move freely within several days of onset. Because transformation of a visibly healthy animal into one exhibiting severe pathological phenotypes starts any time within an ∼2-month interval and always occurs extremely quickly, it was not possible to obtain statistically sound data for any parameters of the disease progression for a cohort of transgenic mice. However, it was possible to predict with absolute confidence that after an individual animal develops the first signs of unstable gait or/and displays slightly compromised performance in the inverted grid test, it will reach the terminal stage of the disease within a maximum of 2 weeks. The lifespan of FUS-TG mice did not exceed 5 months (Fig. 1I).

Bottom Line: Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases.To address this question, we designed a variant of FUS, FUS 1-359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding.These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5-4.5 months and death of affected animals within several days of onset.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom.

ABSTRACT
Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1-359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1-359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5-4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1-359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.

Show MeSH
Related in: MedlinePlus