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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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Characterization of proteinaceous inclusions in FUS-TG mice.A, FUS-positive inclusions in motor neurons of symptomatic FUS-TG mice are resistant to treatment with 200 μg/ml of proteinase K for 30 min (A) or 1 h (A′). B, ubiquitinated inclusions (arrows) in the spinal cord of a symptomatic FUS-TG animal. C, immunofluorescent detection of ubiquitin (green) and FUS (red) in a section through the ventral horns of the spinal cord of a symptomatic FUS-TG mouse. Some FUS-positive inclusions in motor neurons are also ubiquitin-positive, whereas other cells bear FUS-positive/ubiquitin-negative or/and FUS-negative/ubiquitin-positive inclusions (arrows and arrowheads, respectively). D, spinal cord sections of wild type (D) and FUS-TG (D′) mice stained with antibodies against the C terminus (C term) of FUS for detection of the endogenous protein (green) and against the N terminus (N term) of FUS for detection of both endogenous and human FUS 1–359 proteins (red). Endogenous FUS is recruited into nuclear aggregates, formed by human FUS 1–359 (arrowheads). A cytoplasmic FUS 1–359 inclusion (arrow) is negative for endogenous FUS. E, antibody 1482 that specifically recognizes only mouse FUS in both wild type and transgenic animals also detects intranuclear (cell marked by one asterisk) or even cytoplasmic (cell marked by two asterisks and enlarged in E″, arrows) inclusions in a subset of spinal motor neurons of FUS-TG mice (E′). Scale bars: A and A′, 50 μm; B–E′, 30 μm; E″, 10 μm.
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Figure 3: Characterization of proteinaceous inclusions in FUS-TG mice.A, FUS-positive inclusions in motor neurons of symptomatic FUS-TG mice are resistant to treatment with 200 μg/ml of proteinase K for 30 min (A) or 1 h (A′). B, ubiquitinated inclusions (arrows) in the spinal cord of a symptomatic FUS-TG animal. C, immunofluorescent detection of ubiquitin (green) and FUS (red) in a section through the ventral horns of the spinal cord of a symptomatic FUS-TG mouse. Some FUS-positive inclusions in motor neurons are also ubiquitin-positive, whereas other cells bear FUS-positive/ubiquitin-negative or/and FUS-negative/ubiquitin-positive inclusions (arrows and arrowheads, respectively). D, spinal cord sections of wild type (D) and FUS-TG (D′) mice stained with antibodies against the C terminus (C term) of FUS for detection of the endogenous protein (green) and against the N terminus (N term) of FUS for detection of both endogenous and human FUS 1–359 proteins (red). Endogenous FUS is recruited into nuclear aggregates, formed by human FUS 1–359 (arrowheads). A cytoplasmic FUS 1–359 inclusion (arrow) is negative for endogenous FUS. E, antibody 1482 that specifically recognizes only mouse FUS in both wild type and transgenic animals also detects intranuclear (cell marked by one asterisk) or even cytoplasmic (cell marked by two asterisks and enlarged in E″, arrows) inclusions in a subset of spinal motor neurons of FUS-TG mice (E′). Scale bars: A and A′, 50 μm; B–E′, 30 μm; E″, 10 μm.

Mentions: Immunohistochemical analysis of the central nervous system of symptomatic FUS-TG mice using an antibody that recognizes the N-terminal epitope in both mouse and human proteins (Fig. 2, A′–D′) or an antibody specific to the human protein (Fig. 2, F and G) revealed multiple FUS-positive inclusions primarily in the lower motor neuron cell bodies, axons and, in a subset of cells, in the nucleus (Fig. 2A′, inset). Similar inclusions were observed in other neurons throughout the nervous system of symptomatic animals, including upper motor neurons in the motor cortex (Fig. 2D′). In the spinal cord of FUS-TG mice, large eosinophilic inclusions were occasionally detected, which were not seen in non-transgenic littermates (data not shown). Similar to proteinaceous inclusions typical for certain neurodegenerative diseases (19, 20), both cytoplasmic and nuclear inclusions in neurons of FUS-TG mice were resistant to proteinase K treatment (Fig. 3, A and A′). However, they were not stained by the amyloid-detecting dyes Congo Red or thioflavin S, which is also a feature of FUS-positive inclusions in ALS and related diseases (21). Consistent with the presence of ubiquitinated FUS-positive inclusions in these human diseases (14), ubiquitin-positive inclusions of various size and morphology were often observed in motor neurons of FUS-TG mice (Fig. 3B). However, double immunofluorescent staining showed that only a fraction of FUS-positive inclusions was ubiquitinated, and in some cells, non-overlapping FUS and ubiquitin inclusions were present (Fig. 3C). In some neurons that developed intranuclear FUS inclusions, endogenous protein (detected using an antibody that recognizes the C terminus of FUS) became recruited to these structures (Fig. 3D′). Immunostaining with a mouse-specific antibody also revealed nuclear and rarely cytoplasmic (Fig. 3E′ and enlarged in Fig. 3E″) inclusions in spinal motor neurons. Therefore, the truncated form of FUS is able to seed aggregation of normal FUS protein.


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)

Characterization of proteinaceous inclusions in FUS-TG mice.A, FUS-positive inclusions in motor neurons of symptomatic FUS-TG mice are resistant to treatment with 200 μg/ml of proteinase K for 30 min (A) or 1 h (A′). B, ubiquitinated inclusions (arrows) in the spinal cord of a symptomatic FUS-TG animal. C, immunofluorescent detection of ubiquitin (green) and FUS (red) in a section through the ventral horns of the spinal cord of a symptomatic FUS-TG mouse. Some FUS-positive inclusions in motor neurons are also ubiquitin-positive, whereas other cells bear FUS-positive/ubiquitin-negative or/and FUS-negative/ubiquitin-positive inclusions (arrows and arrowheads, respectively). D, spinal cord sections of wild type (D) and FUS-TG (D′) mice stained with antibodies against the C terminus (C term) of FUS for detection of the endogenous protein (green) and against the N terminus (N term) of FUS for detection of both endogenous and human FUS 1–359 proteins (red). Endogenous FUS is recruited into nuclear aggregates, formed by human FUS 1–359 (arrowheads). A cytoplasmic FUS 1–359 inclusion (arrow) is negative for endogenous FUS. E, antibody 1482 that specifically recognizes only mouse FUS in both wild type and transgenic animals also detects intranuclear (cell marked by one asterisk) or even cytoplasmic (cell marked by two asterisks and enlarged in E″, arrows) inclusions in a subset of spinal motor neurons of FUS-TG mice (E′). Scale bars: A and A′, 50 μm; B–E′, 30 μm; E″, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Characterization of proteinaceous inclusions in FUS-TG mice.A, FUS-positive inclusions in motor neurons of symptomatic FUS-TG mice are resistant to treatment with 200 μg/ml of proteinase K for 30 min (A) or 1 h (A′). B, ubiquitinated inclusions (arrows) in the spinal cord of a symptomatic FUS-TG animal. C, immunofluorescent detection of ubiquitin (green) and FUS (red) in a section through the ventral horns of the spinal cord of a symptomatic FUS-TG mouse. Some FUS-positive inclusions in motor neurons are also ubiquitin-positive, whereas other cells bear FUS-positive/ubiquitin-negative or/and FUS-negative/ubiquitin-positive inclusions (arrows and arrowheads, respectively). D, spinal cord sections of wild type (D) and FUS-TG (D′) mice stained with antibodies against the C terminus (C term) of FUS for detection of the endogenous protein (green) and against the N terminus (N term) of FUS for detection of both endogenous and human FUS 1–359 proteins (red). Endogenous FUS is recruited into nuclear aggregates, formed by human FUS 1–359 (arrowheads). A cytoplasmic FUS 1–359 inclusion (arrow) is negative for endogenous FUS. E, antibody 1482 that specifically recognizes only mouse FUS in both wild type and transgenic animals also detects intranuclear (cell marked by one asterisk) or even cytoplasmic (cell marked by two asterisks and enlarged in E″, arrows) inclusions in a subset of spinal motor neurons of FUS-TG mice (E′). Scale bars: A and A′, 50 μm; B–E′, 30 μm; E″, 10 μm.
Mentions: Immunohistochemical analysis of the central nervous system of symptomatic FUS-TG mice using an antibody that recognizes the N-terminal epitope in both mouse and human proteins (Fig. 2, A′–D′) or an antibody specific to the human protein (Fig. 2, F and G) revealed multiple FUS-positive inclusions primarily in the lower motor neuron cell bodies, axons and, in a subset of cells, in the nucleus (Fig. 2A′, inset). Similar inclusions were observed in other neurons throughout the nervous system of symptomatic animals, including upper motor neurons in the motor cortex (Fig. 2D′). In the spinal cord of FUS-TG mice, large eosinophilic inclusions were occasionally detected, which were not seen in non-transgenic littermates (data not shown). Similar to proteinaceous inclusions typical for certain neurodegenerative diseases (19, 20), both cytoplasmic and nuclear inclusions in neurons of FUS-TG mice were resistant to proteinase K treatment (Fig. 3, A and A′). However, they were not stained by the amyloid-detecting dyes Congo Red or thioflavin S, which is also a feature of FUS-positive inclusions in ALS and related diseases (21). Consistent with the presence of ubiquitinated FUS-positive inclusions in these human diseases (14), ubiquitin-positive inclusions of various size and morphology were often observed in motor neurons of FUS-TG mice (Fig. 3B). However, double immunofluorescent staining showed that only a fraction of FUS-positive inclusions was ubiquitinated, and in some cells, non-overlapping FUS and ubiquitin inclusions were present (Fig. 3C). In some neurons that developed intranuclear FUS inclusions, endogenous protein (detected using an antibody that recognizes the C terminus of FUS) became recruited to these structures (Fig. 3D′). Immunostaining with a mouse-specific antibody also revealed nuclear and rarely cytoplasmic (Fig. 3E′ and enlarged in Fig. 3E″) inclusions in spinal motor neurons. Therefore, the truncated form of FUS is able to seed aggregation of normal FUS protein.

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