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A polyalanine tract expansion in Arx forms intranuclear inclusions and results in increased cell death.

Nasrallah IM, Minarcik JC, Golden JA - J. Cell Biol. (2004)

Bottom Line: Transfection of these constructs results in nuclear protein aggregation, filamentous nuclear inclusions, and an increase in cell death.Coexpressing Hsp70 decreases the percentage of cells with nuclear inclusions.Finally, we show that expressing mutant Arx in mouse brains results in neuronal nuclear inclusion formation.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
A growing number of human disorders have been associated with expansions of a tract of a single amino acid. Recently, polyalanine (polyA) tract expansions in the Aristaless-related homeobox (ARX) protein have been identified in a subset of patients with infantile spasms and mental retardation. How alanine expansions in ARX, or any other transcription factor, cause disease have not been determined. We generated a series of polyA expansions in Arx and expressed these in cell culture and brain slices. Transfection of these constructs results in nuclear protein aggregation, filamentous nuclear inclusions, and an increase in cell death. These inclusions are ubiquitinated and recruit Hsp70. Coexpressing Hsp70 decreases the percentage of cells with nuclear inclusions. Finally, we show that expressing mutant Arx in mouse brains results in neuronal nuclear inclusion formation. Our data suggest expansions in one of the ARX polyA tracts results in nuclear protein aggregation and an increase in cell death; likely underlying the pathogenesis of the associated infantile spasms and mental retardation.

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Expanded Arx nuclear inclusions are ubiquitinated. (a and b) COS cells expressing ArxE aggregate ArxE into nuclear inclusions (a). These nuclear inclusions stain positively for ubiquitin (b). (c and d) In contrast, cells expressing wild-type Arx do not form nuclear inclusions (c), nor do they show nuclear ubiquitin staining (d). Bar, 10 μm.
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fig3: Expanded Arx nuclear inclusions are ubiquitinated. (a and b) COS cells expressing ArxE aggregate ArxE into nuclear inclusions (a). These nuclear inclusions stain positively for ubiquitin (b). (c and d) In contrast, cells expressing wild-type Arx do not form nuclear inclusions (c), nor do they show nuclear ubiquitin staining (d). Bar, 10 μm.

Mentions: Untransfected COS cells and those expressing wild-type Arx (Fig. 3 c) show faint ubiquitin staining primarily in the cytoplasm (Fig. 3 d and not depicted). The nuclei of these control cells are generally devoid of ubiquitin reactivity, but occasionally have one or two punctate areas of faint ubiquitin staining that are always located centrally (Fig. 3 d). In contrast, COS cells transfected with ArxE constructs have strong immunoreactivity to ubiquitin at the periphery of the nucleus corresponding to the nuclear inclusions (Fig. 3, a and b). Interestingly, despite ArxE expression throughout the nucleus in cells that do not form inclusions, we only detected ubiquitination when nuclear inclusions form.


A polyalanine tract expansion in Arx forms intranuclear inclusions and results in increased cell death.

Nasrallah IM, Minarcik JC, Golden JA - J. Cell Biol. (2004)

Expanded Arx nuclear inclusions are ubiquitinated. (a and b) COS cells expressing ArxE aggregate ArxE into nuclear inclusions (a). These nuclear inclusions stain positively for ubiquitin (b). (c and d) In contrast, cells expressing wild-type Arx do not form nuclear inclusions (c), nor do they show nuclear ubiquitin staining (d). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172475&req=5

fig3: Expanded Arx nuclear inclusions are ubiquitinated. (a and b) COS cells expressing ArxE aggregate ArxE into nuclear inclusions (a). These nuclear inclusions stain positively for ubiquitin (b). (c and d) In contrast, cells expressing wild-type Arx do not form nuclear inclusions (c), nor do they show nuclear ubiquitin staining (d). Bar, 10 μm.
Mentions: Untransfected COS cells and those expressing wild-type Arx (Fig. 3 c) show faint ubiquitin staining primarily in the cytoplasm (Fig. 3 d and not depicted). The nuclei of these control cells are generally devoid of ubiquitin reactivity, but occasionally have one or two punctate areas of faint ubiquitin staining that are always located centrally (Fig. 3 d). In contrast, COS cells transfected with ArxE constructs have strong immunoreactivity to ubiquitin at the periphery of the nucleus corresponding to the nuclear inclusions (Fig. 3, a and b). Interestingly, despite ArxE expression throughout the nucleus in cells that do not form inclusions, we only detected ubiquitination when nuclear inclusions form.

Bottom Line: Transfection of these constructs results in nuclear protein aggregation, filamentous nuclear inclusions, and an increase in cell death.Coexpressing Hsp70 decreases the percentage of cells with nuclear inclusions.Finally, we show that expressing mutant Arx in mouse brains results in neuronal nuclear inclusion formation.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
A growing number of human disorders have been associated with expansions of a tract of a single amino acid. Recently, polyalanine (polyA) tract expansions in the Aristaless-related homeobox (ARX) protein have been identified in a subset of patients with infantile spasms and mental retardation. How alanine expansions in ARX, or any other transcription factor, cause disease have not been determined. We generated a series of polyA expansions in Arx and expressed these in cell culture and brain slices. Transfection of these constructs results in nuclear protein aggregation, filamentous nuclear inclusions, and an increase in cell death. These inclusions are ubiquitinated and recruit Hsp70. Coexpressing Hsp70 decreases the percentage of cells with nuclear inclusions. Finally, we show that expressing mutant Arx in mouse brains results in neuronal nuclear inclusion formation. Our data suggest expansions in one of the ARX polyA tracts results in nuclear protein aggregation and an increase in cell death; likely underlying the pathogenesis of the associated infantile spasms and mental retardation.

Show MeSH
Related in: MedlinePlus