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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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ArxE causes nuclear inclusion in cortical neurons. Confocal microscopy of cortical neurons electroporated on E14.5 and immunostained for FLAG after 2 d in vitro. (a) Cortical neurons expressing FLAG-tagged wild-type Arx exhibit diffuse nuclear expression. (b) Cells expressing ArxE often form nuclear inclusions (arrows). (c–e) Higher power of one nucleus shows an intensely staining inclusion along with faint diffuse nuclear staining for ArxE. DAPI counterstaining (d) shows that the inclusion is located at the periphery of the nucleus (merge in e). In all frames, the ventricular surface is toward the top. Bar: (a and b) 25 μm; (c–e) 10 μm.
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fig5: ArxE causes nuclear inclusion in cortical neurons. Confocal microscopy of cortical neurons electroporated on E14.5 and immunostained for FLAG after 2 d in vitro. (a) Cortical neurons expressing FLAG-tagged wild-type Arx exhibit diffuse nuclear expression. (b) Cells expressing ArxE often form nuclear inclusions (arrows). (c–e) Higher power of one nucleus shows an intensely staining inclusion along with faint diffuse nuclear staining for ArxE. DAPI counterstaining (d) shows that the inclusion is located at the periphery of the nucleus (merge in e). In all frames, the ventricular surface is toward the top. Bar: (a and b) 25 μm; (c–e) 10 μm.

Mentions: Finally, we sought to determine if ArxE expression in neurons would also result in nuclear inclusion formation. During mammalian embryonic development, Arx is expressed in the forebrain, the floor plate of the spinal cord, and the genital primordial (Miura et al., 1997; Kitamura et al., 2002; Ohira et al., 2002). Based on our in vitro data, we predicted that expression of ArxE in forebrain neurons would result in nuclear protein aggregates and the formation of inclusions during development. By inducing neural dysfunction and cell death, these inclusions may contribute to the phenotype of ISSX and MR. Using whole brain electroporation, we expressed ArxE in cortical neurons. When tagged wild-type Arx is expressed in cortical, subcortical, and hippocampal neurons, we observe diffuse nuclear expression of Arx protein (Fig. 5 a and not depicted). In contrast, expression of ArxE in cortical neural populations results in the formation of nuclear inclusions in 8–22% of transfected neurons (Fig. 5, b–d).


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)

ArxE causes nuclear inclusion in cortical neurons. Confocal microscopy of cortical neurons electroporated on E14.5 and immunostained for FLAG after 2 d in vitro. (a) Cortical neurons expressing FLAG-tagged wild-type Arx exhibit diffuse nuclear expression. (b) Cells expressing ArxE often form nuclear inclusions (arrows). (c–e) Higher power of one nucleus shows an intensely staining inclusion along with faint diffuse nuclear staining for ArxE. DAPI counterstaining (d) shows that the inclusion is located at the periphery of the nucleus (merge in e). In all frames, the ventricular surface is toward the top. Bar: (a and b) 25 μm; (c–e) 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: ArxE causes nuclear inclusion in cortical neurons. Confocal microscopy of cortical neurons electroporated on E14.5 and immunostained for FLAG after 2 d in vitro. (a) Cortical neurons expressing FLAG-tagged wild-type Arx exhibit diffuse nuclear expression. (b) Cells expressing ArxE often form nuclear inclusions (arrows). (c–e) Higher power of one nucleus shows an intensely staining inclusion along with faint diffuse nuclear staining for ArxE. DAPI counterstaining (d) shows that the inclusion is located at the periphery of the nucleus (merge in e). In all frames, the ventricular surface is toward the top. Bar: (a and b) 25 μm; (c–e) 10 μm.
Mentions: Finally, we sought to determine if ArxE expression in neurons would also result in nuclear inclusion formation. During mammalian embryonic development, Arx is expressed in the forebrain, the floor plate of the spinal cord, and the genital primordial (Miura et al., 1997; Kitamura et al., 2002; Ohira et al., 2002). Based on our in vitro data, we predicted that expression of ArxE in forebrain neurons would result in nuclear protein aggregates and the formation of inclusions during development. By inducing neural dysfunction and cell death, these inclusions may contribute to the phenotype of ISSX and MR. Using whole brain electroporation, we expressed ArxE in cortical neurons. When tagged wild-type Arx is expressed in cortical, subcortical, and hippocampal neurons, we observe diffuse nuclear expression of Arx protein (Fig. 5 a and not depicted). In contrast, expression of ArxE in cortical neural populations results in the formation of nuclear inclusions in 8–22% of transfected neurons (Fig. 5, b–d).

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