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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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Expression of ArxE in COS cells leads to the formation of intranuclear inclusions. (a) 5′ FLAG-tagged Arx expression constructs. The location of the homeodomain (HD), aristaless domain (AD) and four polyA repeats (blue) are shown. The first polyA repeat in the ArxE construct has an additional eight residues. The ArxΔ3′ construct contains the complete homeodomain, all four polyA repeats but lacks the aristaless domain. ArxE frequently accumulates into inclusions (b) that deform chromatin (c, arrowhead; d, merge). Wild-type Arx is expressed diffusely within the nucleus (e) overlapping with chromatin (f and g). Compared with untransfected COS cells (h), ultrastructure analysis of ArxE-expressing cells shows that inclusions (j and k, boxed enlarged) are within the nuclear membrane (j, arrowheads). (k) The inclusion shows a fibrillar internal structure. Bar: (b–g) 12 μm; (h and i) 2 μm; and (j and k) 700 nm.
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fig1: Expression of ArxE in COS cells leads to the formation of intranuclear inclusions. (a) 5′ FLAG-tagged Arx expression constructs. The location of the homeodomain (HD), aristaless domain (AD) and four polyA repeats (blue) are shown. The first polyA repeat in the ArxE construct has an additional eight residues. The ArxΔ3′ construct contains the complete homeodomain, all four polyA repeats but lacks the aristaless domain. ArxE frequently accumulates into inclusions (b) that deform chromatin (c, arrowhead; d, merge). Wild-type Arx is expressed diffusely within the nucleus (e) overlapping with chromatin (f and g). Compared with untransfected COS cells (h), ultrastructure analysis of ArxE-expressing cells shows that inclusions (j and k, boxed enlarged) are within the nuclear membrane (j, arrowheads). (k) The inclusion shows a fibrillar internal structure. Bar: (b–g) 12 μm; (h and i) 2 μm; and (j and k) 700 nm.

Mentions: We generated tagged expression constructs that increased the length of the first polyA tract from the normal of 15 (Miura et al., 1997) to 23 (referred to as Arx with expanded polyA repeat [ArxE]), corresponding to the expansion found in patients with ISSX/MR (Kato et al., 2003; Fig. 1 a). Expression of wild-type Arx in COS or 293T cells results in diffuse nuclear expression of the protein (Fig. 1, e–g, and not depicted). In contrast, expression of ArxE results in intranuclear aggregates of mutant protein in 25–35% of transfected cells (Fig. 1, b–d, and not depicted) even with similar levels of proteins expression (Fig. 4 and not depicted). One or two aggregates were found per cell, usually adjacent to the nuclear envelope. Frequently, little ArxE could be detected throughout the remainder of the nucleus (Fig. 1 b). These aggregates were often observed to exclude chromatin (Fig. 1 c). Ultrastructural examination confirmed the intranuclear location of the protein and demonstrated fibril formation characteristic of nuclear inclusions (Fig. 1, h–j). A similar frequency and appearance of nuclear inclusions was observed with different epitope tags located at either the COOH or NH2 terminus (not depicted). Transfected cells not forming nuclear inclusions expressed ArxE in a pattern qualitatively similar to cells transfected with wild-type Arx.


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)

Expression of ArxE in COS cells leads to the formation of intranuclear inclusions. (a) 5′ FLAG-tagged Arx expression constructs. The location of the homeodomain (HD), aristaless domain (AD) and four polyA repeats (blue) are shown. The first polyA repeat in the ArxE construct has an additional eight residues. The ArxΔ3′ construct contains the complete homeodomain, all four polyA repeats but lacks the aristaless domain. ArxE frequently accumulates into inclusions (b) that deform chromatin (c, arrowhead; d, merge). Wild-type Arx is expressed diffusely within the nucleus (e) overlapping with chromatin (f and g). Compared with untransfected COS cells (h), ultrastructure analysis of ArxE-expressing cells shows that inclusions (j and k, boxed enlarged) are within the nuclear membrane (j, arrowheads). (k) The inclusion shows a fibrillar internal structure. Bar: (b–g) 12 μm; (h and i) 2 μm; and (j and k) 700 nm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Expression of ArxE in COS cells leads to the formation of intranuclear inclusions. (a) 5′ FLAG-tagged Arx expression constructs. The location of the homeodomain (HD), aristaless domain (AD) and four polyA repeats (blue) are shown. The first polyA repeat in the ArxE construct has an additional eight residues. The ArxΔ3′ construct contains the complete homeodomain, all four polyA repeats but lacks the aristaless domain. ArxE frequently accumulates into inclusions (b) that deform chromatin (c, arrowhead; d, merge). Wild-type Arx is expressed diffusely within the nucleus (e) overlapping with chromatin (f and g). Compared with untransfected COS cells (h), ultrastructure analysis of ArxE-expressing cells shows that inclusions (j and k, boxed enlarged) are within the nuclear membrane (j, arrowheads). (k) The inclusion shows a fibrillar internal structure. Bar: (b–g) 12 μm; (h and i) 2 μm; and (j and k) 700 nm.
Mentions: We generated tagged expression constructs that increased the length of the first polyA tract from the normal of 15 (Miura et al., 1997) to 23 (referred to as Arx with expanded polyA repeat [ArxE]), corresponding to the expansion found in patients with ISSX/MR (Kato et al., 2003; Fig. 1 a). Expression of wild-type Arx in COS or 293T cells results in diffuse nuclear expression of the protein (Fig. 1, e–g, and not depicted). In contrast, expression of ArxE results in intranuclear aggregates of mutant protein in 25–35% of transfected cells (Fig. 1, b–d, and not depicted) even with similar levels of proteins expression (Fig. 4 and not depicted). One or two aggregates were found per cell, usually adjacent to the nuclear envelope. Frequently, little ArxE could be detected throughout the remainder of the nucleus (Fig. 1 b). These aggregates were often observed to exclude chromatin (Fig. 1 c). Ultrastructural examination confirmed the intranuclear location of the protein and demonstrated fibril formation characteristic of nuclear inclusions (Fig. 1, h–j). A similar frequency and appearance of nuclear inclusions was observed with different epitope tags located at either the COOH or NH2 terminus (not depicted). Transfected cells not forming nuclear inclusions expressed ArxE in a pattern qualitatively similar to cells transfected with wild-type Arx.

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