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

Neuroscience Program, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 12/2004; 167(3):411-6. DOI: 10.1083/jcb.200408091
Source: PubMed


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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Available from: Ilya M Nasrallah, Apr 22, 2015
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    • "Application of these programs for predicting residues involved in protein aggregation were also employed earlier (Sahin et al., 2011), where the predictions match well with the experimental observations. It is also reported that the protein aggregates formed by different polyA proteins such as PABPN1, ARX, etc. can sequester ubiquitin, proteosome and molecular chaperones (Abu-Baker et al., 2003; Nasrallah et al., 2004). Molecular chaperones like HSP70 and HSP40 independently or in conjugation can significantly reduce the aggregation of polyA protein like PABPN1 and its associated cellular toxicity (Abu-Baker et al., 2003; Wang et al., 2005). "
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    ABSTRACT: Presence of polyalanine (polyA) stretches in some proteins is found to be associated with their aggregation, which causes disorders in various developmental processes. In this work, inherent propensities towards aggregation of some residues, which are not part of the polyA stretches, have been identified by using the primary sequences of seven polyA proteins with the help of Betascan, PASTA and Tango programs and explored unambiguously. This provides a basis for proposing molecular mechanism of this type of aggregation. Reported suppression of aggregation of polyA proteins by chaperones like HSP40 and HSP70 is substantiated through molecular docking. The hydrophobic residues of identified aggregating region are found to be interacting with hydrophobic surface of chaperones. This suggests a crucial clue for possible way to inhibit the aggregation of such proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Preview · Article · Nov 2014 · Computational Biology and Chemistry
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    • "Transcriptional effects on PHOX2B promoter activity of co-expressing the PHOX2B wild-type protein and polyalanine expanded mutants As a dominant-negative effect of mutant proteins with alanine expansions has been proposed in other polyalanine diseases (Albrecht et al., 2004; Bruneau et al., 2001; Nasrallah et al., 2004) we tested whether PHOX2B mutant proteins could interfere with the activity of the wild-type protein when expressed in equimolar amounts. This possibility has already been verified for the DBH promoter, and significantly impaired transactivation was observed only in the case of a 10-fold excess of the protein with the longest polyalanine expansion (+13 Ala) (Trochet et al., 2005). "
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    ABSTRACT: The PHOX2B transcription factor plays a crucial role in autonomic nervous system development. In humans, heterozygous mutations of the PHOX2B gene lead to Congenital Central Hypoventilation Syndrome (CCHS), a rare disorder characterized by a broad variety of symptoms of autonomic nervous system dysfunction including inadequate control of breathing. The vast majority of patients with CCHS are heterozygous for a polyalanine repeat expansion mutation involving a polyalanine tract of twenty residues in the C-terminus of PHOX2B. Although several lines of evidence support a dominant-negative mechanism for PHOX2B mutations in CCHS, the molecular effects of PHOX2B mutant proteins on the transcriptional activity of the wild-type protein have not yet been elucidated. As one of the targets of PHOX2B is the PHOX2B gene itself, we tested the transcriptional activity of wild-type and mutant proteins on the PHOX2B gene promoter, and found that the transactivation ability of proteins with polyalanine expansions decreased as a function of the length of the expansion, whereas DNA binding was severely affected only in the case of the mutant with the longest polyalanine tract (+13 alanine). Co-transfection experiments using equimolar amounts of PHOX2B wild-type and mutant proteins in order to simulate a heterozygous state in vitro and four different PHOX2B target gene regulatory regions (PHOX2B, PHOX2A, DBH, TLX2) clearly showed that the polyalanine expanded proteins alter the transcriptional activity of wild-type protein in a promoter-specific manner, without any clear correlation with the length of the expansion. Moreover, although reduced transactivation may be caused by retention of the wild-type protein in the cytoplasm or in nuclear aggregates, this mechanism can only be partially responsible for the pathogenesis of CCHS because of the reduction in cytoplasmic and nuclear accumulation when the +13 alanine mutant is co-expressed with wild-type protein, and the fact that the shortest polyalanine expansions do not form visible cytoplasmic aggregates. Deletion of the C-terminal of PHOX2B leads to a protein that correctly localizes in the nucleus but impairs PHOX2B wild-type transcriptional activity, thus suggesting that protein mislocalization is not the only mechanism leading to CCHS. The results of this study provide novel in vitro experimental evidence of a transcriptional dominant-negative effect of PHOX2B polyalanine mutant proteins on wild-type protein on two different PHOX2B target genes.
    Full-text · Article · Oct 2012 · Neurobiology of Disease
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    • "The pathogenic role of these trinucleotide repeat expansions is currently unknown. However, cell death as a result of ubiquinated nuclear aggregates and filamentous nuclear inclusions may be responsible for the phenotype observed in polyalanine tract expansions as demonstrated by a study that employed tagged expression constructs that increased the length of the first polyalanine tract from the normal of 15–23, corresponding to the expansion found in patients with ISSX/MR (Nasrallah et al., 2004). A 33-bp duplication in exon 2 in two of three unrelated male patients was associated with EIEE/Ohtahara syndrome (Kato et al., 2007). "
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    ABSTRACT: ARX, the aristaless-related homeobox gene, is implicated in cerebral, testicular, and pancreatic development. ARX mutations are associated with various forms of epilepsy, developmental delay, and ambiguous genitalia in humans. A mouse model that recapitulates X-linked lissencephaly with ambiguous genitalia (XLAG) is far from elucidating the substrate for phenotypes that different ARX mutations cause. Moreover, despite phenotypic pleomorphism associated with X-linked dominant ARX mutations, heterozygous female carriers have not been thoroughly studied. Reviewing records of patients with ARX mutations, infantile epilepsies, and psychomotor retardation, we analyzed a family harboring a novel ARX mutation with different phenotypes in males and females, including Ohtahara syndrome. Children's Hospital Boston patient records were retrospectively screened for patients with infantile epileptic encephalopathies who underwent ARX sequencing based on clinical suspicion. Identified families were analyzed for genetic and neuropsychiatric phenomena. The proband was a male with Ohtahara syndrome, ambiguous genitalia, psychomotor delay, and central nervous system dysgenesis due to a novel ARX mutation in exon 5, causing a frameshift in the aristaless domain. Heterozygous females demonstrated neurocognitive/psychiatric phenomena including learning difficulties, anxiety, depression, and schizophrenia. This is the first reported case of Ohtahara syndrome with abnormal genital and psychomotor development in the setting of this novel ARX mutation in exon 5. Based on the unique phenotype of the proband and on the presence of heterozygous females with neurocognitive/psychiatric ailments, this study describes the potential roles for ARX mutations in epilepsy and neuropsychiatric disease, underscoring the importance of ARX in interneuron development, cerebral electrical activity, cognition, and behavior.
    Full-text · Article · Mar 2011 · Epilepsia
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