T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 167, No. 3, November 8, 2004 411–416
The Rockefeller University Press$8.00
A polyalanine tract expansion in Arx forms
intranuclear inclusions and results in increased
Ilya M. Nasrallah,
Jeremy C. Minarcik,
and Jeffrey A. Golden
Neuroscience Program, University of Pennsylvania School of Medicine,
School of Medicine, Philadephia, PA 19104
Department of Pathology, Children’s Hospital of Philadelphia and the University of Pennsylvania
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 con-
structs results in nuclear protein aggregation, filamentous
nuclear inclusions, and an increase in cell death. These
inclusions are ubiquitinated and recruit Hsp70. Coex-
pressing 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.
An increasing number of human diseases have been linked to
the pathological expansion of normal tracts of single amino
acid repeats. The first identified were associated primarily with
polyglutamine repeats (La Spada et al., 1991; HDCRG, 1993;
Zoghbi and Orr, 2000). Although the precise mechanism of
pathogenesis of the expanded polyQ tracts is still unknown,
these diseases share a number of similar characteristics, includ-
ing formation of ubiquitinated inclusions, neural dysfunction,
and cell type–specific cell death (Cummings and Zoghbi, 2000).
More recently, nine disorders have been associated with
the expansion of a polyalanine (polyA) tract. In contrast to
the polyQ repeat disorders, polyA tract expansions are most
common in transcription factors. The one exception is poly-
adenosine binding protein nuclear 1, in which polyA expansions
cause insoluble protein complexes that form nuclear inclusions
in oculopharygeal muscular dystrophy (Brais et al., 1998; Ca-
lado et al., 2000; Fan et al., 2001). The other eight known
polyA expansion disorders are characterized by developmental
malformations ranging from defects in formation of digits
(Synpolydactyly type II; Muragaki et al., 1996) to the central
nervous system (Holoprosencephaly; Brown et al., 2001). Sim-
ilarities between the phenotypes of polyA expansion mutations
and null alleles suggest that the polyA-expanded proteins are at
least partial loss of function mutations (Brown and Brown,
2004). However, the mechanism by which a polyA expansion
in a transcription factor results in cellular dysfunction remains
to be defined.
Expansions of polyA tracts in the
) have recently been identified in children with
various neurological disorders (Stromme et al., 2002; Kato et
is expressed principally within the brain (Bi-
envenu et al., 2002; Kitamura et al., 2002) and contains five
exons with four polyA tracts, a homeodomain, and a conserved
“aristaless” domain (Miura et al., 1997). Deletions and trunca-
tion mutations are associated with X-linked lissencephaly with
ambiguous genitalia (Kitamura et al., 2002; Uyanik et al.,
2003). Patients with X-linked lissencephaly with ambiguous
genitalia have severe structural anomalies of brain development
and suffer from severe epilepsy and mental retardation (Dobyns
et al., 1999; Bonneau et al., 2002). Expansions of the first
polyA tract are associated with infantile spasms syndrome and
mental retardation (ISSX/MR; Stromme et al., 2002; Kato et
al., 2004). A similar phenotype is observed with loss of the 3
aristaless domain (Stromme et al., 2002). Expansions of the
second polyA tract cause a more variable phenotype including
Correspondence to Jeffrey A. Golden: firstname.lastname@example.org
Abbreviations used in this paper:
with expanded polyA repeat; ISSX/MR, infantile spasms syndrome and mental
retardation; polyA, polyalanine.
Arx, Aristaless-related homeobox ; Arx
JCB • VOLUME 167 • NUMBER 3 • 2004412
X-linked mental retardation and dystonia (Bienvenu et al., 2002).
Here, we show expansion of the first polyA-tract in Arx results in
protein accumulation with the formation of nuclear inclusions,
which may be part of the cellular pathogenesis of ISSX/MR in
patients with expansions in the first polyA tract of ARX.
Results and discussion
Expression of polyA-expanded Arx
protein in vitro
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
]), 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, ex-
pression of Arx
results in intranuclear aggregates of mutant
protein in 25–35% of transfected cells (Fig. 1, b–d, and not de-
picted) 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 Arx
could be detected throughout the remainder of the nucleus (Fig.
1 b). These aggregates were often observed to exclude chroma-
tin (Fig. 1 c). Ultrastructural examination confirmed the intra-
nuclear location of the protein and demonstrated fibril forma-
tion 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 NH
terminus (not depicted). Transfected cells not forming nuclear
inclusions expressed Arx
in a pattern qualitatively similar to
cells transfected with wild-type Arx.
Expression of 5
GFP-tagged Arx constructs allowed us
to visualize the formation of nuclear inclusions over time
(Video 1, available at http://www.jcb.org/cgi/content/full/
jcb.200408091/DC1). In these time-lapse series, one can ini-
tially see the steady accumulation of mutant Arx throughout
the nucleus. Inclusions were found to form in cells diffusely
by the progressive consolidation of protein,
first at the edge of the nucleus and next by redistributing all of
the mutant protein over 4–6 h (Video 1).
Expanded Arx protein increases cell
death in vitro
In diseases caused by polyQ expansions and one other polyA
expansion, the formation of nuclear inclusions is accompanied
by an increase in cell death (Cummings and Zoghbi, 2000; Fan
et al., 2001). To test whether expression of Arx
crease in cell death, we determined the rate of cell death in
transfected COS cells (
8 transfections for each construct at
each time point;
70 transfected cells per time point).
Although no significant difference in the rate of cell death
was observed after 24 h, by 48 h an average of 3.6% of cells
transfected with wild-type Arx were TUNEL-positive, whereas
5.2% of cells transfected with Arx
0.048; Fig. 2).
causes an in-
formation of intranuclear inclusions. (a) 5? FLAG-tagged
Arx expression constructs. The location of the homeo-
domain (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
Arx?3? 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 in-
clusion shows a fibrillar internal structure. Bar: (b–g) 12
?m; (h and i) 2 ?m; and (j and k) 700 nm.
Expression of ArxE in COS cells leads to the
POLYALANINE TRACT EXPANSION IN ARX • NASRALLAH ET AL.413
Arx nuclear inclusions are ubiquitinated
and suppressed by overexpression of
Improper protein folding has been implicated in the cellular
dysfunction associated with expanded polyQ diseases. Mis-
folded proteins are believed to aggregate, form inclusions, se-
quester other cellular proteins, and often cause cell death (War-
rick et al., 1998; Evert et al., 1999; Stenoien et al., 1999).
Normally, proteins known as molecular chaperones prevent
protein misfolding while the ubiquitin–proteasome pathway re-
moves old and misfolded proteins. Indeed, studies of polyQ ex-
pansions have found that nuclear inclusions are often ubiquiti-
nated (Paulson et al., 1997b; Cummings and Zoghbi, 2000).
We hypothesized that Arx
within inclusions has a misfolded
structure and is ubiquitinated.
Untransfected COS cells and those expressing wild-type
Arx (Fig. 3 c) show faint ubiquitin staining primarily in the cy-
toplasm (Fig. 3 d and not depicted). The nuclei of these control
cells are generally devoid of ubiquitin reactivity, but occasion-
ally have one or two punctate areas of faint ubiquitin staining
that are always located centrally (Fig. 3 d). In contrast, COS
cells transfected with Arx
constructs have strong immunoreac-
tivity to ubiquitin at the periphery of the nucleus corresponding
to the nuclear inclusions (Fig. 3, a and b). Interestingly, despite
expression throughout the nucleus in cells that do not
form inclusions, we only detected ubiquitination when nuclear
In addition to being ubiquitinated, multiple studies have
found that molecular chaperone proteins, such as Hsp70, cola-
bel with misfolded mutant proteins in the nuclear inclusions
found in HD and SCA (Cummings et al., 1998; Anderson et al.,
1999; Wyttenbach et al., 2000). COS cells expressing Arx
showed minimal colocalization of Arx with Hsp70 (Fig. 4, d–f),
whereas cells transfected with Arx
Hsp70 with nuclear inclusions (Fig. 4, a–c). These data are also
consistent with the hypothesis that protein misfolding may be
responsible for the formation of Arx
Previous studies have shown that overexpression of chap-
erones can prevent the formation of nuclear inclusions (Paulson
et al., 1997a,b; Cummings et al., 1998; Stenoien et al., 1999;
Wyttenbach et al., 2000; Auluck et al., 2002). To determine if
molecular chaperones can prevent Arx
showed colocalization of
-related nuclear inclu-
sions, we cotransfected COS cells with Arx
transfections for each molar ratio of constructs and 70–140
transfected cells per time point per condition; Fig. 4 g). Approx-
imately 30% of COS cells transfected with Arx
with various doses of a control GFP construct show nuclear
inclusion formation (Fig. 4 g). In contrast, overexpression of
Hsp70 resulted in a dose-dependent reduction in the percent of
cells with nuclear inclusions without decreasing the number
of cells expressing Arx
or the level of Arx
(Fig. 4 h and not depicted). Correlating the protein expression data
(Fig. 4 h) with the immunofluorescence data on inclusion forma-
tion (Fig. 1), it is also clear that the expression of high levels of
Arx without the polyA tract expansion cannot form inclusions.
and Hsp70 (
alone or Arx
Nuclear inclusion formation is specific to
Because many different mutations in
ISSX/MR, we wondered whether all mutations predisposed
ARX to form protein aggregates and nuclear inclusions, per-
haps by causing improper protein folding. To test this hypothe-
sis, we generated a 3
responding to a deletion that causes ISSX/MR in humans
(Stromme et al., 2002). Expression of Arx
sulted in no nuclear inclusions despite transfection efficiencies
similar to those for Arx
These data suggest that polyA expansions and 3
ARX likely cause ISSX/MR by distinct mechanisms.
have been linked to
; Fig. 1 a) cor-
in COS cells re-
3 transfections; not depicted).
Expanded Arx causes nuclear inclusion
formation in cortical neurons
Finally, we sought to determine if Arx
would also result in nuclear inclusion formation. During mam-
malian embryonic development, Arx is expressed in the fore-
expression in neurons
were transfected with Arx or ArxE and harvested 24 or 48 h later. At 48 h,
the amount of cell death in transfected neurons between these two groups
was significantly higher. * indicates P ? 0.05.
PolyA expansion of Arx increases cell death in vitro. COS cells
COS cells expressing ArxE aggregate ArxE into nuclear inclusions (a).
These nuclear inclusions stain positively for ubiquitin (b). (c and d) In con-
trast, cells expressing wild-type Arx do not form nuclear inclusions (c), nor
do they show nuclear ubiquitin staining (d). Bar, 10 ?m.
Expanded Arx nuclear inclusions are ubiquitinated. (a and b)
JCB • VOLUME 167 • NUMBER 3 • 2004414
brain, the floor plate of the spinal cord, and the genital primor-
dial (Miura et al., 1997; Kitamura et al., 2002; Ohira et al.,
2002). Based on our in vitro data, we predicted that expression
in forebrain neurons would result in nuclear protein
aggregates and the formation of inclusions during develop-
ment. By inducing neural dysfunction and cell death, these in-
clusions may contribute to the phenotype of ISSX and MR.
Using whole brain electroporation, we expressed Arx
cal neurons. When tagged wild-type
cal, subcortical, and hippocampal neurons, we observe diffuse
nuclear expression of Arx protein (Fig. 5 a and not depicted).
In contrast, expression of Arx
results in the formation of nuclear inclusions in 8–22% of
transfected neurons (Fig. 5, b–d).
Here, we have identified a novel mechanism by which
polyA expansions of ARX may lead to human disease. PolyA
tracts are commonly found in transcription factors; genomics
analysis has identified polyA tracts in
scription factors (Karlin et al., 2002). By folding improperly,
polyA-expanded ARX accumulates in intranuclear inclusions.
Although mutations that eliminate or alter ARX activity and
those that favor it being sequestered in inclusions may both di-
rectly decrease its ability to regulate transcription of as-yet un-
determined targets, misfolded ARX may also coaggregate with
other nuclear proteins and thereby have a “gain of function”
effect. Such an effect is likely, given that one has been found
is expressed in corti-
in cortical neural populations
300 human tran-
for other diseases associated with nuclear inclusions (Ross et
al., 1999). Furthermore, a dominant-negative activity has been
identified in polyA-expanded HoxD13, which causes Synpoly-
dactyly type II in part by altering the function but not expres-
sion of other Hox genes (Bruneau et al., 2001). Such an effect
may be common to other polyA-expansion disorders and me-
diated by the formation of insoluble inclusions. Counter to the
dominant negative hypothesis is the fact that loss of function
mutations are more severe in patients and female carriers are
rarely affected (Kato et al., 2004). Aberrant protein–protein in-
teractions may be relevant to the pathogenesis of polyA expan-
sion disorders even if inclusions are not identified in affected
human cases, as it has been hypothesized that formation of
macroscopic nuclear inclusions is not required for the patho-
genesis of some polyQ disorders (Klement et al., 1998; Ross et
al., 1999; Okazawa, 2003).
Materials and methods
Construction of expression constructs
Wild-type Arx cDNA was obtained from K. Kitamura (Institute of Life Sci-
ences, Tokyo, Japan). Expansion of the first polyA tract was achieved by
adding a short oligonucleotide duplex made by annealing two primers:
(IDT) into a unique NotI site within the region coding for the polyA tract.
Wild-type and expanded
Arx were individually subcloned in frame with
GFP- and 3
V5-tagged expression constructs (pCMV-
Tag2A (Stratagene), pCMV-Myc and pEGFP-C3 (CLONTECH Laborato-
number of cells with nuclear inclusions. (a–c) Endogenous
Hsp70 colocalizes with expanded Arx protein within nu-
clear inclusions in COS cells. Cells that have not formed
nuclear inclusions, as well as cells transfected with wild-
type Arx (d–f), have diffuse cytoplasmic and nuclear
Hsp70 expression. Bar, 10 ?m in a–f. (g) Cotransfection
of ArxE with an Hsp70 expression vector results in a dose-
dependent decrease in transfected cells that have nuclear
inclusions (solid bars) when compared with transfection
of ArxE alone (first bar on graph; ** indicates P ? 0.01),
or cotransfection with a GFP expression vector (open
bars; same DNA concentrations as Hsp70). (h) Western
blot analysis of COS cells transfected with a GFP expres-
sion vector or combinations of Arx or ArxE and HSP70 or
GFP. Expression levels of Arx and ArxE are similar and do
not change when HSP70 is coexpressed. Immunostaining
for GAPDH shows equal protein loading between samples.
Hsp70 colocalizes with ArxE and reduces the
POLYALANINE TRACT EXPANSION IN ARX • NASRALLAH ET AL.415
ries, Inc.), and pCDNA3.1-V5-His (Invitrogen), respectively; Fig. 1 a). To
generate the 3
deleted constructs, Arx
move the 203 3
bp of the 241-bp exon 5, including the aristaless domain
(Fig. 1 a). Hsp70 cDNA was provided by N. Bonini (University of Pennsyl-
vania, Philadelphia, PA) and cloned into pCDNA3.1.
cDNA was digested with SalI to re-
Cell culture and video microscopy
COS and 293T cells were maintained in DME with 10% FBS and trans-
fected using Fugene6 (Roche) and 500 ng of plasmid DNA. The cells
were acid-alcohol fixed 2 d after transfection.
For time-lapse video microscopy, COS cells transfected with GFP-
tagged constructs were placed on a Nikon TE-300 microscope equipped
with a deltaT4 environmental chamber (Bioptechs) and a motorized stage
(Prior) 6–18 h after transfection. A Nikon Plan Fluor ELWD 40
was used for image acquisition. Using a CCD camera (Hamamatsu) con-
trolled by Phase 3 Imaging Software (Media Cybernetics), fields were se-
lected at random and fluorescent images were acquired every 15 min for
12–24 h. Phase 3 Imaging software compiled images into AVI files which
were converted to MOV files in Quicktime Player 6.5.1.
Immunocytochemistry, TUNEL, and immunohistochemistry
Fixed cells were blocked with 10% normal goat serum (Sigma-Aldrich) in
PBS and incubated with primary antibody. Primary antibodies included:
mouse anti-FLAG M2 1:250 (Sigma-Aldrich), mouse anti-Hsp70 (1:300;
StressGen Biotechnologies), mouse anti-myc 1:300 (9E10), and mouse
anti-ubiquitin 1:500 (CHEMICON International Inc.). Secondary antibod-
ies were species-appropriate FITC- or Texas red–conjugated raised in goat
(1:150; Jackson ImmunoResearch Laboratories). Nuclei were counter-
stained with DAPI (Molecular Probes). The percent of transfected cells with
inclusions was determined as the number of cells containing inclusions di-
vided by the total number of FLAG-positive cells.
TUNEL staining was performed as described previously (Minarcik
and Golden, 2003). In brief, fixed COS cells were blocked and incubated
with TdT enzyme and UTP-biotin. Incorporation of UTP-biotin was labeled
with streptavidin-Cy3 (Jackson ImmunoResearch Laboratories), and sam-
ples were immunostained as above. The percent of TUNEL-positivity in
transfected cells was normalized to the percent of TUNEL-positivity in un-
transfected cells in order to control for the background rate of cell death
for each transfection.
For immunohistochemistry of fixed embryonic brain slices, samples
were blocked in PBS with 5% normal goat serum, 0.05% sodium azide,
and 0.3% Triton X-100. They were incubated overnight at 4
antibody diluted in block, then incubated with secondary antibody in PBS.
Nuclei were stained with DAPI.
Images were acquired on a DMR microscope (Leica) equipped with
epifluorescence and an ORCA-ER CCD camera (Hamamatsu) connected
to a Macintosh G4 running Openlab 3.1.5. The images were acquired us-
ing a Plan APO 40
/0.75 objective (Leica). Images were imported into
Adobe Photoshop 7.0 and saved as TIF files. Some images were imported
into Adobe Illustrator 10.0, labeled, and saved as EPS files.
C in primary
Protein was extracted from cells using lysis buffer containing 25 mM Tris,
pH 7.6, 1 mM MgCl
, 1 mM EGTA, 1% Triton X-100, 1% PMSF, 50
ml antipain, 2
g/ml aprotinin, 1
tastatin. The extract was homogenized using a 22-g needle and soluble
protein was quantified with a BCA kit (Pierce Chemical Co.). These
samples were then run on a 10% acrylamide SDS-PAGE gel with a 6%
stacking gel. The separated proteins, including the stacking gel, were
transferred to Immobilon membranes (Millipore). The membranes were
incubated in block (5% NFDM, 0.1% Tween-20) followed by primary anti-
bodies: anti-FLAG (1:1,000; Sigma-Aldrich) and anti-GAPDH (1:7500;
CHEMICON International, Inc.) and HRP-conjugated secondary antibody
(1:1,000; Amersham Biosciences). The blots were incubated in ECL buffer
(Amersham Biosciences) and exposed to Kodak film. Quantification was
performed with ImageJ.
g/ml leupeptin, and 1
Mouse brain electroporation
Whole brains from embryonic day 14.5–15 outbred CD1 mice were em-
bedded in 4% low melting point agarose (Fisher Scientific). DNA was in-
jected into one telencephalic ventricle and electroporated with three
pulses of 40–60 mV using an ECM 830 electroporator and gold “Gene
Paddle” electrodes (BTX). The brains were then cut at 250
ing microtome (model VT1000E; Leica). Brain slices were transferred to
Millicell-CM filters (Millipore) and fed serum-containing medium for 2 h (1:1
DME/F12, Pen/Strep, 6.5
M glucose, 10% FCS). Cultures were then
grown for 2 d in serum-free medium (DME, Pen/Strep, 6.5
m on a vibrat-
Online supplemental material
The dynamics of intranuclear inclusion formation were investigated by
time-lapse video microscopy (Materials and methods). Mutant protein was
first expressed throughout the nucleus at low levels followed by aggrega-
tion into dense inclusions. The development of inclusions was found to re-
quire many hours of mutant protein expression. Interestingly, the inclusions
were dynamic, moving within the nucleus and able to form and disassem-
ble. Video 1 is available at http://www.jcb.org/cgi/content/full/jcb.
We thank the other members of the Golden Lab for their support, discussions,
This work was supported by HD26979 (to J.A. Golden), and institu-
tional training grant GM07170 (to I.M. Nasrallah), and 5 F30 NS 6103-2
(to I.M. Nasrallah).
Submitted: 16 August 2004
Accepted: 29 September 2004
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