Journal of Neuroscience Research 68:442 448 (2002)

Time Course of Polyglutamine Aggregate

Body Formation and Cell Death: Enhanced
Growth in Nucleus and an Interval for
Cell Death
I. Toyoshima,1* M. Sugawara,1 K. Kato,1 C. Wada,1 T. Shimohata,2 R. Koide,2
O. Onodera,2 and S. Tsuji2
1
2

Department of Internal Medicine, Akita University School of Medicine, Akita, Japan

Department of Neurology, Brain Research Institute, Niigata University School of Medicine, Niigata, Japan

Polyglutamine (polyQ) aggregate bodies are a hallmark of

dentatorubral-pallidoluysian atrophy and related neurodegenerative disorders, although the relationship between aggregate body formation and cell death is not
clear. We analyzed the kinetics of polyQ aggregate formation and the time intervals for cell death, tracking
individual cells using fluorescence video microscopy, for
the first time. Expanded polyQ tracts of atrophin-1 with
or without nuclear localization signal (NLS) labeled with
green fluorescent protein (GFP) were constructed,
Q57NLS/GFP and Q56/GFP, respectively. All of the
Q57NLS/GFP aggregate bodies were in nuclei, and all of
the Q56/GFP aggregate bodies were in cytoplasm. Aggregates of Q56/GFP were larger than those of Q57NLS/
GFP. Surprisingly, a kinetic analysis showed that the
latter grew 5.37 times faster than the former. The time
interval between transfection and cell death was shorter
in Q57NLS/GFP, but the time between the end of the
rapid growing phase of aggregation and the start of
the cell death process did not show a significant difference. Aggregate growth was confirmed to correspond to
the accumulated free polyQ by the time of starting aggregation. These findings suggest that aggregate body
formation induced by expanded polyQ stretches is a
self-limiting process and is enhanced by factor(s) in nuclei, whereas it is not tightly bound to the cell death
process. 2002 Wiley-Liss, Inc.
Key words: DRPLA; green fluorescent protein; polyglutamine stretch; neuronal intranuclear inclusion; fluorescence video microscopy

Expanded CAG trinucleotide repeats coding polyglutamine (polyQ) stretches cause neuronal cell death in at
least nine neuronal degenerative diseases: Huntington disease (HD); spinal and bulbar muscular atrophy (SBMA);
spinocerebellar ataxia (SCA)-1, -2, -3/Machado-Joseph
disease (MJD), -6, -7, and -17; and dentatorubralpallidoluysian atrophy (DRPLA; for review see Zoghbi
and Orr, 2000; Gusella and MacDonald, 2000; Orr, 2001).
2002 Wiley-Liss, Inc.

Neuronal intranuclear inclusions (NIIs) were found to be

critical for neuronal cell death in patients or transgenic
mice with HD (Davies et al., 1997; DiFiglia et al., 1997),
SBMA (Li et al., 1998), SCA1 (Skinner et al., 1997),
SCA2 (Koyano et al., 1999), SCA3/MJD (Paulson et al.,
1997), SCA7 (Holmberg et al., 1998), SCA17 (Nakamura
et al., 2001), and DRPLA (Igarashi et al., 1998). Contrary
to this, SCA6 has exclusively cytoplasmic inclusions (Ishikawa et al., 1999). It is still not clear whether full-length
proteins or polyQ stretches are responsible for neuronal
cell death. Full-length ataxin-1 is reported to be effective
for NII formation (Skinner et al., 1997), but 3% of the
N-terminus region of huntingtin is enough for NIIs in
HD (Davies et al., 1997), and only truncated ataxin-3
resulted in neurodegeneration in SCA3/MJD (Ikeda et al.,
1996).
DRPLA is an autosomal dominant neurodegenerative disease characterized by myoclonus epilepsy, choreoathetosis, and dementia (Naito and Oyanagi, 1982). Unstable expansion of CAG coding polyQ was detected in
the DRPLA gene of patients (Koide et al., 1994; Nagafuchi et al., 1994; Tsuji, 1999). Expanded polyQ stretches
with shorter flanking regions induced aggregates, whereas
full-length atrophin-1, the gene product of DRPLA, containing expanded polyQ did not result in aggregate formation in nuclei or cytoplasm (Igarashi et al., 1998).
Green fluorescent protein (GFP)-tagged polyQ stretches
of atrophin-1 developed polyQ length- and timedependent aggregate formation and cell death in rat cerebellar granule cells (Moulder et al., 1999). Transgenic
Contract grant sponsor: Ministry of Education, Culture, Sports, Science and
Technology, Japan.
*Correspondence to: Itaru Toyoshima, MD, PhD, Department of Internal
Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita
010-8543, Japan. E-mail: toyoshim@med.akita-u.ac.jp
Received 8 November 2001; Revised 27 December 2001; Accepted 18
January 2002
Published online 16 April 2002 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jnr.10233

Time Course of PolyQ Aggregate Formation

mice expressing human atrophin-1 showed massive deposition of 120 kDa fragment harboring the N-terminus
portion and polyQ tract in nuclei (Schilling et al., 1999).
Transgenic mice with 129 repeats of glutamine showed
neurological symptoms similar to those in human disease
(Sato et al., 1999).
Recent experiments using yeast two-hybrid revealed
preferential association of expanded polyQ in atrophin-1
with human TATA binding protein (TBP)-associated factor, TAFII130 (Shimohata et al., 2000). Reports on the
association of polyQ with nuclear proteins have been
made for polyQ diseases (Fernandez-Funez et al., 2000;
McCampbell et al., 2000; Shibata et al., 2000; Wood et al.,
2000; Holbert et al., 2001; Nucifora et al., 2001).
In this study, we tested the significance of the site of
aggregate body formation induced by expanded polyQ
stretches with short flanking regions of atrophin-1. We
analyzed the time course of polyQ aggregate formation
and cell death, tracking individual cells using GFP tag and
fluorescence video microscopy, and found a different time
course and kinetics of aggregate formation and cell death
between nuclear and cytoplasmic aggregate bodies.
MATERIALS AND METHODS
DNA Construct
A part of the DRPLA gene containing a polyQ stretch
with short flanking regions of both sides was inserted into GFP
vector pEGFPN1 (Clontech, Palo Alto, CA) as previously reported (Onodera et al., 1997; Igarashi et al., 1998). Q19GFP had
a normal length of polyQ, 19, and Q56GFP had 56 repeats of
glutamine. Nuclear localization signal (NLS) of SV40 T antigen
(PKKKRKV) was added to Q57NLS/GFP with 57 repeats of
glutamine (Shimohata et al., 2000).
Cell Culture and Lipofection
COS-7 cells were plated in Daigo-T medium without
phenol red (Wako, Tokyo, Japan) supplemented with 10% fetal
calf serum (Hyclone, Logan, UT) 24 hr before transfection at
105 cells per glass-bottomed Microwell dish (MatTek, Ashland,
MA). Lipofection of 0.5 g plasmid DNA with SuperFect
(Qiagen, Hilden, Germany) was carried out according to the
manufacturers instructions.
Fluorescence Video Microscopy
Most of an inverted microscope (Axiovert; Carl Zeiss,
Tokyo, Japan) was covered by a chamber with the temperature
adjusted to 37C 0.2C. Transfected cells in the culture dish
were kept in a small chamber with a continuous flow of 5%
carbon dioxide. Emitted light for fluorescence microscopy from
a mercury lamp was attenuated by AttoArc (Carl Zeiss) to 1%
and further with a neutral filter. Final attenuation of the light
source was to 0.003% of original. This enabled us to track
individual cells for 48 hr without obvious photobleaching of
GFP aggregates or photodamage to cell function. An air-cooled
3CCD video camera (Hamamatsu Photonics, Hamamatsu, Japan) captured emitted light and accumulated photons on tip for
45 sec. Each frame was stored in a time-lapse S-VHS video
recorder (Matsushita, Tokyo, Japan) at a rate of 1/240, 1 frame

443

by 4 sec. Dual use of fluorescence and phase microscopy was

employed to visualize cell morphology other than GFPilluminated structures.
Morphometry
Volume of aggregate was estimated under the assumption
that the aggregates form ellipsoids of revolution. Long and short
axes were calculated with morphometric software, NIH Image
version 1.62 (free NIH software), after loading images of illuminated aggregates on S-VHS tapes to a Macintosh computer
through a video capture board, CG7 (Scion, Frederick, MD).
Three-dimensional reconstruction of aggregates was carried out
using a confocal microscope (Carl Zeiss) to confirm that the
volume of aggregates was estimated as ellipsoids of revolution.
Nuclei were visualized with Syto16 (Molecular Probes, Eugene,
OR), a red dye staining DNA.
The time intervals between lipofection, initiation of aggregation, end of rapid growing phase of aggregate bodies, and
start of cell death process were measured by reading the time
records on video frames generated by an SVHS time-lapse video
recorder (Matsushita, Tokyo, Japan).
Time Course of Free PolyQ in Nuclei
Mean fluorescence density in the nuclei other than aggregates was measured with NIH Image between the start of
aggregation (time 0) and the end of the rapid-growth phase
(time 100) in the case of intranuclear Q57NLS/GFP aggregate
formation. The highest density was adjusted to 100 and the
lowest to 0. Time course of aggregate growing was normalized
from time 0 to time 100 and size 0 to 100 and plotted onto the
same format. Mean and standard deviation in each 10% successive time interval were calculated.
Statistical Analysis
Difference in each parameter was analyzed by two-tailed
Students t-test.

RESULTS
Fluorescence Video Microscopy
PolyQ aggregates were well visualized and captured
by a cooled CCD video camera. Illuminated aggregates
with GFP tag (Fig. 1a) were also recognized on phase
contrast microscopy (Fig. 1b). Differential interference
contrast microscopy revealed fuzzy and spreading fringes
from aggregates (Fig. 1c). Confocal microscopy showed a
revoluted spheroidal structure. Most aggregate bodies both
in nuclei and in cytoplasm were attached to or were close
to the nuclear membrane on three-dimensional analysis
(Fig. 1d).
Parameters of Aggregate Body Formation
Profiles of the time course of aggregate body formation were obtained from 24 Q57NLS/GFP aggregates and
22 Q56/GFP aggregates. All of the Q57NLS/GFP aggregates were in nuclei, and all of the Q56/GFP aggregate
bodies were in cytoplasm. Only solitary aggregates were
analyzed.
Figure 2 shows one of the typical records of aggregate formation. At time 0, an illuminated aggregate ap-

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Toyoshima et al.

rapidly than Q56/GFP aggregates. Only Q57NLS/GFP

aggregates showed a speed over 10 al/min.
Both Q57NLS/GFP and Q56/GFP aggregates
showed a correlation between maximal volume and enlargement speed with two different regression lines (Fig.
4c). This indicates that Q57NLS/GFP bodies grew 5.37
times faster than Q56/GFP aggregates after compensation
for maximal size.

Fig. 1. Images of aggregate bodies of Q56/GFP (a c) and Q57NLS/

GFP (d) were obtained by fluorescence (a), phase contrast (b), differential interference contrast (DIC; c), and confocal (d) microscopy. The
fluorescent image well reflects the shape of an aggregate body visualized
with a DIC microscope. Confocal image confirmed the tangentially
round shape of an aggregate body in the nucleus. Scale bars 10 m
in a (for a c); 10 m in d.

peared in the nucleus and rapidly grew for 30 min. After

that, the size of the aggregate was stationary, and abrupt
detachment, a sign of cell death, occurred at 152 min.
Figure 3 shows a plot of the calculated volume of an
aggregate body in sequential video frames against the time
after start of aggregate formation. Maximal growing rate
was calculated from the plot. End point of aggregate
growing was calculated as the crossing point of the rapidgrowth phase and the stationary phase. The rapid-growth
phase was fitted to a three-degree polynomial equation
and the stationary phase to a double reciprocal line with
the assistance of the statistical software StatView version
4.0 (Abacus, Berkeley, CA) and KaleidaGraph version 3.0
(Synergy, Reading, PA; Fig. 3).
Volume of Aggregate Bodies
Maximal volumes of aggregate bodies with
Q57NLS/GFP and Q56/GFP transfection were 146
122 attoliters (al) and 241 140 al, respectively (Fig. 4a).
Mean size of Q56/GFP aggregates was 1.65 times greater
than that of Q57NLS/GFP aggregates. Volume of nuclei
of COS-7 was estimated as 1,240 667 al.
Speed of Aggregate Body Enlargement
Maximal speeds of enlargement of Q57NLS/GFP
aggregates and Q56/GFP aggregates were 4.93 4.67
al/min and 2.31 1.31 al/min, respectively (Fig. 4b).
Q57NLS/GFP aggregate bodies grew 2.02 times more

Time Course of Aggregate Body Formation

Time interval between lipofection and the start of
aggregate body formation with Q57NLS/GFP was significantly shorter than that in Q56/GFP aggregation;
Q57NLS/GFP aggregation was 2,430 1,300 min, and
Q56/GFP aggregation was 3,270 1,480 min (Fig. 5a).
The shortest time was 10 hr in both, but some aggregates
started had already at that time. Time from lipofection to
cell death in Q57NLS/GFP was shorter than that in
Q56/GFP; the former was 3,190 1,260, and the latter
was 3,880 1,350 min.
The duration of the rapid-growth phase in
Q57NLS/GFP and Q56/GFP aggregates was 57
64 min and 126 70 min, respectively (Fig. 5b), the latter
being longer than the former. The interval between the
end of the aggregate growing phase and the start of the cell
death process in Q57NLS/GFP and Q56/GFP was 519
322 min and 400 416 min, respectively (Fig. 5c). Time
from initiation of aggregation to cell death also did not
show a significant difference; 578 325 min in Q57NLS/
GFP and 529 405 min in Q56/GFP. These intervals did
not correlate with any parameters in this study.
Time Course of Free PolyQ in Nuclei
To confirm that the accumulated polyQ makes aggregates, we analyzed the mean fluorescence density in the
nuclei other than aggregates, in the case of intranuclear
Q57NLS/GFP aggregate body formation (Fig. 6). Normalized fluorescence densities in nuclei clearly showed the
corresponding decrease in aggregate body growth as a
sigmoid curve.
DISCUSSION
Here we have shown the time course of polyQ
aggregate body formation and cell death with or without
NLS. Both showed sigmoid curves for aggregate body
volume against time. After the end of the growing phase,
aggregates maintained constant size until the start of the
cell death process. The presence of this stationary period
allows the chance to observe aggregate bodies in living
cells.
COS-7 cells harboring intranuclear aggregate bodies
with Q57NLS/GFP showed a shorter time for cell death
than that with cytoplasmic aggregate bodies of Q56/GFP.
A major contribution to the shorter period for cell death is
a shorter incubation time for the start of aggregation from
lipofection. The time lag between lipofection and the
appearance of aggregate bodies might be explained by
local concentration of free polyQ for the start of aggregate
formation. Amyloid fibril formation in Alzheimers disease

Time Course of PolyQ Aggregate Formation

445

Fig. 2. Time sequence of an aggregate body formation. An aggregate body of Q57NLS/GFP

appeared in the nucleus at time 0, rapidly grew by 32 min, and was stationary in the subsequent
periods. al, Attoliters. Scale bar 20 m

Fig. 3. Parameters of aggregate body formation.

(Harper and Lansbury, 1997) and polyQ aggregates in HD

(Scherzinger et al., 1999) are reported to be dependent on
the concentration of soluble -amyloid protein and free
polyQ, respectively. If the production rates of Q57NLS/
GFP and Q56/GFP in the cells are similar, the NLS
promotes the transport of Q57NLS/GFP peptides from
cytoplasm to the nucleus to accumulate polyQ in the small
compartment, whereas Q56/GFP is distributed diffusely
in the whole cell, with a lower concentration of free
polyQ.
Nucleation-dependent in vitro fibril formation proceeds in a nonlinear sigmoid curve, indicating a selflimiting and thermoequilibrating process (Harper and
Lansbury, 1997). Aggregate growing in a sigmoid curve
and the corresponding decline of accumulated free polyQ
by the time of start of aggregation was confirmed in this
study. These findings suggest for the first time that aggregate body formation induced by expanded polyQ stretches
is a self-limiting process in living cells.

Seeding enhances fibril formation (Harper and Lansbury, 1997); there might be an enhancement factor for
nucleation and seed formation in nuclei. Aggregate bodyassociated proteins have the potential for enhancement of
aggregation, leucine-rich acidic nuclear protein (LANP) in
SCA-1 (Matilla et al., 1997), ubiquitin-proteasome pathway (Cummings et al., 1998), and CREB binding protein
(CBP) in HD (Kazantsev et al., 1999), although inhibition
of proteasome function resulted in enhancement of aggregation (Chai et al., 1999).
A higher growing rate relative to maximal size of
intranuclear aggregate bodies was revealed in this study.
This may suggest a different acceleration mechanism of
aggregate growing between intranuclear and cytoplasmic inclusion. TAFII130 is a major binding protein of
polyQ in nuclei that preferentially binds expanded
polyQ (Shimohata et al., 2000). All the aggregate bodies
in nuclei and cytoplasm were reported to colocalize
with TAFII130, although they were exclusively distributed in nuclei in the control. The preferential distribution of TAFII130 in nuclei may enhance seeding and
growth of aggregates.
The difference in maximal volume of aggregate bodies between Q57NLS/GFP and Q56/GFP may be the
difference in the locus of aggregate bodies. Accumulated
free polyQ by the time of start of aggregation may correspond with the maximal size of aggregation, but no correlation was observed between maximal size and time lag
by the start aggregation. Rigidity of nuclear matrix that is
resistant to high-salt treatment (Skinner et al., 1997) may
suppress the growth of aggregates. The limit of transpor-

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Fig. 4. Maximal volume (a), growth rate (b), and correlation of maximal volume and growth rate of
aggregate bodies (c). Bars indicate standard deviation. al, Attoliters. P values were obtained by
Students t-test.

Fig. 5. Intervals of aggregate body formation; periods between lipofection and the appearance of
aggregate body (a), the rapid growing phase (b), and the stationary period until the start of cell death
(c). Bars indicate standard deviation; n.s., not significant.

tation of polyQ through nuclear pores may determine the

end of the rapid-growth phase, because all the proteins in
nuclei are synthesized in cytoplasm. Close appearance of
aggregate bodies to the nuclear membrane with Q57NLS/
GFP might be explained by a higher local concentration of
polyQ close to nuclear pores, through which all the polyQ
synthesized in cytoplasm goes.
Apparent stationary periods of various lengths after
rapid growing of aggregates were observed in this study.
This may indicate that polyQ aggregates induce the cessation of protein production. Alternatively, production of

GFP-labeled polyQ may continue too slowly to be recognized even after the rapid phase of aggregate body
growth (Fig. 7). The long period between the end of
aggregate growth and cell death suggests the presence of
multiple steps for cell death from aggregate formation,
including depletion of associated proteins essential for cell
function, such as GAPDH (Burke et al., 1996), CBP
(Kazantsev et al., 1999; Steffan et al., 2000), and
TAFII130. Overexpression of TAFII130 (Shimohata et al.,
2000) and CBP (Nucifora et al., 2001) rescued cells from
polyQ-induced toxicity.

Time Course of PolyQ Aggregate Formation

447

in the biological effects on neuronal function. If polyQ

tracts with short flanking regions are produced in neuronal
cells, they must be highly toxic to induce cell death
resulting in rapid disappearance, which is hard to recognize in human neuropathology.
ACKNOWLEDGMENTS
We thank Y. Watanabe and T. Sasaki for technical
assistance.
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Fig. 6. Density of nuclear background and time course were normalized and plotted. Normalized aggregate body formation is displayed by
gray density. Bars indicate standard deviation of the density of free
polyQ and the volume of aggregates in each successive period.

Fig. 7. Relationship of production of fluorescently labeled polyQ,

aggregate body formation, and cell death. Time scale is illustrated
proportionally.

There is controversy regarding whether large aggregates directly cause cell death or small invisible aggregates
induce cell death (Zoghbi and Orr, 2000). Recent transgenic experiments using genetically modified sequences
with inhibition of aggregate formation indicate that the
presence of an expanded polyQ tract in nuclei is critical for
the pathogenic process (Klement et al., 1998). Cytotoxicity of intranuclear expanded polyQ stretches but not
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original molecule and are deposited in the DRPLA brain
and in other polyQ diseases. There might be differences in
half-life and amount between truncated proteins because
of the variation in the activities of processing enzymes and

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