From the a Programs on Aging and Apoptosis, The Burnham Institute, La Jolla, California 92037, the b Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V52 4H4, Canada, the e Department of Psychiatry, Johns Hopkins University Medical School, Baltimore, Maryland 21205, and the i Department of Neuroscience, University of California, San Diego, California 92093
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ABSTRACT |
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Dentatorubropallidoluysian atrophy (DRPLA) is one
of eight autosomal dominant neurodegenerative disorders characterized
by an abnormal CAG repeat expansion which results in the expression of
a protein with a polyglutamine stretch of excessive length. We have
reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with
these open reading frame triplet repeat expansions are substrates for
the cysteine protease cell death executioners, the caspases. This led
us to hypothesize that caspase cleavage of these proteins may represent
a common step in the pathogenesis of each of these four
neurodegenerative diseases. Here we present evidence that caspase
cleavage of atrophin-1 modulates cytotoxicity and aggregate formation.
Cleavage of atrophin-1 at Asp109 by caspases is
critical for cytotoxicity because a mutant atrophin-1 that is resistant
to caspase cleavage is associated with significantly decreased
toxicity. Further, the altered cellular localization within the nucleus
and aggregate formation associated with the expanded form of atrophin-1
are completely suppressed by mutation of the caspase cleavage site at
Asp109. These results provide support for the toxic
fragment hypothesis whereby cleavage of atrophin-1 by caspases may be
an important step in the pathogenesis of DRPLA. Therefore, inhibiting
caspase cleavage of the polyglutamine-containing proteins may be a
feasible therapeutic strategy to prevent cell death.
To date, eight different dominantly inherited neurodegenerative
diseases have been shown to be associated with polyglutamine tract
expansions in their respective proteins (1-3). These include Huntington disease, spinal and bulbar muscular atrophy (Kennedy's disease), Machado-Joseph disease (SCA-3), dentatorubropallidoluysian atrophy (DRPLA),1 and
spinocerebellar ataxia types 1, 2, 6, and 7 (SCA-1, SCA-2, SCA-6,
SCA-7) (4-12). Expansion of the polyglutamine repeat in these disease
proteins results in selective death of neurons in different regions of
the brain.
Because all of these disease-associated proteins share a similar
mutation, i.e. CAG expansion in the coding region causing expansion of a polyglutamine stretch, they may have a common
pathological mechanism leading to neuronal cytotoxicity. Except for the
polyglutamine tract, it is generally believed that the eight disease
proteins are unrelated because their amino acid sequences bear no
discernible sequence homology. However, recent evidence from our
laboratories (13-15)2
suggest that seven of the eight
identified polyglutamine repeat proteins involved in CAG expansion
diseases contain caspase consensus cleavage sites (i.e.
DXXD). This would imply that a second common feature of at
least seven of the polyglutamine expansion disease proteins may be
their involvement in the apoptotic cell death pathway as cellular
substrates for the caspases. This finding has important implications
because studies in vitro and in vivo indicate
that the truncated forms of these proteins lead to the formation of
intracellular aggregates, and thus caspase cleavage of the full-length
proteins could in part explain how these cytotoxic truncated proteins
are formed (16-23).
To date, we have characterized the caspase cleavage of four
of the polyglutamine repeat disease proteins: huntingtin,
the androgen receptor, atrophin-1 (DRPLA), and ataxin-3
(Machado-Joseph disease) (13-15). These initial studies
suggested that a caspase-dependent apoptotic pathway may be
a critical factor in the generation of truncated proteins in some of
these polyglutamine repeat disease proteins and raised a number of
questions that warrant further investigation: Is the proteolytic
pathway involving caspases required to explain a common mechanism of
cytotoxicity of these proteins with expanded polyglutamine stretches?
How are these caspase substrates involved in the apoptotic process and
neurodegeneration? Are any of the functional domains within these
proteins activated or inactivated by caspase cleavage? Does the
proteolytic processing by caspases form the basis for selective
neuronal loss characteristic of these neurodegenerative diseases? Does
the distinct cellular localization of each of these structurally and
functionally unrelated proteins determine which caspase family member
cleaves them? Do other proteolytic pathways contribute to the
cytotoxicity of these proteins?
Given our recent findings, we wished to address the following specific
questions: Is caspase cleavage required for the cytotoxicity exhibited
by these proteins? In other words, is the formation of a truncated
protein containing the polyglutamine stretch via a
caspase-dependent pathway required for cytotoxicity?
Additionally, we wished to address whether the caspase cleavage site is
required for the formation of the protein aggregates and/or altered
cellular localization characteristic of these diseases. Of the four
proteins we reported recently as caspase substrates, atrophin-1 is a
particularly attractive candidate for our initial investigation because
in all likelihood it contains only one caspase cleavage site within the
entire protein. This is in contrast to ataxin-3, huntingtin, and
androgen receptor, which are cleaved at multiple sites within the
protein. Here we provide in vitro evidence that caspase
cleavage of atrophin-1 modulates cytotoxicity, formation of protein
aggregates, and its subcellular localization.
Culture and Transfection of Cells--
Cells from the human
embryonic kidney cell line 293T were cultured in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum with 1%
penicillin/streptomycin. Transient transfection was carried out with
pcDNA3, pcDNA3-DRPLA26, pRc/CMV-LacZ, pcDNA3-DRPLA65, pcDNA3-DRPLA26D109N, and pcDNA3-DRPLA65 D109N (24). Preparation of the atrophin-1 constructs has been described previously (14, 25).
Using pRc/CMV-LacZ, transfection efficiency was determined by staining
for the expression of In Vitro Translation Reactions--
Plasmids pcDNA3,
pcDNA3-DRPLA26, pRc/CMV-LacZ, pcDNA3-DRPLA65,
pcDNA3-DRPLA26D109N, and pcDNA3-DRPLA65 D109N were transcribed using T7 polymerase and then translated using the TNT system (Promega) in the presence of [35S]methionine. Translations (2.5 µl) were incubated with 10-50 ng caspase-3 for 2 h in the
following buffer (10 µl): 20 mM PIPES, 100 mM
NaCl, 1% CHAPS, 10% sucrose, 10 mM dithiothreitol, and 0.1 mM EDTA, pH 7.2 at 37 °C.
Purification of Caspase--
His-tagged caspase-3 was purified
by nickel affinity chromatography as described previously (27-29).
Caspase Western Analysis--
Western blots were carried out as
described previously using anti-caspase-3 mouse monoclonal antibody
(Transduction Laboratories) (26).
Atrophin-1 Western Blotting--
293T cells were transiently
transfected at 40% confluence using a modified calcium phosphate
protocol by mixing Qiagen-prepared DNA (Qiagen, Chatsworth, CA) with
2.5 mM CaCl2 and 2 × BBS (50 mM BES, 280 mM NaCl, 1.5 mM
Na2HPO4, pH 7.0) and adding the mixture to
cells immediately. After a 3-h incubation, the media were removed and
replaced with fresh growth media. At 24 h post-transfection, cells
were either treated with 35 µM tamoxifen (Sigma) for
4 h or left untreated. Cells were harvested by gentle scraping
into the growth media and centrifugation at 4,000 × g
for 4 min. Samples were washed once with PBS, centrifuged as before,
and suspended in lysis buffer (20 mM HEPES, 5 mM MgCl2, 0.5 mM EDTA, 0.01% (w/v) sucrose, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml
leupeptin, and 10 mg/ml aprotinin). Equal amounts of total cellular
protein were mixed with 5 × Laemmli sample buffer, denatured at
95 °C for 5 min, and separated on 7.5% SDS-polyacrylamide gels.
Protein was transferred electrophoretically to polyvinylidene
difluoride membrane, immunoblotted with anti-atrophin-1 antibody, and
detected using enhanced chemoluminescence (Amersham Pharmacia Biotech).
Site-directed Mutagenesis and Plasmid Construction--
Human
DRPLA26D109N and DRPLA65D109N were created using the QuikChange
site-directed mutagenesis system from Stratagene. pcDNA3-DRPLA constructs were used as templates with the following two synthetic primers according to manufacturer instructions:
5'-CCGATCTGGATAGCTTGAACGGGCGGAGCCTTAATG-3' and
5'-CATTAAGGCTCCGCCCGTTCAAGCTATCCAGATCGG -3'.
Mapping of Caspase Cleavage Sites by
Radiosequencing--
Radiosequencing was performed as described
previously (27, 30). Plasmid pcDNA3-DRPLA26 was transcribed and
translated with T7 polymerase using the TNT system (Promega) with
either [35S]methionine or [3H]leucine. The
translation was treated with caspase-3, separated by SDS-polyacrylamide
gel electrophoresis, and electroblotted onto a polyvinylidene
difluoride membrane. After autoradiography, the position of the
[35S]methionine-labeled atrophin-1 fragments was used to
cut out the [3H]leucine atrophin-1 bands from the
polyvinylidene difluoride membrane. The samples were subjected to
automated sequencing using an Applied Biosystems 476A sequencer, and
the anilinothiazolinone derivatives in each cycle were counted in a
scintillation counter. Comparison of the known positions of leucines
relative to the caspase cleavage site aspartate allowed identification
of the atrophin-1 cleavage site.
Immunofluorescence Microscopy--
293T cells were grown on
glass coverslips and transiently transfected with the indicated DRPLA
construct as described above. At 36 h post-transfection, the cells
were treated with 35 µM tamoxifen for 45 min. After
fixation in 4% paraformaldehyde and PBS solution for 20 min, the cells
were washed and then permeabilized in 0.5% Triton X-100 PBS for 5 min.
The DRPLA antibody utilized in these studies has been described by Wood
et al. (31) and was raised in rabbits against synthetic
peptide DRPLA425 (residues 425-439 of atrophin-1). The cells were
washed twice, incubated at room temperature with anti-DRPLA antibody
(1:200) for 1 h, washed three times with PBS, and then incubated
in Texas red-conjugated anti-rabbit antibody (1:1,000) for 20 min.
Cells were washed three times with PBS and then mounted onto slides
with DAPI (4',6'-diamindino-2-phenylindole, Sigma, 0.05 µg/ml) in
90% glycerol and PBS as a nuclear counterstain. Immunofluorescence was
observed using a Zeiss confocal microscope. Control experiments were
performed, including incubation with secondary antibody only, and
immunofluorescence of cells transfected with control plasmids.
Analysis of the Atrophin-1 Caspase Cleavage Site--
DRPLA is one
of eight autosomal dominant neurodegenerative diseases with expansion
of CAG trinucleotide repeats encoding polyglutamine stretches (32).
This neurodegenerative disorder is characterized by progressive
dementia, myoclonic epilepsy, cerebellar ataxia, and choreoathetotic
movements. Like many of these disease-associated proteins, atrophin-1
is expressed ubiquitously in the central nervous system (33), and thus
its expression pattern offers little clue to the relative
susceptibility or resistance of certain neuronal populations of cells
to undergo neurodegeneration.
We and others have demonstrated recently that atrophin-1 is cleaved by
caspases (14, 34). Atrophin-1 is one of at least 40 cellular caspase
substrates identified, and its function, as well as its contribution to
the apoptotic process, is unknown. Atrophin-1 contains a consensus
caspase-3 cleavage site (14, 35) near the NH2 terminus of
the protein (106DSLD109) (Fig.
1A), and the polyglutamine
tract is located in the middle of the protein (Fig. 1A). The
cleavage products generated during caspase-3 cleavage of in
vitro translated atrophin-1 migrated at 145 and 150 kDa for
constructs with 26 and 65 glutamines, respectively (Fig. 1B,
lanes 2 and 6). These COOH-terminal fragments
contain the polyglutamine tract and would be expected to lack the
predicted nuclear targeting sequence located at the NH2
terminus of the protein at amino acids 16-32 (see Fig. 1B).
NH2-terminal sequencing of the DRPLA cleavage product
confirmed that atrophin-1 was cleaved at Asp109.
To analyze the functional significance of caspase cleavage we prepared
constructs of DRPLA without a caspase cleavage site. Mutation of the
caspase P1 residue in atrophin-1 from Asp109 to Asn
abolished the processing of the in vitro translated
atrophin-1 (Fig. 1B, lanes 4 and 8) by
caspases. Because we have shown previously that atrophin-1 can be
cleaved by caspase-1, caspase-7, and caspase-8 in addition to
caspase-3, we evaluated whether the Asp109 mutation
abolished cleavage by multiple caspases present in transfected cells by
Western blotting (Fig. 1C). Caspase cleavage products were
observed in 293T cells transiently transfected with DRPLA26 or DRPLA65
after tamoxifen treatment, showing that tamoxifen challenge induced
caspase activation in cells transfected with these constructs. In
contrast, no cleavage products were generated in cells transfected with
DRPLA26D109N or DRPLA65D109N after tamoxifen treatment. These results
show that we have prepared atrophin-1 proteins resistant to caspase
cleavage in vitro and in transfected cells. Therefore, we
utilized these constructs to test whether this site influences the
cytotoxicity of atrophin-1 in cell culture.
Increased Cytotoxicity of DRPLA Mutant Protein--
We have
recently developed an in vitro tissue culture model to
investigate the cellular toxicity of polyglutamine repeat expansion disease proteins (15, 17). In this system, a sublethal stress is
induced by tamoxifen in transiently transfected human embryonic kidney
293T cells. A sublethal stress in these studies is defined as a stress
delivered by a concentration of a pro-apoptotic agent that does not
result in the processing of caspase-3 in plasmid control transfected
cells. As shown in Fig. 2A,
treatment of 293T cells with tamoxifen at a concentration of 35 µM does not lead to processing of caspase-3 over a 24-h
period of time. Higher concentrations of tamoxifen (>40
µM) result in apoptotic cell death based on acridine
orange/ethidium bromide staining of the cells (data not shown) and the
processing of caspase-3 (Fig. 2B).
To investigate the cytotoxicity of atrophin-1, we transiently
transfected 293T cells with expression constructs encoding the human
atrophin-1 gene with a normal CAG repeat length (DRPLA26) and an
expanded CAG repeat length (DRPLA65). These cells were then treated
with sublethal concentrations of tamoxifen (35 µM) 36 h after transfection. Untransfected 293T cells do not express atrophin-1, and lack of expression in transfected cells was verified by
immunofluorescence and Western analysis. 293T cells expressing DRPLA26
induced significantly higher proportions of apoptotic cell death than
vector controls (Fig. 3A) when
cells were exposed to tamoxifen. Furthermore, there was a statistically
significant increase in cell death and corresponding caspase activity
(Fig. 3B) when the polyglutamine repeat length was expanded
(DRPLA26 versus DRPLA65), indicating a gain of function for
the disease-associated form of atrophin-1 protein. Western analysis
verified equal expression of each of the atrophin-1 proteins.
Interestingly, the expression of DRPLA26 is pro-apoptotic when exposed
to an apoptotic stress. Because the function of atrophin-1 protein is
unknown, it is difficult to speculate how overexpression of normal
atrophin-1 enhances cellular death. However, there is a growing body of
literature suggesting that many of the caspase substrates can act to
enhance or block apoptotic cell death upon cleavage by caspases. For
example, cleavage of presenilin-2 results in the generation of an
anti-apoptotic cleavage product (36). In contrast, expression of the
caspase substrate mitogen-activated protein kinase kinase results in
the generation of a pro-apoptotic fragment that enhances caspase
activation through a positive feedback loop (27). Furthermore, we have
reported recently that the truncated fragment of Huntington disease
containing the normal polyglutamine repeat is pro-apoptotic (17). Our
results demonstrate that DRPLA65 is more pro-apoptotic than DRPLA26,
which indicates that the gain of function related to CAG length may
influence downstream events in apoptosis.
The Pro-apoptotic Effect of Atrophin-1 Requires
Cleavage--
Next, we assessed the effect of blocking caspase
cleavage of DRPLA26 and DRPLA65 on the pro-apoptotic effects of these
proteins in culture. As shown in Fig. 3, transient transfection of
DRPLA26D109N and DRPLA65D109N resulted in almost complete suppression
of apoptotic cell death when compared with DRPLA26 and DRPLA65.
Therefore, proteins with the same CAG length which are no longer
cleaved by caspases have decreased toxicity.
Increased Cytotoxicity of Atrophin-1 Correlates with Formation of
Aggregates and Altered Nuclear Localization--
Because our
cytotoxicity studies indicated that the caspase cleavage site is
crucial to the pro-apoptotic effect of atrophin-1, we investigated
whether apoptosis induction with tamoxifen modulated the formation of
aggregates. Intracellular neuronal inclusions may be a common property
for glutamine repeat expansion diseases (37). Aggregates have been
reported recently in the brains of patients with DRPLA and are similar
to those observed in Huntington disease (16, 20, 38).
Immunofluorescence analysis of transiently transfected 293T cells or
COS-7 cells indicated that atrophin-1 is localized at the outer border
of the nucleus, consistent with its putative nuclear localization
signal (Fig. 4A). This result differs from earlier work that has suggested that atrophin-1 is localized to the cytoplasm (20, 39). Some of these earlier studies used
epitope-tagged proteins in which the epitope may have interfered with
the nuclear targeting signal. Expression of DRPLA65, but not DRPLA26,
led to formation of densely stained nuclei (granular in appearance)
with altered nuclear distribution upon apoptosis induction compared
with the controls (atrophin-1-transfected cells not treated with
tamoxifen), as determined by confocal microscopy (Fig. 4, A
and C). Normal atrophin-1 protein is localized to the outer
edge of the nuclei before and after apoptotic stimulation, whereas the
disease-associated form of atrophin-1 leads to dense, particulate
staining throughout the nuclei during apoptotic stimulation. Further analysis (Fig. 5) of the
disease-associated form of atrophin-1 demonstrates that atrophin-1
colocalizes with DAPI-stained nuclei during apoptotic stimulation with
tamoxifen (Fig. 5, A-C), and confocal images under high
magnification show that the aggregates are nuclear (Fig. 5,
D-F). Thus, the disease-associated form of atrophin-1 shows
an altered pattern of nuclear distribution compared with the normal
form of atrophin-1. Modulation of aggregate formation did not occur in
the absence of apoptosis induction for DRPLA65-transfected cells (data
not shown) during the first 48 h after transfection. Interestingly, longer periods of time after transfection (>72 h)
resulted in increased formation of cytoplasmic aggregates. These
aggregates were larger in the DRPLA65-transfected cells and occurred
with higher frequency (Fig. 6).
Because aggregate formation was modulated by the stimulation of
apoptosis, we next assessed whether mutation of the caspase cleavage
site in DRPLA65 blocks formation of aggregates. Cells transfected
with pcDNA3-DRPLA26 expressed atrophin-1 protein that was localized
to the outer edge of the nucleus with a homogeneous pattern during
apoptotic cell death with tamoxifen (Fig. 4A). Cells
transfected with pcDNA3-DRPLA65 showed granular dense
straining throughout the nucleus (Fig. 4C) during apoptotic
cell death. In sharp contrast, cells transfected with
pcDNA3-DRPLA65D109N did not show altered nuclear distribution and
granular dense staining, suggesting that, at least in this system,
caspase cleavage of DRPLA is required for aggregation (Fig. 4,
B and D). It is of relevance to compare our
results with those found for SCA1, given that it is also a nuclear
protein yet does not appear to be cleaved by caspases. Interestingly,
recent studies on SCA1 show that the subcellular localization of
wild-type ataxin-1 differs from the mutant ataxin-1 both in
vitro and in vivo. Wild-type ataxin-1 localizes to the
nucleus in COS-1 cells (40), whereas mutant ataxin-1 shows a specific
redistribution or disruption of the nuclear structure. In these studies
there was no evidence that apoptosis modulated the formation of
aggregates, which is consistent with our finding that ataxin-1 is not a
caspase substrate.2 Ataxin-1 redistribution may be
important for the pathogenic mechanism in this disease, and additional
studies will determine whether this is similarly the case for
atrophin-1.
In this study, we show that cells transfected with expression
constructs encoding atrophin-1 undergo enhanced apoptotic cell death
that is mediated by a pro-apoptotic caspase cleavage product. In
vitro mutagenesis of caspase cleavage site Asp109
blocks production of this pro-apoptotic fragment and reduces cellular
toxicity dramatically. This is consistent with our recent findings that
expression of truncated huntingtin fragments resulted in significantly
more cell death than the full-length huntingtin (17, 22). Our results
suggest that caspase cleavage is required for modulation of aggregate
formation but does not determine whether aggregation is required for
cellular toxicity. Recent work on ataxin-1 indicates that nuclear
localization is critical for pathogenesis but not aggregation (41).
The results described here, along with our recent work (13-15, 17),
suggest that one common feature shared among at least seven of the
polyglutamine repeat disease proteins is that they are cleaved by
caspases to produce pro-apoptotic fragments. In this model, initial
cleavage by caspases or other proteases would produce a toxic fragment
with a gain of toxic function, e.g. aggregation or altered
protein-protein interactions. This generation of a toxic fragment would
lead to increased activation of caspases through a feedback loop. In
other words, the toxic fragments may function as caspase amplifiers.
This amplification loop would be highly dependent upon the cellular
context such as caspase/inhibitor distribution within the cell as well
as protein-protein and/or protein-ligand interaction with each type of
polyglutamine repeat protein (14). Further, amplification would also be
dependent upon the ability of a particular cell type to evoke
proteolytic pathways that remove this toxic caspase-amplifying
fragment. These experiments have not addressed what is the initial
trigger for caspase activation in the disease process but suggest that
proteolysis is important for cytotoxicity. However, physiological
stresses that are otherwise sublethal may in the presence of a caspase amplification mechanism lead to cell death. In addition, the results in
this study do not exclude additional mechanisms for proteolytic cleavage of atrophin-1 or the other CAG-containing gene products generating a smaller toxic fragment.
Furthermore, the results do not offer an explanation for the specific
pattern of neuronal loss in CAG repeat diseases. It is possible that
alterations in caspase expression, caspase inhibitors, partner
proteins, or downstream targets may determine the selective vulnerability for each of the CAG repeat diseases. Further studies directed at identifying the specific caspase family members that process the CAG repeat disease proteins, as well as a study of the
regional specificity of the caspases in the brain, should shed light on
this question. Finally, because blocking the cleavage of atrophin-1
inhibits its pro-apoptotic effect, such a strategy may prove useful for
the treatment of neurodegenerative diseases associated with
polyglutamine repeat expansions. Extension of these results to animal
models of polyglutamine expansion diseases should prove useful.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-galactosidase. Cell death was measured by
trypan blue exclusion, acridine orange/ethidium bromide, and LacZ
reporter gene cotransfection. Death was established as apoptotic based
on acridine orange/ethidium bromide staining and assessment of
caspase-3 activation. Cell death was measured 36-50 h after
transfection. Cellular death in confluent cells was induced with
tamoxifen citrate at a concentration of 35 µM 36-48 h
after transfection (26). Data were collected for three to five
experiments and then compared by Student's t test for statistical significance. Apoptosis was also monitored with the ApoAlert caspase assay kit according to the manufacturer's
instructions with the Ac-DEVD-AFC substrate
(CLONTECH).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Panel A, primary structure of normal and
mutant human atrophin-1. Caspases cleave atrophin-1 in the
NH2-terminal region of the protein at Asp109.
The predicted nuclear localization signal sequence is also located in
the NH2-terminal region of the protein and released from
the polyglutamine-containing region of the protein upon caspase
cleavage. Panel B, in vitro translated DRPLA26
(lanes 1 and 2), DRPLA26D109N (lanes 3 and 4), DRPLA65 (lanes 5 and 6), and
DRPLA65D109N (lanes 7 and 8) before treatment
( ) or after treatment (+) with caspase-3. Panel C, Western
blot of 293T cells transiently transfected with DRPLA26, DRPLA26D109N,
DRPLA65, and DRPLA65D109N without (
) or with (+) a 4-h treatment with
35 µM tamoxifen. wt, wild type.
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Fig. 2.
Effect of tamoxifen on caspase-3 processing
in 293T cells analyzed by Western blot. Panel A,
proteolytic profile of caspase-3 when treated with sublethal
concentrations of tamoxifen (35 µM) for the indicated
times. This concentration of tamoxifen is sublethal in nontransfected
cells, as indicated by no capase-3 processing. Panel B,
proteolytic profile of caspase-3 processing during tamoxifen-induced
cell death (50 µM), forming the active 17-kDa
fragment.
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Fig. 3.
Expansion of the polyglutamine repeat within
atrophin-1 from 26 to 65 glutamines enhances the pro-apoptotic effect
of DRPLA. Panel A, mutation of the
NH2-terminal caspase cleavage site inhibits the cytotoxic
effect of both wild-type and expanded atrophin-1. 293T cells were
transiently transfected with plasmids pcDNA3, pcDNA3-DRPLA26,
pRc/CMV-LacZ, pcDNA3-DRPLA65, pcDNA3-DRPLA26D109N, and
pcDNA3-DRPLA65 D109N at 25% confluence. Death was induced 48 h after transfection with 35 µM tamoxifen, and the
percent of apoptotic cells was measured with trypan blue. Panel
B, caspase activity assays of 293T cells transfected
with the indicated constructs and treated with 35 µM
tamoxifen for 2.5 h. The difference in cell death of
pcDNA3-DRPLA26- and pcDNA3-DRPLA65-transfected cells was
statistically significant (p < 0.01).
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Fig. 4.
Immunofluorescence of atrophin-1 with the
expanded polyglutamine repeat shows altered nuclear distribution/and or
aggregates in 293T cells during apoptotic stimulation. Panel
A, localization of atrophin-1 with 26 repeats (DRPLA26) in 293T
cells after tamoxifen treatment (1 h, 35 µM) shows
homogeneous staining on the outer edge of the nucleus. Panel
B, localization of the caspase cleavage site mutant atrophin-1
(DRPLA26 D109N) in 293T cells after tamoxifen treatment (1 h, 35 µM). Panel C, localization of
atrophin-1 with 65 repeats (DRPLA65) in 293T cells after tamoxifen
treatment (1 h, 35 µM) shows an altered subcellular
distribution and particulate granular staining. Panel D,
localization of caspase cleavage site mutant atrophin-1 (DRPLA65 D109N)
in 293T cells after tamoxifen treatment (1 h, 35 µM).
Images were collected with a confocal microscope.
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Fig. 5.
Subcellular localization of atrophin-1 with
expanded repeats in transfected 293T cells treated with tamoxifen.
Panel A, immunofluorescence of atrophin-1 with expanded
repeats localizes with (panel B) DAPI-stained nuclei
demonstrating a nuclear distribution. Panel C,
phase-contrast microscopy of the same cells. The images in panels
A, B, and C were collected at a
magnification of × 400 on a Zeiss inverted microscope.
Panel D, confocal image of atrophin-1 stained with
atrophin-1 antibody demonstrates substantial aggregation with nuclear
localization. Panel E, phase-contrast of the same cells.
Panel F, overlay of panels D and E.
The images in panels D, E, and F were
collected using a confocal microscope at a magnification of × 1000.
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Fig. 6.
Immunofluorescence of atrophin-1 normal
(DRPLA26) and disease causing (DRPLA65) 3 days after transfection in
293T cells without apoptotic stimulation. Cells expressing DRPLA65
(bottom panel) have cytoplasmic aggregates
(arrows) that are found at much higher frequency and are
larger than those found in cells expressing DRPLA26 (top
panel). Images were collected with a confocal microscope.
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FOOTNOTES |
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* This work was supported in part by Grants AG12282 and CA69381 from the National Institutes of Health (to D. E. B) and by the Glendorn Foundation (Catherine Dorn, trustee).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
c Hereditary Disease Foundation postdoctoral fellow.
d Medical Research Council of Canada postdoctoral fellow.
f Supported by Grants NS34172 and NS16375 from the NINDS, National Institutes of Health.
g Supported by Grant NS37878 from the National Institutes of Health.
h Supported by operating grants from the Canadian Networks of Centers of Excellence (NCE-Genetics) and Medical Research Council of Canada. Established investigator of the British Columbia Children's Hospital.
j To whom correspondence should be addressed: Program on Aging, The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-455-6480; Fax: 619-646-3192; E-mail: dbredesen{at}ljcrf.edu.
2 L. M. Ellerby, R. L. Andrusiak, C. L. Wellington, A. S. Hackam, S. S. Propp, J. D. Wood, A. H. Sharp, R. L. Margolis, C. A. Ross, G. S. Salvesen, M. R. Hayden, and D. E. Bredesen, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: DRPLA, dentatorubropallidoluysian atrophy; SCA, spinocerebellar ataxia; CMV, cytomegalovirus; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; PBS, phosphate-buffered saline; DAPI, 4',6'-diamindino-2-phenylindole.
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