From the a Division of Neurobiology, Department of Psychiatry, b The Program in Cellular and Molecular Medicine, and the Departments of g Neurology, h Neuroscience, and i Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, the c Buck Institute for Age Research, Novato, California 94945, the e Center for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia and Children's and Women's Hospital, Vancouver, British Columbia V5H 4H4, Canada, and f The University of Sheffield, Academic Neurology Unit E Floor, Medical School, Beech Hill Road, Sheffield S10 2RX, United Kingdom
Received for publication, November 4, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dentatorubral and pallidoluysian atrophy (DRPLA)
is an autosomal dominant neurodegenerative disorder similar to
Huntington's disease, with clinical manifestations including chorea,
incoordination, ataxia, and dementia. It is caused by an expansion of a
CAG trinucleotide repeat encoding polyglutamine in the atrophin-1 gene.
Both patients and DRPLA transgenic mice have nuclear accumulation of
atrophin-1, especially an ~120-kDa fragment, which appears to
represent a cleavage product. We now show that this is an N-terminal
fragment that does not correspond to the previously described caspase-3 fragment, or any other known caspase cleavage product. The atrophin-1 sequence contains a putative nuclear localization signal in the N
terminus of the protein and a putative nuclear export signal in the C
terminus. We have tested the hypothesis that endogenous localization
signals are functional in atrophin-1, and that nuclear localization and
proteolytic cleavage contribute to atrophin-1 cell toxicity. In
transient cell transfection experiments using a neuroblastoma cell
line, full-length atrophin-1 with 26 (normal) or 65 (expanded)
glutamines localized to both nucleus and cytoplasm, with no significant
difference in toxicity between the normal and mutant proteins. A
construct with 65 glutamine repeats encoding an N-terminal fragment
(which removes an NES) of atrophin-1 similar in size to the truncation
product in DRPLA patient tissue, showed increased nuclear labeling, and
an increase in cellular toxicity, compared with a similar fragment with
26 glutamines. Full-length atrophin-1 with 65 polyglutamine
repeats and mutations inactivating the NES also yielded increased
nuclear localization and increased toxicity. These data suggest that
truncation enhances cellular toxicity of the mutant protein, and that
the NES is a relevant region deleted during truncation. Furthermore,
mutating the NLS in the truncated protein shifted atrophin-1 more to
the cytoplasm and eliminated the increased toxicity, consistent with
the idea that nuclear localization enhances toxicity. In none of the
experiments were inclusions visible in the nucleus or cytoplasm
suggesting that inclusion formation is unrelated to cell death. These
data indicate that truncation of atrophin-1 may alter its ability to shuttle between the nucleus and cytoplasm, leading to abnormal nuclear
interactions and cell toxicity.
Dentatorubral and pallidoluysian atrophy
(DRPLA)1 is a progressive
neurodegenerative disorder, characterized by movement, cognitive, and
emotional abnormalities (1-3), and caused by an expansion beyond 49 consecutive CAGs in the atrophin-1 gene on chromosome 12p (4-8). DRPLA
is one of at least eight disorders, including Huntington's disease and
several forms of spino-cerebellar ataxia (SCA), involving a
polyglutamine expansion. These disorders are thought to occur through a
genetic "gain of adverse function" mechanism (9-11). Of these
diseases, DRPLA is most similar to Huntington's disease.
Atrophin-1, the DRPLA gene product, encodes a hydrophilic 1184-amino
acid protein (12, 13) with several simple repetitive motifs, including
a serine-rich region, a variable length polyglutamine tract, a
polyproline tract, and a region of alternating acidic and basic
residues. The sequence of atrophin-1 contains a putative NLS in its N
terminus and a putative NES in its C terminus. The atrophin-1 protein
normally localizes to both the nucleus and cytoplasm of cells and its
function is unknown (12, 14, 15). Atrophin-1 is expressed widely in
brains of normal and affected individuals (8, 13, 14). Degeneration and
cell death in DRPLA occurs mainly in the dentate nucleus of the
cerebellum, red nucleus, globus pallidus, and subthalamic nucleus.
Neurons in these regions show atrophin-1 containing inclusions and
diffuse nuclear labeling. Therefore atrophin-1 expression levels alone do not explain the pathogenesis.
The cellular site of toxicity in HD and DRPLA is controversial.
Inclusions are predominantly nuclear in DRPLA (17, 18), and both
nuclear and cytoplasmic in HD (19-21), but the presence of inclusions
does not correlate well with toxicity. In some cell model studies of
HD, an exogenous NLS or an exogenous NES was fused to the huntingtin
protein, and cellular toxicity was correlated with nuclear location
(22, 23), although not all studies have come to similar conclusions
(24).
Cleavage of atrophin-1 may also be relevant to pathogenesis, although
the nature of the relevant cleavage product is uncertain. Igarashi
et al. (25) generated several atrophin-1 constructs with
varying lengths for all model studies. In transient transfection of
COS-7 cells, small truncated constructs of atrophin-1 with expanded
repeats caused cellular toxicity, whereas full-length or long fragments
of atrophin-1 with normal or expanded repeats did not (25). These data
suggest that generation of a short truncation fragment (which could
diffuse into the nucleus) can lead to the formation of nuclear
inclusions and cellular toxicity. Studies in HD have also indicated
that short truncations of huntingtin are toxic (23, 26, 27). However,
the DRPLA disease model mice and DRPLA patient tissue show a relatively
large cleavage fragment of mutant atrophin-1, running in a gel at about
120 kDa (28, 29). This fragment would be too large to enter the nucleus via passive diffusion. It is not known whether a fragment of this length could be toxic. Caspase cleavage of atrophin-1 at
Asp109 can generate a large C-terminal fragment (30-32);
however, whether this is present in vivo is uncertain.
We have made several constructs to study the role of nuclear targeting
and truncation of atrophin-1 in toxicity related to DRPLA. Our goal was
to determine whether the putative endogenous NLS and NES sequences in
atrophin-1 are functional, and whether a truncation fragment of the
approximate size and site of cleavage seen in patients and transgenic
mice enhances nuclear transport and cellular toxicity.
Atrophin-1 Constructs--
Full-length constructs were prepared
previously (13) and are in pcDNA-3 (Invitrogen). At-N917-26Q and
At-N917-65Q were generated by restriction digest at unique
BsrGI and XbaI sites blunt-ending, recircularizing, and deleting the region from BsrGI to
XbaI. The NLS (amino acids 16-32) in atrophin-1 was mutated
using PCR-based mutagenesis and designated At-N917-26Q-mNLS or
At-N917-65Q-mNLS and the NES (amino acids 1033-1041) was mutated
using PCR-based mutagenesis (33) and designated At-FL-26Q-mNES or
At-FL-65Q-mNES.
Immunofluorescence--
Mouse Neuro-2a (N2a) cells were cultured
in 1:1 Dulbecco's modified Eagle's medium:Opti-MEM I supplemented
with 5% fetal bovine serum and antibiotics, plated at a density of
1.5 × 105 per well of one-well chamber slides (Nunc),
cultured overnight, and transfected with 0.5 µg of DNA (At-FL-26Q,
At-FL-65Q, At-N917-26Q, At-N917-65Q, At-N917-26Q-mNLS,
At-N917-65Q-mNLS, At-FL-26Q-mNES, or At-FL-65Q-mNES) using
LipofectAMINE Plus (Invitrogen) as recommended by the
manufacturer's protocol. At 48 h post-transfection, cells were
fixed with methanol at In Vitro Translation Reaction--
Atrophin-1 constructs were
transcribed using T7 polymerase and then translated using the
TNT system (Promega) in the presence of
[35S]methionine. Translations were incubated with 10-50
ng of each of the caspases for 2 h in the following buffer: 20 mM PIPES, 100 mM NaCl, 1% CHAPS, 10% sucrose,
10 mM dithiothreitol, and 0.1 mM EDTA, pH 7.2, at 37 °C.
Immunoblot Analysis--
Protein concentrations were assayed by
the dye-binding method (Pierce) with bovine serum albumin as standard.
Samples were solubilized in SDS-PAGE loading buffer and then sonicated
for 20 s, and microcentrifuged for 1 min prior to loading.
Proteins were resolved by SDS-PAGE in a 6% polyacrylamide-SDS gel and
then transferred to a nitrocellulose membrane (Schleicher and Schuell). Transfer buffer was 25 mM Tris, 192 mM glycine,
0.1% SDS, and 10% methanol; and the blots were probed with
anti-atrophin-1 antibody AP142, AP302, or Apg840 (34). Bound antibodies
were visualized with horseradish peroxidase-conjugated secondary
antibodies and enhanced chemiluminescence (Renaissance; PerkinElmer
Life Sciences).
Quantitation of Atrophin-1 Distribution--
N2a cells were
cultured and transfected and immunofluorescence was performed as
described above. N2a cells transfected with each of the atrophin-1
constructs were assessed by randomly selected fields and at least 100 cells per condition were counted. Cells were scored based on nuclear,
nuclear and cytoplasmic, or cytoplasmic staining using standard
epifluorescence on a Zeiss microscope. Each condition was assessed at
least three times.
Cell Toxicity Assay--
N2a cells were seeded in six-well
plates at a density of 1.5 × 105 and cotransfected
with 0.5 µg of DNA for GFP and 0.5 µg of DNA for LacZ or
atrophin-1 constructs (At-FL-26Q, At-FL-65Q, At-N917-26Q, At-N917-65Q, At-N917-26Q-mNLS, At-N917-65Q-mNLS, At-FL-26Q-mNES, or
At-FL-65Q-mNES). GFP positive fluorescent cells were counted 48 h
later by capture on a Zeiss Axiovert 135 inverted microscope via IP
Spectrum Labs image analysis software. Each experiment contains cells
from 10 randomly selected fields from each of three wells. Mean ± standard deviations are taken from at least three different
experiments. As a measure of cell loss, GFP positive cells were counted
in each condition and standardized to the LacZ condition (22,
35). We have previously shown that this measure of cell loss correlates
with cell death as measured by propidium iodide to label dead cells and
Hoechst 33342 to label total cells (22) or Hoeschst for shrunken and
dead nuclei (35). Plasmids purified in separate preparations gave
similar results.
The atrophin-1 protein with sequences relevant to this study is
shown at the top of Fig. 1.
Atrophin-1 is a 1184-amino acid protein with a glutamine repeat near
the middle of the protein. It contains a putative NLS in its N terminus
(amino acids 16-32) and a putative NES in its C terminus (amino acids
1033-1041) (see below). The recognition site for antibody AP302 is
amino acids 1-15 and for antibody AP142 or APG840 is amino acids
425-439, which is near the glutamine repeat on the N-terminal side
(34). To identify which broad regions of atrophin-1 determine its
cellular localization, four deletion constructs were generated: one
coding for only the N-terminal 455 amino acids (which contains the
putative NLS), two coding for only amino acids 416-731 (with either 26 or 65 glutamines), and one coding for amino acids 640-1184 (which contains the putative NES) (Fig. 1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C for 10 min. Cells were rinsed three
times in phosphate-buffered saline and blocked for 1 h in 5%
normal goat serum. Rabbit polyclonal antibody AP 142 was used to detect
the atrophin-1 or an anti-Myc antibody was used to detect the Myc
epitope tag at the N terminus of the atrophin-1 constructs. Cells were
incubated with antibody for 3 h at 37 °C. Washed cells were
incubated with fluorescein isothiocyanate or Cy3-conjugated secondary
antibody (Jackson Laboratories) diluted 1:250. ToTo-3 (Molecular
Probes) was used as a nuclear marker. Coverslips were mounted with
Vectashield (Vector Laboratories). Cells were viewed on a Noran
confocal microscope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
[in a new window]
Fig. 1.
Schematic diagram of atrophin-1 protein,
antibody binding sites, and initial set of constructs. Atrophin-1
encodes a hydrophilic 1184-amino acid protein. It contains a putative
NLS in its N terminus, a polymorphic polyglutamine tract near the
middle of the protein, and a putative NES in its C terminus. The
antibody AP302 binds to amino acids 1-15 and the antibodies AP142 and
APG840 bind to amino acids 425-439. Four constructs were generated
spanning the entire protein. The midportion could have either normal or
mutant polyglutamine.
N2a cells were transfected and immunofluoresence was performed 48 h later. The N-terminal construct localized to the nucleus, whereas the
other three constructs were cytoplasmic (Fig.
2), suggesting the existence of a
functional NLS in the N terminus of the protein, and consistent with an
NES in the C terminus of the protein.
|
Because DRPLA forms nuclear aggregates and the putative NLS is in the N
terminus, we performed a Western blot analysis on nuclear extracts from
transgenic mice to confirm that the truncation product contains the N
terminus, using an antibody to the N terminus (AP302) and an antibody
to an epitope near the glutamine repeat (APG840). Fig.
3 demonstrates the full-length protein of
both alleles in the transgenic mice migrating at ~190-200 kDa, and a
band representing the truncation product, running at about 120 kDa
(28). The full-length band runs between 190 and 220 kDa depending on
gel conditions. The truncation band is broader than the full-length
bands, consistent with our previous observation that the fragment runs
as a doublet, and that there is cleavage product accumulation over time
(28). A C-terminal antibody does not react with the truncation product
(not shown). Thus this truncation product contains the N terminus and
lacks the C terminus. This is consistent with the N-terminal NLS being
relevant for the abnormal nuclear localization seen in DRPLA.
|
To address the question of whether the truncation product is caused by
caspase cleavage, atrophin-1 protein was generated by in
vitro translation and incubated with caspase 1-10 (Fig. 4). Consistent with previous data,
caspase-2, -3, -6, -7, and -8 can cleave wild type atrophin-1 (30-32).
When the putative caspase-3 site at Asp109 is mutated, all
but caspase-6 cleavage is abrogated (Fig. 4). This suggests that
caspase-2, -3, -6, -7, and -8 cleave at Asp109 and that
caspase-6 also cleaves at another site, which is currently unknown.
|
We then compared the fragment in the DRPLA mouse model with the caspase
fragments. When 293T extracts expressing full-length atrophin-1 with 65 glutamines were subjected to caspase-3, -6, or -7 treatment, the
Western blot showed a band corresponding to full-length atrophin-1 in
the absence of any caspase enzymes and a band migrating faster in the
presence of these caspase enzymes, representing a cleavage product
(Fig. 5). However, the product in each
caspase condition is larger than the truncation seen in the transgenic
mice, indicating that the cleavage product does not arise from any of
the caspases known to cleave atrophin-1.
|
Next we generated constructs to determine whether the putative NLS and
putative NES are functional, and to determine the effect of a
truncation similar in size and location of truncation to the cleavage
product seen in DRPLA transgenic mice and patient postmortem tissue. A
consensus bipartite NLS is 2 basic amino acids, a 10-amino acid spacer,
followed by 3-5 basic amino acids. A consensus NES is a hydrophobic
amino acid, followed by 2-4 amino acids, a hydrophobic amino acid, 2 amino acids, a leucine, an amino acid, and a hydrophobic amino acid.
Consensus NLS and NES sequences are present in atrophin-1 (Fig
6).
|
Two full-length constructs with 26 or 65 repeats (At-FL-26Q and
At-FL-65Q, respectively) (Fig. 7) were
generated to determine whether the putative localization signals
functioned in the context of atrophin-1 and to determine whether the
localization of the protein influenced toxicity.
|
We generated two constructs truncated at amino acid 917 with 26 or 65 repeats (At-N917-26Q and At-N917-65Q, respectively) (Fig. 7). This
length was chosen because it removed the NES and should produce a
product at least as large as, or slightly larger than, the 120-kDa
fragment of mutant atrophin-1 seen in mice and DRPLA patients (Fig.
8). Fig. 8 shows At-N917-65Q running at
~120 kDa, demonstrating that At-N917-65Q is slightly larger than the truncation product seen in the mice and human patient tissue, and thus
also too large to diffuse passively into the nucleus. Therefore,
At-N917-65Q is reasonable to study in cell model experiments.
|
Two constructs truncated at amino acid 917, and with the NLS mutated (At-N917-26Q-mNLS and At-N917-65Q-mNLS), were generated, to determine whether the NLS is functional and if nuclear localization is related to toxicity (Fig. 7). Finally, two full-length constructs with the NES mutated were generated (At-FL-26Q mNES and At-FL-65Q mNES), to determine whether the NES is functional and whether eliminating it would enhance nuclear localization and cellular toxicity (Fig. 7).
Immunofluorescence and subcellular localization of these constructs in
N2a cells transfected with full-length atrophin-1, At-FL-26Q or
At-FL-65Q was quantified to determine the effect of altering the
endogenous targeting signals (Figs. 9 and
10). Staining in most cells was in both
the nucleus and cytoplasm. When atrophin-1 was truncated, removing the
NES, At-N917-26Q, and At-N917-65Q showed predominately nuclear
staining. Similar to the results from the truncation products, when the
NES was mutated, At-FL-26Q-mNES or At-Fl-65Q-mNES showed predominately nuclear staining. When the NLS was mutated in the truncated product (At-N917-26Q-mNLS or At-N917-65Q-mNLS) most cells showed cytoplasmic staining (Figs. 9 and 10). In none of these experiments were there inclusion bodies or other large aggregates of atrophin-1, either in the
nucleus or cytoplasm. These data suggest that both the putative NLS and
NES are functional in the context of atrophin-1.
|
|
Cell toxicity assays were performed using each of the constructs
described above to determine the relationship between subcellular localization and cell death. Western blots using antibody AP 142 showed
that each of these constructs yields expression at approximately the
same levels (Fig. 8 and data not shown) and therefore, we can directly
compare each construct for cellular toxicity. We did not detect any
aggregation at the top of the Western blot, consistent with the lack of
nuclear inclusions or other visible aggregates in our transient cell
transfection assay. Cell viability was determined by co-transfecting an
atrophin-1 construct with GFP as a marker for transfected cells (22,
35). GFP positive cells were counted 48 h later. Baseline toxicity
in this transfection assay is 10-18%. Cells transfected with
At-FL-26Q, At-FL-65Q, or At-N916-26Q showed essentially no difference
in cell viability. However, At-N917-65Q caused an ~2-fold increase
in cell loss (Fig. 11).
|
Because truncation removes a functional NES and increases nuclear
localization, we performed the cell loss assay for At-FL-65Q-mNES, and
cell loss was comparable with that seen with the truncation. Because
truncation and mutation of the NES changed the localization of the
protein from both nuclear and cytoplasmic to mostly nuclear, this
suggests that nuclear localization enhances toxicity. To further test
if nuclear localization is a factor in this enhanced toxicity we
mutated the NLS in At-N917-65Q (which shifted the protein mostly to
the cytoplasm), and this decreased toxicity to control levels. These
data suggest that an effect of the truncation is to relieve atrophin-1
of its NES, concentrating atrophin-1 into the nucleus through its
functional NLS, and leading to enhanced cellular toxicity.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data indicate that endogenous localization signals are functional in atrophin-1, and that nuclear localization and proteolytic cleavage to an N-terminal fragment containing an NLS contribute to atrophin-1 cell toxicity. Transfection of At-FL-65Q had no increased toxicity compared with At-FL-26Q. By contrast, both At-N917-65Q (which removes the NES) and At-FL-65Q-mNES showed increased nuclear labeling and an increase in cellular toxicity, compared with similar constructs with 26 glutamines or full-length atrophin-1. These data suggest that truncation and nuclear localization enhance cellular toxicity of the mutant protein, and that the C terminus with its NES is a relevant region deleted during truncation. Mutating the NLS in the truncated protein shifted atrophin-1 to the cytoplasm and eliminated the increased toxicity, confirming that nuclear localization is relevant to enhanced toxicity. By contrast, there were no inclusions or other visible aggregation in the transfected cells, indicating that inclusion formation is not necessary for toxicity.
Most N2a cells transiently transfected with full-length atrophin-1 (either normal or expanded) showed both nuclear and cytoplasmic staining. Truncation (which removes the NES) or mutation of the NES increases the number of cells with nuclear localization of the protein. By contrast, mutating the NLS in the truncated product shifts the protein more to the cytoplasm. These data are consistent with previous data that atrophin-1 contains a functional endogenous NLS in its N terminus (30, 36) and with the idea that atrophin-1 contains a functional endogenous NES in its C terminus, and can shuttle between nucleus and cytoplasm.
Other regions of the protein may also influence localization, as the mutation in the NLS did not abolish nuclear localization in every cell, and neither truncation (which removes the NES) nor the mutation of the NES yielded complete nuclear localization in every cell. Comparable data have been reported for ataxin-1, the SCA1 gene product (38). Ataxin-1 can also translocate from the cytoplasm to the nucleus. Nuclear localization of ataxin-1 is mediated by a C-terminal NLS. When the NLS in ataxin-1 was mutated and that construct transfected into COS-7 cells, some of the protein still entered the nucleus, but in transgenic mice made with this construct the protein was mainly cytoplasmic (38). Thus nuclear and cytoplasmic localization signals may be most effective in vivo. In addition nuclear and cytoplasmic localization may be influenced by phosphorylation (39) of atrophin-1, or by other cell signaling pathways.
The site of cellular toxicity in the polyglutamine diseases has been controversial. Two cell model studies of HD found that the addition of an exogenous NLS increased cellular toxicity, and the addition of an exogenous NES reduced or blocked toxicity (22, 23). By contrast, another study suggested that cellular toxicity can also occur with cytoplasmic localization. Hackam et al. (24) found that the addition of exogenous NLS or NES sequences did shift huntingtin aggregates to either the nucleus or cytoplasm but did not alter toxicity. However, their model focused on shifts in aggregate location and not diffuse appearing nuclear staining, which may be more relevant to cellular toxicity. Furthermore, that study used 293T cells and induced toxicity with tamoxifen plus huntingtin. SCA1 transgenic mice with a mutation in the endogenous nuclear localization signal did not develop the disease (38), indicating a critical role for nuclear location in ataxin-1 toxicity.
For atrophin-1, we find that modifications that enhance nuclear localization enhance cellular toxicity. These data add to the growing evidence that nuclear localization is relevant to toxicity for polyglutamine disorders. However, nuclear localization alone is not likely to be the only determinant of toxicity. There is partial nuclear localization with the expanded full-length constructs, which are not toxic. It is possible that truncation may disrupt the normal regulated pattern of shuttling of atrophin between the nucleus and cytoplasm. The truncation product may accumulate to a higher concentration in the nucleus (28), or may localize to a different nuclear compartment and interact with different proteins within the nucleus compared with the full-length mutant protein (see below).
Previous studies have shown that DRPLA transgenic mice and patient brain tissue show accumulation of a 120-kDa cleavage fragment specific to mutant atrophin-1 (28). Immunoblotting experiments in the present study indicate that the 120-kDa fragment seen in DRPLA transgenic mice contains the N terminus of the protein, but lacks the C terminus. We thus have generated a construct comparable in cleavage site and length to the product identified in transgenic mice and DRPLA patient tissue. The protein product of the At-N917-65Q construct migrates similarly to the human and mouse truncation product or slightly slower; thus it is at least the same length, and no shorter.
Several studies have shown that caspases can cleave atrophin-1 at Asp109 (30-32) in the N terminus of the protein. Our current data indicate that it is unlikely that any of the caspases-1-10 generate the cleavage product seen in human patient or mouse tissue. The in vitro translation and Western analysis data presented here show that the caspase cleavage products are different from the cleavage product seen in the DRPLA transgenic mice. Our antibody data indicate that the cleavage product in patient and transgenic mice retains the N terminus and the NLS and deletes the C terminus of the protein. By contrast the caspase-3 cleavage product does not contain the N terminus or the NLS (and is a different size). Based on our in vitro translation data and Western blots from brain nuclei, the cleavage product is not generated from any of the other known caspase enzymes studied here.
It was not previously known whether a fragment as large as studied in the current experiments could cause toxicity. Igarashi et al. (25) reported that short truncations of atrophin-1, but not longer truncations, were toxic. However, their long fragments lacked the NLS, and therefore would not be expected to enter the nucleus. By contrast smaller truncations may have been able to diffuse into the nucleus without a nuclear targeting signal. Our truncation fragment is relatively large, at least as large as the truncation seen in transgenic mice and DRPLA patients, and causes cellular toxicity. Because mutating the NES causes comparable toxicity to truncating the protein, we hypothesize that truncation relieves atrophin-1 of its NES and concentrates it in the nucleus, interfering with its shuttling between the nucleus and cytoplasm.
Truncation may also be important in other trinucleotide repeat diseases. In HD nuclear inclusions in postmortem patient tissues contain detectable N-terminal huntingtin epitopes (19-21) and the polyglutamine expansion. C-terminal epitopes are generally not detected, and, if present, could represent secondary recruitment of full-length huntingtin. In HD cell model experiments, truncations containing the N terminus and the polyglutamine expansion are generally more toxic than mutant full-length protein. In mouse models made using full-length huntingtin, it has been controversial whether huntingtin in inclusions is a cleaved fragment, full-length huntingtin, or full-length huntingtin with C-terminal epitopes masked (40-45). In vivo evidence of cleavage of huntingtin by caspase-3 at specific sites and cleavage of huntingtin by calpain has been demonstrated (46-48). In addition smaller fragments, not likely to be generated by either caspases or calpain, have been reported (49-52). Recent studies in both cell models and postmortem tissue have suggested that there may be several sequential cleavage steps by both caspase and non-caspase steps (52).
It remains unclear whether aggregation of polyglutamine protein contributes to pathogenesis. Because large inclusions were not seen by immunofluorescence in our studies (although microaggregates may be present), it appears that inclusions are not necessary for atrophin-1 cellular toxicity. This is consistent with studies of other diseases. Saudou et al. (23) found that in a cell model of HD, inclusions were not related to toxicity. Klement et al. (38) abrogated aggregation in a mouse model of ataxin-1, but the mice still developed ataxia. It is possible that an earlier step in a multistep process leading from conformational change to microaggregates to inclusions might be toxic. This is consistent with recent studies indicating that oligomeric aggregation in intermediates are most likely to be the toxic species (53-55). Oligomeric aggregation intermediates have recently been described for huntingtin exon-1 with an expanded repeat (56).
Thus, our data suggest that a truncation event that preserves the NLS and removes the NES is relevant for cellular toxicity, and that a relatively large fragment can cause cell death. Truncation may interfere with normal cycling between the nucleus and cytoplasm, leading to nuclear accumulation, as we have previously shown in the mouse model (28). When this fragment accumulates in the nucleus it is possible that it binds abnormally to nuclear proteins, subsequently leading to cell death. It has also recently been shown that abnormal interactions between atrophin-1 and the nuclear matrix may be relevant to the disease process (57).
Our results are consistent with emerging data that polyglutamine pathogenesis relates to abnormal interactions between polyglutamine proteins and other proteins within cells, especially in the cell nucleus (45, 58). Several polyglutamine proteins normally interact with nuclear constituents. Huntingtin binds to the nuclear co-repressor protein N-CoR (60), and the strength of this interaction is dependent on polyglutamine repeat length. Atrophin-1 binds to even-skipped in Drosophila, and both human and Drosophila atrophin-1 represses transcription in vivo (61). Atrophin-1 binds to the ETO protein, which is also a member of nuclear co-repressor complexes (57). Huntington and atrophin-1 and other polyglutamine proteins associate with TAFII130 (62) and Sp1 (63) and interactions between DRPLA inclusions and promyelocytic leukemia protein have been identified (64, 65). Ataxin-1 alters nuclear matrix domains containing promyelocytic leukemia protein (66) and p53 (67). HD and DRPLA transgenic mice have altered gene expression compared with control mice (68, 69).
We have demonstrated that atrophin-1 and huntingtin with expanded
repeats, but not with normal repeats, can interact with cAMP-response
element-binding protein (CREB)-binding protein (CBP) and interfere with
CBP-mediated transcription, yielding toxicity. Full-length atrophin-1
with a normal or expanded repeat did not alter CBP-mediated
transcription or cause cellular toxicity. By contrast, the At-N917-65Q
truncated construct used in the current study interfered with
CBP-mediated transcription and caused toxicity (35). There was no
effect on CBP transcription and no toxicity when this atrophin
truncation construct had a normal repeat. Therefore, interference with
CBP transcriptional co-activation is specific for the truncation with
an expanded repeat, and may explain at least in part why this construct
preferentially causes toxicity. This effect is dependent on alteration
of CBP and removal from its normal site of action, and would not
necessarily require the formation of inclusion bodies, although
aggregation intermediates may be involved. Wyttenbach et al.
(70) have also recently showed that mutant huntingtin impairs
CRE-mediated transcription (70). Steffan et al. (59, 71)
have also found abnormal interaction between huntingtin and several
transcription factors including CBP and p53, and have recently shown
that histone deacetylase inhibitors can interfere with HD
neurodegenration in Drosophila. The interactions with
TAFII130 and Sp1 may also be important for pathogenesis
(62, 63) related to altered transcription. One of the downstream genes
may be BDNF, which is a neuronal survival factor regulated by CBP (37).
Thus, taking the recent data together, we hypothesize that truncation,
nuclear localization, and abnormal interactions with nuclear components
of the transcriptional complexes may be key steps in atrophin-1
toxicity, and more generally (58) in the pathogenesis of glutamine
repeat diseases.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank David Borchelt and Gabriele Schilling for helpful discussions. We thank Mike Delanoy for expertise with the confocal microscope. We thank Paul McHugh and the Department of Psychiatry for support.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Huntington's Disease Society of America "Coalition for the Cure," Hereditary Disease Foundation "Cure HD Initiative," and National Institutes of Health Grants NS16375, NS34172, and NS38144 (to C. A. R.) and NS37090 (to T. M. D.).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.
d Supported by National Institutes of Health Grant NS40251, the Huntington's Disease Society of America, and the Hereditary Disease Foundation.
j To whom correspondence should be addressed: Ross Research Bldg., Rm. 618, 720 Rutland Ave., Baltimore, MD 21205-2196. E-mail: caross@jhu.edu.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M211224200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: DRPLA, dentatorubral and pallidoluysian atrophy; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GFP, green fluorescent protein; CBP, cAMP-response element-binding protein (CREB)-binding protein; SCA, spino-cerebellar ataxia.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Smith, J. K., Gonda, V. E., and Malamud, N. (1958) Neurology 8, 205-209[Medline] [Order article via Infotrieve] |
2. | Naito, H., and Oyanagi, S. (1982) Neurology 32, 798-807[Medline] [Order article via Infotrieve] |
3. | Ross, C. A., Margolis, R. L., Rosenblatt, A., Ranen, N. G., Becher, M. W., and Aylward, E. (1997) Medicine 76, 305-338[CrossRef][Medline] [Order article via Infotrieve] |
4. | Koide, R., Ikeuchi, T., Onodera, O., Tanaka, H., Igarashi, S., Endo, K., Takahashi, H., Kondo, R., Ishikawa, A., Hayashi, T., et al.. (1994) Nat. Genet. 6, 13-19 |
5. | Ikeuchi, T., Koide, R., Tanaka, H., Onodera, O., Igarashi, S., Takahashi, H., Kondo, R., Ishikawa, A., Tomoda, A., Miike, T., et al.. (1995) Ann. Neurol. 37, 769-775[Medline] [Order article via Infotrieve] |
6. | Komure, O., Sano, A., Nishino, N., Yamauchi, N., Ueno, S., Kondoh, K., Sano, N., Takahashi, M., Murayama, N., Kondo, I., Nagafuchi, S., Yamada, M., and Kanazawa, I. (1995) Neurology 45, 143-149[Abstract] |
7. | Li, S. H., McInnis, M. G., Margolis, R. L., Antonarakis, S. E., and Ross, C. A. (1993) Genomics 16, 572-579[CrossRef][Medline] [Order article via Infotrieve] |
8. | Nagafuchi, S., Yanagisawa, H., Ohsaki, E., Shirayama, T., Tadokoro, K., Inoue, T., and Yamada, M. (1994) Nat. Genet. 8, 177-182[Medline] [Order article via Infotrieve] |
9. | Housman, D. (1995) Nat. Genet. 10, 3-4[Medline] [Order article via Infotrieve] |
10. | MacDonald, M. E., and Gusella, J. F. (1996) Curr. Opin. Neurobiol. 6, 638-643[CrossRef][Medline] [Order article via Infotrieve] |
11. | Margolis, R. L., and Ross, C. A. (2001) Trends Mol. Med. 7, 479-482[CrossRef][Medline] [Order article via Infotrieve] |
12. | Yazawa, I., Nukina, N., Hashida, H., Goto, J., Yamada, M., and Kanazawa, I. (1995) Nat. Genet. 10, 99-103[CrossRef][Medline] [Order article via Infotrieve] |
13. | Margolis, R. L., Li, S-H., Young, W. S., Wagster, M. V., Stine, O. C., Kidwai, A. S., Ashworth, R. G., and Ross, C. A. (1996) Mol. Brain Res. 36, 219-226[CrossRef][Medline] [Order article via Infotrieve] |
14. | Yazawa, I., Nukina, N., Goto, J., Kurisaki, H., Hebisawa, A., and Kanazawa, I. (1997) Neurosci. Lett. 225, 53-56[CrossRef][Medline] [Order article via Infotrieve] |
15. | Knight, S. P., Richardson, M. M., Osmand, A. P., Stakkestad, A., and Potter, N. T. (1997) J. Neurol. Sci. 146, 19-26[CrossRef][Medline] [Order article via Infotrieve] |
16. | Deleted in proof |
17. | Becher, M. W., and Ross, C. A. (1998) Movement Disorders 13, 852-853[Medline] [Order article via Infotrieve] |
18. |
Sato, A.,
Shimohata, T.,
Koide, R.,
Takano, H.,
Sato, T.,
Oyake, M.,
Igarashi, S.,
Tanaka, K.,
Inuzuka, T.,
Nawa, H.,
and Tsuji, S.
(1999)
Hum. Mol. Genet.
8,
997-1006 |
19. |
DiFiglia, M.,
Sapp, E.,
Chase, K. O.,
Davies, S. W.,
Bates, G. P.,
Vonsattel, J. P.,
and Aronin, N.
(1997)
Science
277,
1990-1993 |
20. | Becher, M. W., Kotzuk, J. A., Sharp, A. H., Davies, S. W., Bates, G. P., Price, D. L., and Ross, C. A. (1998) Neurobiol. Dis. 4, 387-397[CrossRef][Medline] [Order article via Infotrieve] |
21. | Davies, S. W., Beardsall, K., Turmaine, M., DiFiglia, M., Aronin, N., and Bates, G. P. (1998) Lancet 351, 131-133[CrossRef][Medline] [Order article via Infotrieve] |
22. | Peters, M. F., Nucifora, F. C., Jr., Kushi, J., Seaman, H. J., Cooper, J. K., Herring, W. J., Dawson, V. L., Dawson, T. M., and Ross, C. A. (1999) Mol. Cell. Neurosci. 14, 121-128[CrossRef][Medline] [Order article via Infotrieve] |
23. | Saudou, F., Finkbeiner, S., Devys, D., and Greenberg, M. E. (1998) Cell 95, 55-66[Medline] [Order article via Infotrieve] |
24. |
Hackam, A. S.,
Singaraja, R.,
Zhang, T.,
Gan, L.,
and Hayden, M. R.
(1999)
Hum. Mol. Genet.
8,
25-33 |
25. | Igarashi, S., Koide, R., Shimohata, T., Yamada, M., Hayashi, Y., Takano, H., Date, H., Oyake, M., Sato, T., Sato, A., Egawa, S., Ikeuchi, T., Tanaka, H., Nakano, R., Tanaka, K., Hozumi, I., Inuzuka, T., Takahashi, H., and Tsuji, S. (1998) Nat. Genet. 18, 111-117[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Hackam, A. S.,
Singaraja, R.,
Wellington, C. L.,
Metzler, M.,
McCutcheon, K.,
Zhang, T.,
Kalchman, M.,
and Hayden, M. R.
(1998)
J. Cell Biol.
141,
1097-1105 |
27. |
Cooper, J. K.,
Schilling, G.,
Peters, M. F.,
Herring, W. J.,
Sharp, A. H.,
Kaminsky, Z.,
Masone, J.,
Khan, F. A.,
Delanoy, M.,
Borchelt, D. R.,
Dawson, V. L.,
Dawson, T. M.,
and Ross, C. A.
(1998)
Hum. Mol. Genet.
7,
783-790 |
28. | Schilling, G., Wood, J. D., Duan, K., Slunt, H. H., Gonzales, V., Yamada, M., Cooper, J. K., Margolis, R. L., Jenkins, N. A., Copeland, N. G., Takahashi, H., Tsuji, S., Price, D. L., Borchelt, D. R., and Ross, C. A. (1999) Neuron 24, 275-286[Medline] [Order article via Infotrieve] |
29. | Yamada, M., Wood, J. D., Shimohata, T., Hayashi, S., Tsuji, S., and Ross, C. A. (2001) Ann. Neurol. 49, 14-23[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Miyashita, T.,
Miyashita, T.,
Okamura-Oho, Y.,
Mito, Y.,
Nagafuchi, S.,
and Yamada, M.
(1997)
J. Biol. Chem.
272,
29238-29242 |
31. |
Wellington, C. L.,
Ellerby, L. M.,
Hackam, A. S.,
Margolis, R. L.,
Trifiro, M. A.,
Singaraja, R.,
McCutcheon, K.,
Salvesen, G. S.,
Propp, S. S.,
Bromm, M.,
Rowland, K. J.,
Zhang, T.,
Rasper, D.,
Roy, S.,
Thornberry, N.,
Pinsky, L.,
Kakizuka, A.,
Ross, C. A.,
Nicholson, D. W.,
Bredesen, D. E.,
and Hayden, M. R.
(1998)
J. Biol. Chem.
273,
9158-9167 |
32. |
Ellerby, L. M.,
Andrusiak, R. L.,
Wellington, C. L.,
Hackam, A. S.,
Propp, S. S.,
Wood, J. D.,
Sharp, A. H.,
Margolis, R. L.,
Ross, C. A.,
Salvesen, G. S.,
Hayden, M. R.,
and Bredesen, D. E.
(1999)
J. Biol. Chem.
274,
8730-8736 |
33. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
34. | Wood, J. D., Yuan, J., Margolis, R. L., Colomer, V., Duan, K., Kushi, J., Kaminsky, Z., Kleiderlein, J., Sharp, A. H., and Ross, C. A. (1998) Mol. Cell. Neurosci. 11, 149-160[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Nucifora, F. C., Jr.,
Sasaki, M.,
Peters, M. F.,
Huang, H.,
Cooper, J. K.,
Yamada, M.,
Takahashi, H.,
Tsuji, S.,
Troncosco, J.,
Dawson, V. L.,
Dawson, T. M.,
and Ross, C. A.
(2001)
Science
291,
2423-2428 |
36. |
Okamura-Oho, Y.,
Miyashita, T.,
Ohmi, K.,
and Yamada, M.
(1999)
Hum. Mol. Genet.
8,
947-957 |
37. |
Zuccato, C.,
Ciammola, A.,
Rigamonti, D.,
Leavitt, B. R.,
Goffredo, D.,
Conti, L.,
MacDonald, M. E.,
Friedlander, R. M.,
Silani, V.,
Hayden, M. R.,
Timmusk, T.,
Sipione, S.,
and Cattaneo, E.
(2001)
Science
293,
493-498 |
38. | Klement, I. A., Skinner, P. J., Kaytor, M. D., Yi, H., Hersch, S. M., Clark, H. B., Zoghbi, H. Y., and Orr, H. T. (1998) Cell 95, 41-53[Medline] [Order article via Infotrieve] |
39. | Yazawa, I. (2000) Biochem. J. 351, 587-593[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Wheeler, V. C.,
Auerbach, W.,
White, J. K.,
Srinidhi, J.,
Auerbach, A.,
Ryan, A.,
Duyao, M. P.,
Vrbanac, V.,
Weaver, M.,
Gusella, J. F.,
Joyner, A. L.,
and MacDonald, M. E.
(1999)
Hum. Mol. Genet.
8,
115-122 |
41. |
Mende-Mueller, L. M.,
Toneff, T.,
Hwang, S. R.,
Chesselet, M. F.,
and Hook, V. Y.
(2001)
J. Neurosci.
21,
1830-1837 |
42. | Dyer, R. B., and McMurray, M. E. (2001) Nat. Genet. 29, 270-278[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Wheeler, V. C.,
White, J. K.,
Gutekunst, C. A.,
Vrbanac, V.,
Weaver, M.,
Li, X. J.,
Li, S. H.,
Yi, H.,
Vonsattel, J. P.,
Gusella, J. F.,
Hersch, S.,
Auerbach, W.,
Joyner, A. L.,
and MacDonald, M. E.
(2000)
Hum. Mol. Genet.
9,
503-513 |
44. | Reddy, P. H., Williams, M., Charles, V., Garrett, L., Pike-Buchanan, L., Whetsell, W. O., Jr., Miller, G., and Tagle, D. A. (1998) Nat. Genet. 20, 198-202[CrossRef][Medline] [Order article via Infotrieve] |
45. | Hodgson, J. G., Agopyan, N., Gutekunst, C. A., Leavitt, B. R., LePiane, F., Singaraja, R., Smith, D. J., Bissada, N., McCutcheon, K., Nasir, J., Jamot, L., Li, X. J., Stevens, M. E., Rosemond, E., Roder, J. C., Phillips, A. G., Rubin, E. M., Hersch, S. M., and Hayden, M. R. (1999) Neuron 23, 181-192[Medline] [Order article via Infotrieve] |
46. |
Gafni, J.,
and Ellerby, L. M.
(2002)
J. Neurosci.
22,
4842-4849 |
47. |
Wellington, C. L.,
Ellerby, L. M.,
Gutekunst, C. A.,
Rogers, D.,
Warby, S.,
Graham, R. K.,
Loubser, O.,
van Raamsdonk, J.,
Singaraja, R.,
Yang, Y. Z.,
Gafni, J.,
Bredesen, D.,
Hersch, S. M.,
Leavitt, B. R.,
Roy, S.,
Nicholson, D. W.,
and Hayden, M. R.
(2002)
J. Neurosci.
22,
7862-7872 |
48. |
Kim, Y. J.,
Yi, Y.,
Sapp, E.,
Wang, Y.,
Cuiffo, B.,
Kegel, K. B.,
Qin, Z. H.,
Aronin, N.,
and DiFiglia, M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
12784-12789 |
49. | Li, H., Johnston, H., Shelbourne, P. F., and Li, X. J. (2000) Nat. Genet. 25, 385-389[CrossRef][Medline] [Order article via Infotrieve] |
50. | Tanaka, Y., Igarashi, S., Morita, H., Schilling, G., Wanner, T., Bennett, K. M., Wood, J. D., Borchelt, D. R., and Ross, C. A. (2001) Soc. Neurosci. Abstr. 863.13 |
51. | Sun, B., Fan, W., Balciunas, A., Cooper, J. K., Bitan, G., Steavenson, S., Denis, P. E., Young, Y., Adler, B., Daughterty, L., Manoukian, R., Elliott, G., Shen, W., Talvenheimo, J., Teplow, D. B., Haniu, M., Haldankar, R., Wypych, J., Ross, C. A., Citron, M., and Richards, W. G. (2002) Neurobiol Dis. 11, 111-122[CrossRef][Medline] [Order article via Infotrieve] |
52. | Lunkes, A., Lindenberg, K. S., Ben-Haiem, L., Weber, C., Devys, D., Landwehrmeyer, G. B., Mandel, J. L., and Trottier, Y. (2002) Mol. Cell 10, 259-269[Medline] [Order article via Infotrieve] |
53. | Ellis, R. J., and Pinheiro, T. J. T. (2002) Nature 416, 535-539[CrossRef][Medline] [Order article via Infotrieve] |
54. | Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., and Stefani, M. (2002) Nature 416, 507-511[CrossRef][Medline] [Order article via Infotrieve] |
55. | Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002) Nature 416, 535-539[CrossRef][Medline] [Order article via Infotrieve] |
56. |
Poirier, M. A.,
Li, H.,
Macosko, J.,
Cai, S.,
Amzel, M.,
and Ross, C. A.
(2002)
J. Biol. Chem.
277,
41032-41037 |
57. |
Wood, J. D.,
Nucifora, F. C., Jr.,
Duan, K.,
Zhang, C.,
Wang, J.,
Kim, Y.,
Schilling, G.,
Sacchi, N. J.,
Liu, J. M.,
and Ross, C. A.
(2000)
J. Cell Biol.
150,
939-948 |
58. | Ross, C. A. (2002) Neuron 35, 819-822[Medline] [Order article via Infotrieve] |
59. | Steffan, J. S., Bodai, L., Pallos, J., Poelman, M., McCampbell, A., Apostol, B. L., Kazantsev, A., Schmidt, E., Zhu, Y. Z., Greenwald, M., Kurokawa, R., Housman, D. E., Jackson, G. R., Marsh, J. L., and Thompson, L. M. (2001) Nature 413, 739-743[CrossRef][Medline] [Order article via Infotrieve] |
60. |
Boutell, J. M.,
Thomas, P.,
Neal, J. W.,
Weston, V. J.,
Duce, J.,
Harper, P. S.,
and Jones, A. L.
(1999)
Hum. Mol. Genet.
8,
1647-1655 |
61. | Zhang, S., Xu, L., Lee, J., and Xu, T. (2001) Cell 108, 45-56 |
62. | Shimohata, T., Nakajima, T., Yamada, M., Uchida, C., Onodera, O., Naruse, S., Kimura, T., Koide, R., Nozaki, K., Sano, Y., Ishiguro, H., Sakoe, K., Ooshima, T., Sato, A., Ikeuchi, T., Oyake, M., Sato, T., Aoyagi, Y., Hozumi, I., Nagatsu, T., Takiyama, Y., Nishizawa, M., Goto, J., Kanazawa, I., Davidson, I., Tanese, N., Takahashi, H., and Tsuji, S. (2000) Nat. Genet. 26, 29-36[CrossRef][Medline] [Order article via Infotrieve] |
63. |
Dunah, A. W.,
Jeong, H.,
Griffin, A.,
Kim, Y. M.,
Standaert, D. G.,
Hersch, S. M.,
Mouradian, M. M.,
Young, A. B.,
Tanese, N.,
and Krainc, D.
(2002)
Science
296,
2238-2243 |
64. |
Waerner, T.,
Gardellin, P.,
Pfizenmaier, K.,
Weith, A.,
and Kraut, N.
(2001)
Cell Growth Differ.
12,
201-210 |
65. |
Yamada, M.,
Sato, T.,
Shimohata, T.,
Hayashi, S.,
Igarashi, S.,
Tsuji, S.,
and Takahashi, H.
(2001)
Am. J. Pathol.
159,
1785-1795 |
66. | Skinner, P. J., Koshy, B. T., Cummings, C. J., Klement, I. A., Helin, K., Servadio, A., Zoghbi, H. Y., and Orr, H. T. (1997) Nature 389, 971-974[CrossRef][Medline] [Order article via Infotrieve] |
67. | Shahbazian, M. D., Orr, H. T., and Zoghbi, H. Y. (2001) Neurobiol. Dis. 8, 874-981 |
68. |
Luthi-Carter, R.,
Strand, A.,
Peters, N. L.,
Solano, S. M.,
Hollingsworth, Z. R.,
Menon, A. S.,
Frey, A. S.,
Spektor, B. S.,
Penney, E. B.,
Schilling, G.,
Ross, C. A.,
Borchelt, D. R.,
Tapscott, S. J.,
Young, A. B.,
Cha, J-H. J.,
and Olson, J. M.
(2000)
Hum. Mol. Genet.
9,
1259-1271 |
69. |
Luthi-Carter, R.,
Strand, A. D.,
Hanson, S. A.,
Kooperberg, C.,
Schilling, G.,
La Spada, A. R.,
Merry, D. E.,
Young, A. B.,
Ross, C. A.,
Borchelt, D. R.,
and Olson, J. M.
(2002)
Hum. Mol. Genet.
11,
1927-1937 |
70. |
Wyttenbach, A.,
Swartz, J.,
Kita, H.,
Thykjaer, T.,
Carmichael, J.,
Bradley, J.,
Brown, R.,
Maxwell, M.,
Schapira, A.,
Orntoft, T. F.,
Kato, K.,
and Rubinsztein, D. C.
(2001)
Hum. Mol. Genet.
10,
1829-1845 |
71. |
Steffan, J. S.,
Kazantsev, A.,
Spasic-Boskovic, O.,
Greenwald, M.,
Zhu, Y. Z.,
Gohler, H.,
Wanker, E. E.,
Bates, G. P.,
Housman, D. E.,
and Thompson, L. M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6763-6768 |