 |
 Previous Article | Next Article 
The Journal of Neuroscience, June 15, 2002, 22(12):4897-4905
Polyglutamine-Expanded Ataxin-7 Promotes Non-Cell-Autonomous
Purkinje Cell Degeneration and Displays Proteolytic Cleavage in Ataxic
Transgenic Mice
Gwenn A.
Garden1,
Randell T.
Libby2,
Ying-Hui
Fu7,
Yoshito
Kinoshita3,
Jing
Huang4,
Daniel E.
Possin4,
Annette C.
Smith2,
Refugio A.
Martinez2,
Gabriel C.
Fine1,
Sara K.
Grote1, 2,
Carol B.
Ware5,
David D.
Einum8,
Richard S.
Morrison3,
Louis J.
Ptacek8, 9,
Bryce L.
Sopher2, and
Albert R. La
Spada1, 2, 6
Departments of 1 Neurology, 2 Laboratory
Medicine, 3 Neurological Surgery,
4 Ophthalmology, 5 Comparative Medicine, and
6 Medicine (Division of Medical Genetics), University of
Washington Medical Center, Seattle, Washington 98195-7110, Departments
of 7 Neurobiology and Anatomy and 8 Human
Genetics, and 9 Howard Hughes Medical Institute,
University of Utah, Salt Lake City, Utah 84112
 |
ABSTRACT |
Spinocerebellar ataxia (SCA) type 7 is an inherited
neurodegenerative disorder caused by expansion of a polyglutamine tract within the ataxin-7 protein. To determine the molecular basis of
polyglutamine neurotoxicity in this and other related disorders, we
produced SCA7 transgenic mice that express ataxin-7 with 24 or 92 glutamines in all neurons of the CNS, except for Purkinje cells.
Transgenic mice expressing ataxin-7 with 92 glutamines (92Q) developed
a dramatic neurological phenotype presenting as a gait ataxia and
culminating in premature death. Despite the absence of expression of
polyglutamine-expanded ataxin-7 in Purkinje cells, we documented severe
Purkinje cell degeneration in 92Q SCA7 transgenic mice. We also
detected an N-terminal truncation fragment of ataxin-7 in
transgenic mice and in SCA7 patient material with both anti-ataxin-7
and anti-polyglutamine specific antibodies. The appearance of truncated
ataxin-7 in nuclear aggregates correlates with the onset of a disease
phenotype in the SCA7 mice, suggesting that nuclear localization and
proteolytic cleavage may be important features of SCA7 pathogenesis.
The non-cell-autonomous nature of the Purkinje cell degeneration in our
SCA7 mouse model indicates that polyglutamine-induced dysfunction in
adjacent or connecting cell types contributes to the neurodegeneration.
Key words:
polyglutamine; ataxin-7; neurodegeneration; Purkinje
cell; non-cell autonomous; truncation; proteolytic cleavage
| |
INTRODUCTION |
The spinocerebellar ataxias (SCAs)
are a group of inherited neurological disorders that share the common
feature of cerebellar degeneration. Spinocerebellar ataxia type 7 (SCA7) is classified as an autosomal dominant cerebellar ataxia type II
(ADCA II), because affected patients display retinal and brainstem
degeneration (Enevoldson et al., 1994 ). Neuropathological examination
corroborates the clinical picture, revealing marked atrophy of the
cerebellar vermis, the inferior olivary nucleus, and the dentate
nucleus (Weiner et al., 1967). Neuronal loss and degenerative changes are consequently greatest in the Purkinje cell layer of the cerebellum and in the inferior olivary complex.
A compelling feature of SCA7 is genetic anticipation (Enevoldson et
al., 1994). On the basis of this observation, a repeat expansion was
sought as the cause of SCA7, and an expanded CAG tract, ranging from 38 to >250 triplets, was identified (David et al., 1997). The CAG repeat
encodes a run of glutamines in the ataxin-7 protein, placing SCA7 in a
category of diseases caused by polyglutamine tract expansions. The
CAG/polyglutamine repeat disease group consists of at least nine
inherited neurodegenerative disorders, including spinal and bulbar
muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA),
Huntington's disease (HD), and six types of spinocerebellar ataxia
(SCA1, -2, -3, -6, -7, and -17) (Zoghbi and Orr, 2000; Nakamura et al.,
2001).
With the exception of SCA6, all of the polyglutamine repeat diseases
involve expansion beyond a threshold of ~35 glutamines, resulting in
a change in the conformation of the protein. The transition to a novel
conformation may be detected by antibodies directed against extended
glutamine runs (Trottier et al., 1995), and the adoption of an altered
conformation is hypothesized to underlie a toxic gain-of-function
effect. Studies done on animals and in cell culture suggest that the
mutant polyglutamine-containing protein (or a peptide fragment thereof)
must localize to the nucleus to initiate pathogenesis (Klement et al.,
1998; Saudou et al., 1998). Indeed, our recent work on the molecular
basis of SCA7 retinal degeneration supports such a model (La Spada et
al., 2001). Once in the nucleus, the polyglutamine-expanded protein or
peptide fragment can interfere with the function of transcription
factors or coactivators (McCampbell et al., 2000; Orr, 2001). Expanded polyglutamine tracts are also relatively resistant to proteasomal degradation via the ubiquitin pathway (Paulson, 1999), and this leads
to the accumulation of polyglutamine-containing peptide fragments
within the cell. In SBMA, HD, DRPLA, SCA1, SCA3, and SCA7, aggregates
of polyglutamine-containing fragments form nuclear inclusions (NIs)
within neurons (Paulson et al., 2000).
To advance our understanding of polyglutamine neurodegeneration, we
have generated a mouse model for SCA7. Lines of transgenic mice
expressing ataxin-7 with 92 glutamines developed a neurological phenotype presenting as a gait ataxia. Despite the absence of expression of polyglutamine-expanded ataxin-7 in Purkinje cells, we
documented severe degeneration of cerebellar Purkinje cells in these
mice. We observed NIs consisting of N-terminal truncations of
polyglutamine-expanded ataxin-7 and were able to detect an N-terminal
truncation fragment of ~55 kDa in protein lysates from affected
tissues. The appearance of NIs in cerebellar neurons coincides with the
onset of a coordination defect in the SCA7 transgenic mice, suggesting
that nuclear localization of the truncated mutant ataxin-7 correlates
with the ability to initiate pathogenesis. The non-cell-autonomous
nature of the Purkinje cell degeneration in this model implies that
dysfunction in adjacent cell types may play a prominent role in SCA7.
| |
MATERIALS AND METHODS |
Transgenic mice. The generation of the SCA7
transgenic constructs and the production of the PrP-SCA7-c92Q and
PrP-SCA7-c24Q transgenic mice have been described previously (La Spada
et al., 2001).
Immunohistochemistry. Deeply anesthetized mice were perfused
transcardially with 4% paraformaldehyde in 0.1 M
phosphate buffer (PB), pH 7.4. The brain was removed, placed in
paraformaldehyde for 4 hr, and cryoprotected in 10% sucrose and then
30% sucrose in PB. Parasagittal frozen sections were cut at 30 µm
thickness on a sliding microtome. Immunohistochemistry (IHC) was
performed on free-floating sections after blocking in 10% goat serum,
1% BSA, and 0.3% Triton X-100 in PBS, and the primary antibody was diluted in 0.3% Triton X-100 in PBS. The antibodies used and their respective dilutions are as follows: anti-ataxin-7 antibody A, 1:300
(La Spada et al., 2001); anti-ataxin-7 antibody K, 1:2000 (La Spada et
al., 2001); expanded polyglutamine tract antibody 1C2, 1:1000
(Chemicon); HDJ-2, 1:500 (NeoMarkers); and Hsc-70, 1:200 (StressGen).
Primary antibody staining was visualized with the ABC Vector elite kit
(Vector Laboratories) using diaminobenzidine as a chromagen.
Western blot analysis. Protein lysates were obtained by
homogenizing tissues in sample buffer (62.5 mM
Tris-HCl, pH 6.8, 4% SDS, 200 mM dithiothreitol,
10% glycerol, 0.001% Bromophenol blue) at a ratio of 1:10 (w/v) and
then boiling. Nuclear extracts were prepared using the NE-PER Nuclear
and Cytoplasmic Extraction Reagents Kit (Pierce). Protein samples were
resolved by SDS-PAGE (8% acrylamide), transferred to nitrocellulose,
and probed with antibody K (La Spada et al., 2001) or 1C2 antibody
(Dako) or p44/42 MAP kinase antibody (Cell Signaling Technologies) at
1:1000 dilution in TBS containing 0.1% Tween 20 and 5% BSA, after
membrane blocking in TBS with 0.1% Tween 20 and 5% non-fat dry milk.
Antibody 1598, produced by Yvert et al. (2000), was kindly provided by
J.-L. Mandel (Institut de Génétique et de Biologie
Moléculaire et Cellulaire, Institute National de la Santé
et de la Recherche Médicale, Strasbourg, France) and was also
used at a dilution of 1:1000. The primary antibody was visualized with
horseradish peroxidase-coupled anti-rabbit antibodies (Amersham) at
1:2000 dilution and Enhanced Chemiluminescence (Amersham).
Behavioral analysis. Mice of varying ages and genotype were
assessed with four different behavioral tests in a blinded manner by
investigators over the course of four successive days. The "clasping
test" assessed whether mice would clasp their forelimbs and hindlimbs
into their bodies or would splay their limbs when suspended by their
tails (Lin et al., 2001). The "activity test" measured the activity
of mice after removal of their cage lid. Normal "active" mice
typically explore their cages when the lid is removed, whereas
neurologically affected mice remain inactive or show enhanced
exploration of their surroundings (Lin et al., 2001). The
"ledge test" (which we developed) involved placing a mouse on the
ledge of a cage and observing the response of the mouse to this
predicament. Although normal mice either scurry along the bar of the
cage and jump down without a problem, mice with neurological
dysfunction typically freeze in place or fall into their cages.
Finally, we determined the coordination and motor functions of the mice
by performing rotarod testing with an Economex Accelerating Rotarod
apparatus (Columbus Instruments). The speed of the rod was set to 4 rpm
and increased to a final speed of 40 rpm at a rate of 0.1 rpm/sec, and
latency to fall was measured in the best four of five daily trails over
the course of 4 d. Performance on the rotarod was statistically
compared by two-factor ANOVA (Microsoft EXCEL, Office 98).
Light microscopy. Mice were killed, and the brain was
removed and immersed in a fixative consisting of 1.6%
paraformaldehyde, 2.5% glutaraldehyde, 0.05%
MgCl2 · 6H20, and 0.04 M sucrose in 0.08 M PIPES
buffer at pH 7. After washing, brains were secondarily fixed with 1%
osmium tetroxide for 1 hr. A graded alcohol series (methanol or
ethanol) was then used to dehydrate the brains: 35, 70, 95, and 100%,
each 15 min per change. After three propylene oxide transition
washings, brains were embedded in epoxy, and the propylene oxide was
allowed to evaporate from the mixture overnight on a rotating shaker.
The embedded brains were transferred to molds, and after hardening, the
resulting blocks were sectioned on an ultramicrotome.
One-micrometer-thick sections were cut and mounted on glass slides
after immersion in 50% (v/v) Richardson's stain (sodium borate,
Methylene blue, and Azure II, each at 1% w/v) in 0.1 M dibasic sodium phosphate solution, pH 8.8.
Confocal microscopy. Free-floating 40 µm brain sections
were blocked with 10% goat serum, 1% BSA, and 0.3% Triton X-100 in PBS for 1 hr and then incubated with antibody K (1:2000) and
anti-calbindin (1:1000) in PBS containing 0.3% Triton X-100 for 48 hr
at 4°C. After three washes with PBS, Alexa Fluor 488 anti-mouse IgG
(Molecular Probes) at 1:100 dilution, Cy3 anti-rabbit IgG (Jackson
ImmunoResearch) at 1:200 dilution, and 4',6'-diamidino-2-phenylindole
dihydrochloride (DAPI; Molecular Probes) at 1:10,000 dilution were
applied to the sections and incubated together overnight at 4°C.
After washing, the sections were mounted on glass slides and
coverslipped in a medium of 90% glycerol and 10% phosphate buffer
with n-propyl gallate added at a final concentration of 0.5% to reduce
fluorescent label bleaching. The sections were viewed, and digital
scans were recorded using a Zeiss 510 multiphoton nonlinear
optics confocal microscope system. Digital image
z-stacks were created, and projections were made from them
using the Zeiss AIM LSM software and the NIH Image J program
(http://rsb.info.nih.gov/ij/).
| |
RESULTS |
Characterization of the SCA7 transgenic mice: expression pattern
and aggregates
To produce a mouse model of SCA7, we inserted the ataxin-7 coding
region with either 24 or 92 CAGs into the MoPrP expression vector to
yield two transgenic constructs: PrP-SCA7-c24Q and PrP-SCA7-c92Q. PCR
screening identified five founders (one PrP-SCA7-c24Q mouse and four
PrP-SCA7-c92Q mice). As described previously, we determined the
expression level of the ataxin-7 transgene in brain RNA samples from
representative individuals for each available line and confirmed expression of full-length ataxin-7 protein (La Spada et al., 2001).
To study the expression pattern of ataxin-7 in the CNS, we
immunostained frozen sections from our transgenic mice. We found that
ataxin-7 is widely expressed in neurons throughout the CNS in the 92Q
transgenic mice, but we observed no appreciable increase in ataxin-7
immunostaining in 92Q cerebellar Purkinje cells in comparison with 24Q
and non-transgenic control mice (Fig. 1)
(data not shown). Lack of transgene expression in Purkinje cells is a
feature of the MoPrP construct and has been observed repeatedly (Borchelt et al., 1996; Schilling et al., 1999a,b). In the 92Q transgenic mice, there was intense nuclear staining of neurons, with
especially dense staining noted in the granule cell layer of the
cerebellum (Fig. 1A,B), pyramidal
neurons, and dentate gyrus granule neurons of the hippocampus (Fig.
1C,D), the pontine nucleus (Fig.
1E), and the inferior olivary nucleus (Fig.
1F). At higher magnification, this nuclear staining
is clearly punctate with usually one or two dense regions of
immunoreactivity (Fig. 1G,H). These
densely stained structures resemble the NIs reported in other
polyglutamine repeat diseases and mouse models (Ross, 1997) and could
be detected with antibodies directed against polyglutamine expansion
tracts or heat shock chaperones (1C2, HDJ2, Hsc-70; data not shown). We
observed variation in the intensity of ataxin-7 immunoreactivity in NIs
between transgenic individuals of the same line and of different lines
(data not shown). The intensity of anti-ataxin-7 antibody staining
increases with transgene expression level and with age, suggesting that
ataxin-7 protein is accumulating in NIs over time.
View larger version (140K):
[in this window]
[in a new window]
|
Figure 1.
Immunohistochemical staining of CNS sections from
a 19-week-old line 6076 PrP-SCA7-c92Q mouse and a 19-week-old line 6129 PrP-SCA7-c24Q mouse. After paraformaldehye perfusion, tissues were
frozen, cut into 30 µm sections, and immunostained with antibody K
(A-D, G,
H) or antibody A (E,
F). A, B, NIs form
in cerebellar neurons in ataxin-7 92Q mice but not in ataxin-7 24Q
mice. The cerebellum of the ataxin-7 92Q mouse
(A) reveals intense punctate nuclear staining of
neurons in the granule cell layer and the molecular layer, whereas no
such staining is observed in the ataxin-7 24Q transgenic control
(B). Nonspecific cytoplasmic Purkinje cell
immunoreactivity is similar in both genotypes (as shown here) and in
non-transgenic littermate controls (data not shown). C,
D, Regions of the cortex show intense immunoreactivity
in the ataxin-7 92Q transgenic mice. Neuronal nuclei of the hippocampus
stain strongly with the ataxin-7 antibody in the 92Q mouse
(C), whereas the staining is much less intense in
the 24Q transgenic control (D). E,
F, The basis pontis (E) and the
inferior olivary nucleus (F) show expression of
ataxin-7 in the PrP-SCA7-92Q transgenic mouse. G,
H, Extensive NI formation occurs in PrP-SCA7-c92Q mice.
At high-power magnification, dense nuclear staining is apparent in the
ataxin-7 92Q mouse with prominent inclusions (G). The
immunostaining in the ataxin-7 24Q mouse
(H) is much less intense, and no evidence
of nuclear inclusion formation is observed. Scale bars are as marked to
indicate magnification.
|
|
PrP-SCA7-c92Q mice develop ataxia
The first sign of a neurological dysfunction in the PrP-SCA7-c92Q
mice is an unsteady gait beginning at 8-15 weeks of age (Table
1). The gait ataxia gradually progresses
until the mice display a wide-based stride with occasional falling. The
movement disorder then becomes more complicated, and the mice start to shudder and startle easily. The mice become less explorative and a
chronic whole-body tremor eventually ensues. By this time, the PrP-SCA7-c92Q mice are significantly smaller in size than their non-transgenic littermates. Such phenotypically affected transgenic mice show decreased survival, with the most severely ill individuals of
the highest expressing line (i.e., 6080) dying or requiring euthanasia
at 13 weeks of age (Table 1). Despite high level expression of the
ataxin-7 transgene in heart and kidney, necropsies performed on these
affected mice found no non-neural pathology or obvious cause of death
(data not shown). In contrast, the eldest PrP-SCA7-c24Q transgenic mice
(1 year of age) show no visible signs of neurological disease and
display no increased mortality.
To determine the nature of the behavioral abnormality and the timing of
its onset in the SCA7 transgenic mice, we performed a series of four
tests to gauge neurological function: clasping test, activity test,
ledge test, and rotarod test (see Materials and Methods). Because of
the difficulty in maintaining lines 6080 and 6561, we focused our
behavioral testing on one high-expressing PrP-SCA7-c92Q line (i.e.,
6076), one low-expressing PrP-SCA7-c92Q line (i.e., 6529), and the
high-expressing PrP-SCA7-c24Q line (i.e., 6129). Although all tested
mice performed all four paradigms at 4 weeks of age without significant
differences, 92Q transgene-positive mice from line 6076 exhibited a
significant impairment in rotarod function by 8 weeks of age (Fig.
2). At 12 weeks of age, performance on
the rotarod considerably worsened for line 6076 mice, and the performance difference became even more apparent. This was accompanied by abnormalities in the other testing paradigms, with 50% of the 12-week-old line 6076 92Q mice being classified as "claspers," "inactive," or failing the ledge test (data not shown). No
functional deficits were noted in these three testing paradigms for
comparably aged non-transgenic littermates or for younger (i.e., 4 and
8 week old) line 6076 92Q mice. Once line 6076 mice become visibly ataxic at 13-15 weeks of age, virtually all such mice display abnormal
behaviors in all three of these paradigms. Line 6529 mice that express
the ataxin-7 92Q transgene at levels less than endogenous, however,
continued to perform comparably on the rotarod to aged-matched
littermate controls when tested at ages beyond 1 year, as did the line
6129 PrP-SCA7-c24Q mice (data not shown).
View larger version (12K):
[in this window]
[in a new window]
|
Figure 2.
Rotarod analysis of the SCA7 transgenic mice.
Groups of six to eight mice were tested over the course of 4 d on
an accelerating rotarod. Mean latency to fall (in seconds) was obtained
for each group (PrP-SCA7-c92Q mice from line 6076 and their age-matched
non-transgenic littermates) and compared statistically by ANOVA. Error
bars represent the SD from the mean. A, At 4 weeks of
age, SCA7 transgenic mice perform comparably to non-transgenic controls
on the rotarod (p = 0.29). B,
By 8 weeks of age, SCA7 transgenic mice begin to show a significant
impairment in rotarod performance in comparison with non-transgenic
control mice (p < 0.05). C,
At 12 weeks of age, the impairment in rotarod performance in the SCA7
transgenic mice becomes even more pronounced as the coordination
deficit progresses (p < 0.001).
|
|
Neurodegenerative changes in the cerebellum occur in the absence of
apoptosis or significant neuronal loss
Because the neurological dysfunction in the SCA7 mice resembles
the presentation of patients with cerebellar ataxia, we evaluated the
CNS of our transgenic mice for neurodegenerative changes with particular emphasis on neuroanatomical analysis of the cerebellum. Using a basic dye stain (Richardson's), we examined the cerebella of
PrP-SCA7-c92Q mice from line 6076 at 20 weeks of age and found marked
histopathology and degenerative changes (Fig.
3). Although the cerebella of
non-transgenic littermate controls displayed Purkinje cells of typical
morphology, the PrP-SCA7-c92Q cerebella consisted of Purkinje cells
that were much smaller and shrunken by comparison. Instead of a normal
cuboid appearance, the Purkinje cells of the line 6076 PrP-SCA7-c92Q
mice were flattened and exhibited less dendritic arborization. The
nuclei of the 92Q Purkinje cells revealed occasional invaginations and
appeared granular. Despite the observation of degenerative changes in
cerebella of PrP-SCA7-c92Q animals killed at end-stage disease, granule
cells and Purkinje cells were not labeled by the terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end
labeling method of apoptosis detection (data not shown). Furthermore,
there was no obvious difference in the number of neurons within
PrP-SCA7-c92Q cerebella immunoreactive for NeuN or
microtubule-associated protein 2 (data not shown). These findings
suggest that functional deficits develop in PrP-SCA7-c92Q mice without
significant neuronal loss.
View larger version (147K):
[in this window]
[in a new window]
|
Figure 3.
Neuroanatomical analysis of the SCA7
transgenic mice reveals marked histopathology and degenerative changes.
Using a basic dye stain (Richardson's), we examined the cerebella of
PrP-SCA7-c92Q transgenic mice from line 6076 (Tg) and
non-transgenic littermate controls (Nt) at 20 weeks of
age. Although the cerebella of Nt controls displayed Purkinje cells of
typical morphology, Tg cerebella contained shrunken Purkinje cells with
diminished dendritic arborization. The nuclei of the Tg ataxin-7 92Q
Purkinje cells also revealed occasional invaginations and appeared
granular. gcl, Granule cell layer; pcl,
Purkinje cell layer; ml, molecular layer.
|
|
To determine the timing of aggregate formation relative to the onset of
neurological dysfunction, cerebellar sections from SCA7 transgenic mice
at 4, 8, and 13 weeks of age were double labeled with antibodies
recognizing ataxin-7 and the Purkinje cell-specific calcium binding
protein calbindin. Although 4-week-old PrP-SCA7-c92Q mice from line
6076 do not display ataxin-7-positive aggregates in cerebellar neurons,
NIs are clearly visible in cerebellar granule cell neurons at 13 weeks
of age in c92Q mice from line 6076 (Fig.
4). Although NIs can be detected in
non-Purkinje cell neurons of the molecular layer (such as stellate,
basket, and Golgi cells), NIs are not visible in the Purkinje cells
themselves, confirming that the MoPrP promoter does not drive
expression in this cell type. Interestingly, detection of NIs in the
granule cell layer and the molecular cell layer of the cerebella of the line 6076 mice first becomes apparent at 8 weeks of age (Fig. 4). The
NIs are few in number at this age and accompanied by diffuse nuclear
staining in a subset of cerebellar granule neurons, suggesting that
accumulation of the polyglutamine-expanded ataxin-7 first becomes
appreciable around this time. Despite the absence of expression of
polyglutamine-expanded ataxin-7 in Purkinje cells, calbindin immunoreactivity in Purkinje cell dendrites was markedly decreased in
both 8- and 13-week-old line 6076 transgenic mice (Fig. 4). These
findings demonstrate that ataxin-7-containing NIs and
non-cell-autonomous Purkinje cell abnormalities develop coincident with
the onset of neurological dysfunction in the line 6076 SCA7 mice (Fig.
1). IHC analysis of line 6129 ataxin-7 24Q mice and of low-expressing line 6529 ataxin-7 92Q mice revealed no loss of calbindin
immunoreactivity or NI formation in individuals at 1 year of age (data
not shown).
View larger version (120K):
[in this window]
[in a new window]
|
Figure 4.
Nuclear aggregate formation coincides with the
onset of Purkinje cell degeneration in SCA7 transgenic mice. IHC
analysis of cerebellar sections from SCA7 transgenic mice at 4, 8, and
13 weeks of age was performed by staining with antibody K
(magenta), a calbindin antibody
(green), and DAPI (blue). Both
4-week-old PrP-SCA7-c92Q transgenic mice from line 6076 (A) and age-matched non-transgenic littermate
controls (B) do not display aggregates in
cerebellar granule neurons and other non-Purkinje cell neurons.
Calbindin immunostaining indicates a substantial loss of healthy
dendrites from the molecular layer of the cerebellum in 8-week-old
ataxin-7-92Q mice (C), whereas age-matched
non-transgenic littermate controls show extensive dendritic
arborization in the ML (D). Loss of calbindin
immunostaining in 8-week-old PrP-SCA7-c92Q transgenic mice from line
6076 is accompanied by ataxin-7 immunoreactivity in a subset of granule
cells in the GCL and neurons in the ML (C). Note
that at this age ataxin-7 immunostaining is diffuse in certain cells
and punctate in other cells, indicating that aggregate formation is
just beginning. E, At 13 weeks of age, NIs are apparent
in numerous, if not all, cerebellar granule cell neurons in
PrP-SCA7-c92Q transgenic mice from line 6076. Although NIs can be
detected in numerous non-Purkinje cell neurons of the ML (such as
stellate, basket, and Golgi cells), NIs are not seen in the PCL,
confirming absence of expression of polyglutamine-expanded ataxin-7 in
Purkinje cells. Calbindin staining nonetheless reveals a further
decrease in dendritic arbors from the Purkinje cells in the ML.
F, Age-matched non-transgenic littermate controls again
show normal cerebellar cytoarchitecture and dendritic arborization.
ML, Molecular layer; PCL, Purkinje cell
layer; GCL, granule cell layer.
|
|
Ataxin-7 undergoes proteolytic cleavage
As we reported previously, affected PrP-SCA7-c92Q mice produce
full-length ataxin-7 protein with 92 glutamines, but the soluble, monomeric form of polyglutamine-expanded ataxin-7 is only readily detectable before the onset of a phenotype (La Spada et al., 2001). With advancing age and neurological dysfunction, little of the full-length monomeric form of ataxin-7 is seen on Western blot analysis, but instead, insoluble immunostaining material tends to be
seen in the stacking portion of polyacrylamide gels (La Spada et al.,
2001). Inspection of Western blots, which we probed with antibody K
raised against the N-terminal portion of ataxin-7, also reveals intense
bands migrating at 50-60 kDa (Fig.
5A). These bands appear
specific to SCA7 transgenic mice that express the ataxin-7 transgene
with either 24 or 92 glutamines, because they are not observed in
non-transgenic littermate controls.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 5.
Proteolytic cleavage of ataxin-7 92Q yields an
~55 kDa fragment. A, Western blot analysis of SCA7
transgenic mice probed with anti-ataxin-7 antibody K. Nuclear extracts
prepared from non-transgenic (nt), PrP-SCA7-c24Q
(24Q), and PrP-SCA7-c92Q (92Q) mice were
immunoblotted with antibody K at a dilution of 1:10,000. Although
full-length, soluble, monomeric ataxin-7 protein is detected for the
24Q transgenic mouse migrating at ~115 kDa, little soluble, monomeric
protein is detected for the 92Q transgenic mouse. A soluble, truncated
form of ataxin-7, however, is detected for both the 24Q and 92Q mice.
B, Western blot analysis of SCA7 transgenic mice and
patients probed with anti-expanded polyglutamine antibody 1C2. To
confirm the existence of the truncation fragment in the PrP-SCA7-c92Q
mice, protein lysates were generated from various tissues from a line
6076 individual (tg1-4) and a
non-transgenic control (c1-4) and
probed with the 1C2 antibody after immunoblotting. Mouse tissues are as
follows: 1 = liver, 2 = heart,
3 = brain, 4 = kidney. The 1C2
antibody specifically identifies a truncation fragment of ~55 kDa in
the SCA7 transgenic mice. Furthermore, protein lysates from the
fibroblasts of a juvenile-onset SCA7 patient with 85 CAG repeats
(Pt) reveal a fragment of similar size when probed with
the 1C2 antibody, whereas fibroblast lysates from two human controls
(C1, C2) yields no such fragment.
|
|
To confirm the existence of this truncation fragment in the
PrP-SCA7-c92Q mice, protein lysates were generated from various tissues
from a line 6076 individual and a non-transgenic control and probed
with the 1C2 antibody after immunoblotting. The 1C2 antibody, which is
directed against expanded polyglutamine tracts and has already been
shown to successfully detect polyglutamine-expanded ataxin-7 (Trottier
et al., 1995; Lindenberg et al., 2000), also identifies an ~55 kDa
fragment in the PrP-SCA7-c92Q mice (Fig. 5B). Using the 1C2
antibody, no such fragments are detected in non-transgenic littermate
controls (as shown) or in PrP-SCA7-c24Q mice (data not shown).
Furthermore, protein lysates from the fibroblasts of an affected SCA7
patient reveal a fragment of similar size when probed with the 1C2
antibody, whereas fibroblast lysates from two human controls yield no
such fragment. An N-terminal ataxin-7 truncation fragment of comparable
mobility is also apparent in protein lysates obtained from the
occipital lobe of an unrelated SCA7 patient (data not shown).
Interestingly, the amount of detectable fragment in the line 6076 SCA7
transgenic mice varies according to tissue, with heart showing the
largest quantity. To verify that ataxin-7 does undergo proteolytic
cleavage, we performed IHC analysis on brain sections from
PrP-SCA7-c92Q mice with antibody 1598 (Yvert et al., 2000) directed
against a C-terminal epitope of the ataxin-7 protein. Although brain
sections immunostained with antibody K reveal obvious NIs (Figs. 1, 4),
antibody 1598 did not detect NIs or show appreciable staining in
transgene-positive mice beyond 4 weeks of age (data not shown). When
used to probe Western blots, antibody 1598 could detect full-length
ataxin-7 protein but did not identify a fragment specific to SCA7
transgenic mice (data not shown). Because a Northern blot survey of
ataxin-7 expression revealed only a single 7.5 kb mRNA species in
humans (David et al., 1997), independent detection of a 50-60 kDa
protein with polyglutamine-directed and ataxin-7 N-terminal-directed
antibodies suggests that this protein product is the result of
proteolytic degradation. However, until the proteolytic enzyme is
identified or the truncation fragment is peptide sequenced, other
possible explanations, including alternative splicing, cannot be excluded.
| |
DISCUSSION |
We have generated a representative mouse model of SCA7 by
expressing a human ataxin-7 transgene with 92 glutamines in the CNS.
The behavioral phenotype observed in the PrP-SCA7-c92Q mice includes
features common to SCA7 patients, including prominent gait ataxia. The
neurological phenotype progresses to include tremors and hypokinesis.
Despite the complexity of the phenotype, the SCA7 transgenic mice
display expression of mutant ataxin-7 in the cerebellum, basis pontis,
and inferior olivary nucleus, tissues of primary pathological
importance in SCA7. The PrP-SCA7-c92Q mice show intense nuclear
staining in neurons expressing the ataxin-7 transgene. In humans,
ataxin-7 is normally expressed in both the nucleus and cytoplasm of
neurons in the pons, inferior olive, cerebellum, thalamus, and striatum
(Lindenberg et al., 2000). Studies of SCA7 patient material, however,
suggest that this cytoplasmic staining gives way to intense nuclear
staining and nuclear aggregate formation, perhaps reflecting
relocalization of the polyglutamine-expanded ataxin-7 to the nucleus
(Holmberg et al., 1998; Einum et al., 2001). The anti-ataxin-7 antibody
used primarily in this study, antibody K, appears specific for human
ataxin-7 because it fails to recognize murine ataxin-7. Antibody K
immunostaining of transgenic brain sections primarily yields a nuclear
pattern, suggesting that the PrP-SCA7-c92Q mice recapitulate the
nuclear relocalization of polyglutamine-expanded ataxin-7 observed in
SCA7 patients.
In SCA1 transgenic mice and in a striatal neuron model of HD,
localization of polyglutamine-expanded protein to the nucleus is
required for pathogenesis (Klement et al., 1998; Saudou et al., 1998).
In SCA7 patients, however, NIs occur at high frequency in both affected
and unaffected brain regions (Holmberg et al., 1998), suggesting that
although nuclear localization is necessary to initiate pathogenesis, it
is by no means sufficient. Lack of correlation between NI distribution
and neurodegeneration has been established similarly for other
polyglutamine repeat diseases (Ross et al., 1997; Becher et al., 1998).
Additional studies report onset of neuronal dysfunction in the absence
of NIs (Klement et al., 1998; Saudou et al., 1998; Hodgson et al.,
1999), leading some workers to suggest that nuclear aggregate formation
is a protective response by the cell (Sisodia, 1998). Indeed, impaired ubiquitin-proteasome function yielded fewer NIs but accelerated disease
pathology in a mouse model of SCA1 (Cummings et al., 1999). Other
investigators nonetheless argue that the process of aggregate formation
is pathogenic, because development of NIs typically precedes onset of
neuronal dysfunction (Perutz and Windle, 2001). In the PrP-SCA7-c92Q
transgenic mice, nuclear aggregate formation in granule cell neurons
and in neurons in the molecular layer is prominent, with virtually all
of these neurons showing NIs when a visibly ataxic phenotype is
detectable (Fig. 4). Furthermore, although presymptomatic line 6076 mice lack detectable aggregates, the development of NIs in these mice
occurs at 8 weeks of age and coincides with the onset of impaired
performance on the rotarod. Thus, it is the accumulation of
polyglutamine-expanded ataxin-7 within neuronal nuclei that best
correlates with the onset of neurological dysfunction in our model,
indicating that once a sufficient quantity of the altered ataxin-7
conformer is present in the nucleus, the disease process is under way.
Formation of nuclear aggregates corresponds to the achievement of this
threshold and may serve as a useful marker for disease progression.
Whether NIs themselves cause or contribute to the disease process or
are solely a protective cellular response in our SCA7 transgenic mice and in polyglutamine disease pathogenesis, however, remains to be determined.
A feature of the PrP-SCA7-c92Q mouse model that appears relevant to the
human disease is the production of an N-terminal ataxin-7 truncation
fragment. Previous studies of HD, DRPLA, and SBMA have yielded evidence
that N-terminal truncation fragments accumulate in the nuclei of
neurons from brain and spinal cord tissues in affected patients (Li et
al., 1998; Schilling et al., 1999a,b). For HD, DRPLA, and SCA7,
analysis of transgenic mouse models by immunoblotting and
immunostaining suggests that N-terminal truncation fragments are
produced from the full-length proteins (Schilling et al., 1999a,b;
Yvert et al., 2000). In the present study, we detected N-terminal
truncation fragments of ataxin-7 in PrP-SCA7-c92Q and PrP-SCA7-c24Q
mice with an ataxin-7-specific antibody. We and others (Yvert et al.,
2000) have found that ataxin-7-positive NIs in transgenic mice lack
C-terminal epitopes, indicating that N-terminal truncated forms are
accumulating in the nucleus. We, however, additionally used the
polyglutamine-specific 1C2 antibody to probe protein lysates and were
able to detect an N-terminal truncation product in the ataxin-7 92Q
mice. Interestingly, with this antibody, we also detected an N-terminal
truncation fragment of ataxin-7 in SCA7 patient material, migrating at
the same molecular weight position as the truncation fragment present
in the SCA7 transgenic mice. The presence of an N-terminal truncation
fragment of ataxin-7 in transgenic mice and in SCA7 patients is
consistent with the detection of an N-terminal truncation fragment in
DRPLA transgenic mice and patients by Schilling et al. (1999b).
Of the polyglutamine-expanded proteins responsible for human disease,
huntingtin, the androgen receptor, atrophin-1, ataxin-1, and ataxin-3
can each be cleaved by caspases to generate proteolytic truncation
fragments (Wellington et al., 1998). Caspases are
cysteine-dependent aspartyl-directed proteases involved in both signal
transduction and the execution of the apoptotic cascade (Stennicke and
Salvesen, 1998). In some cases, the cytotoxicity of the
polyglutamine-expanded protein appears to be dependent on caspase
cleavage (Wellington et al., 1998; Ellerby et al., 1999).
Interestingly, tissue culture cells expressing polyglutamine-expanded
ataxin-7 recruit the active form of caspase-3 into NIs, and CNS tissue
from SCA7 patients appears immunoreactive for the activated form of
caspase-3 (Zander et al., 2001). This observation suggests that
proteolytic processing of ataxin-7 may also be mediated by caspases.
However, although the ataxin-7 protein contains several caspase
consensus cleavage sites (DXXD), none of these optimal sites is located
in a region of the protein that corresponds to the molecular weight of
the N-terminal truncation fragment observed in SCA7 transgenic mice and
patient material. Indeed, whether proteolysis of ataxin-7 is required
for toxicity is not yet known. The goal of future studies will be to
determine how the ataxin-7 truncation product is generated and what its
role in cerebellar injury and dysfunction is.
In a recent study, Dyer and McMurray (2001) sought to determine whether
the huntingtin N-terminal truncation product is generated from
polyglutamine-expanded huntingtin or from the normal huntingtin protein
product in human HD brains. Using the 1C2 antibody, these workers
reported that polyglutamine-expanded huntingtin remains full length,
whereas the normal huntingtin protein product is subject to proteolytic
cleavage. Although the N-terminal truncation fragment of huntingtin
appears to be generated from the normal huntingtin protein, our data
and that of others respectively suggest that N-terminal truncation
fragments of ataxin-7 and atrophin-1 do derive from the
polyglutamine-expanded versions of these proteins (Schilling et al.,
1999b; Yvert et al., 2000). Thus, although loss of normal huntingtin
function caused by enhanced proteolytic degradation may contribute to
the pathogenesis of HD, differences in protein metabolism in the non-HD
polyglutamine repeat diseases may indicate that a corollary process of
loss of normal function does not occur in all polyglutamine repeat diseases.
A significant feature of the PrP-SCA7-c92Q mouse model is the
degeneration of cerebellar Purkinje cells despite the absence of
expression of polyglutamine-expanded ataxin-7 in this cell type. To
assist in the interpretation of this result, it is useful to consider
previous studies of SCA7. Although Purkinje cell degeneration and loss
are hallmarks of SCA7, immunostaining of human SCA7 cerebella from both
juvenile and late onset patients reveals that few, if any, surviving
Purkinje cells contain ubiquitinated NIs (Holmberg et al., 1998; Einum
et al., 2001). Furthermore, when Yvert et al. (2000) generated SCA7
transgenic mice expressing ataxin-7 90Q protein at high levels
exclusively in Purkinje cells, these mice remained normal beyond 1 year
of age, ultimately showing a phenotype and neurodegeneration at 16 months of age. When this group directed high-level expression of
polyglutamine-expanded ataxin-7 throughout the cerebellum and brainstem
with the platelet-derived growth factor B promoter, however, a motor
incoordination phenotype appeared at 3 months of age (Yvert et
al., 2000). Our results, taken together with these studies, indicate
that expression of polyglutamine-expanded ataxin-7 in neurons that
synapse with or receive input from Purkinje cells may underlie the
Purkinje cell degeneration in SCA7. Ubiquitinated NIs are most frequent
in brainstem neurons in the basis pontis and inferior olive of SCA7
patients (Holmberg et al., 1998). As shown in Figure 1, these brainstem regions contain numerous NIs in our PrP-SCA7-c92Q mice.
What then is the mechanism of Purkinje cell degeneration in the
PrP-SCA7-c92Q mice? A well recognized function of synaptic connections
between communicating neurons is to provide neurotrophic support
(Sofroniew et al., 2001). Members of the neurotrophin family, including
brain-derived neurotrophic factor (BDNF), are believed to perform this
function in both a retrograde and an anterograde manner (Nawa and
Takei, 2001). In HD, a reduction in BDNF levels in corticostriatal
projection neurons was found in transgenic mice and patients,
suggesting that loss of striatal neurons in HD may involve withdrawal
of trophic support (Zuccato et al., 2001). The non-cell-autonomous
nature of Purkinje cell degeneration in our SCA7 mouse model could
similarly stem from a loss of trophic support involving cerebellar
synaptic interconnections. Inferior olivary neurons as well as deep
cerebellar and brainstem nuclei are among the principal populations of
neurons with which Purkinje cells synapse. Granule cell neurons and
Bergmann glia (in the Purkinje cell layer) also show expression of
polyglutamine-expanded ataxin-7 in the PrP-SCA7-c92Q mice and could
similarly be involved in Purkinje cell degeneration. Whatever the
molecular basis of this phenomenon, our SCA7 mouse model clearly
demonstrates that Purkinje cell abnormalities develop without intrinsic
expression of a polyglutamine-expanded protein, supporting the
hypothesis that "murder" by adjacent, dysfunctional neurons or glia
does occur in the polyglutamine repeat expansion diseases.
| |
FOOTNOTES |
Received Feb. 7, 2002; revised April 4, 2002; accepted April 5, 2002.
This work was supported by research funds from the University of
Washington, by a generous gift from a Utah SCA7 family (Y.-H.F.), and
in part by National Institutes of Health Grant NS31775 to R.S.M. We
thank G. Nicholson, J. Christodoulou, and T. Bird for patient
samples and D. Borchelt for the MoPrP vector.
Correspondence should be addressed to Dr. Albert R. La Spada,
Department of Laboratory Medicine, University of Washington Medical
Center, Box 357110, Room NW 120, Seattle, WA 98195-7110. E-mail:
laspada{at}u.washington.edu.
| |
REFERENCES |
-
Becher MW,
Kotzuk JA,
Sharp AH,
Davies SW,
Bates GP,
Price DL,
Ross CA
(1998)
Intranuclear neuronal inclusions in Huntington's disease and dentatorubral and pallidoluysian atrophy: correlation between the density of inclusions and IT15 CAG triplet repeat length.
Neurobiol Dis
4:387-397[Web of Science][Medline].
-
Borchelt DR,
Davis J,
Fischer M,
Lee MK,
Slunt HH,
Ratovitsky T,
Regard J,
Copeland NG,
Jenkins NA,
Sisodia SS,
Price DL
(1996)
A vector for expressing foreign genes in the brains and hearts of transgenic mice.
Genet Anal
13:159-163[Medline].
-
Cummings CJ,
Reinstein E,
Sun Y,
Antalffy B,
Jiang Y,
Ciechanover A,
Orr HT,
Beaudet AL,
Zoghbi HY
(1999)
Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice.
Neuron
24:879-892[Web of Science][Medline].
-
David G,
Abbas N,
Stevanin G,
Durr A,
Yvert G,
Cancel G,
Weber C,
Imbert G,
Saudou F,
Antoniou E,
Drabkin H,
Gemmill R,
Giunti P,
Benomar A,
Wood N,
Ruberg M,
Agid Y,
Mandel JL,
Brice A
(1997)
Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion.
Nat Genet
17:65-70[Web of Science][Medline].
-
Dyer RB,
McMurray CT
(2001)
Mutant protein in Huntington disease is resistant to proteolysis in affected brain.
Nat Genet
29:270-278[Web of Science][Medline].
-
Einum DD,
Townsend JJ,
Ptacek LJ,
Fu YH
(2001)
Ataxin-7 expression analysis in controls and spinocerebellar ataxia type 7 patients.
Neurogenetics
3:83-90[Web of Science][Medline].
-
Ellerby LM,
Hackam AS,
Propp SS,
Ellerby HM,
Rabizadeh S,
Cashman NR,
Trifiro MA,
Pinsky L,
Wellington CL,
Salvesen GS,
Hayden MR,
Bredesen DE
(1999)
Kennedy's disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity.
J Neurochem
72:185-195[Web of Science][Medline].
-
Enevoldson TP,
Sanders MD,
Harding AE
(1994)
Autosomal dominant cerebellar ataxia with pigmentary macular dystrophy. A clinical and genetic study of eight families.
Brain
117:445-460[Abstract/Free Full Text].
-
Hodgson JG,
Agopyan N,
Gutekunst CA,
Leavitt BR,
LePiane F,
Singaraja R,
Smith DJ,
Bissada N,
McCutcheon K,
Nasir J,
Jamot L,
Li XJ,
Stevens ME,
Rosemond E,
Roder JC,
Phillips AG,
Rubin EM,
Hersch SM,
Hayden MR
(1999)
A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.
Neuron
23:181-192[Web of Science][Medline].
-
Holmberg M,
Duyckaerts C,
Durr A,
Cancel G,
Gourfinkel-An I,
Damier P,
Faucheux B,
Trottier Y,
Hirsch EC,
Agid Y,
Brice A
(1998)
Spinocerebellar ataxia type 7 (SCA7): a neurodegenerative disorder with neuronal intranuclear inclusions.
Hum Mol Genet
7:913-918[Abstract/Free Full Text].
-
Klement IA,
Skinner PJ,
Kaytor MD,
Yi H,
Hersch SM,
Clark HB,
Zoghbi HY,
Orr HT
(1998)
Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice.
Cell
95:41-53[Web of Science][Medline].
-
La Spada AR,
Fu Y,
Sopher BL,
Libby RT,
Wang X,
Li LY,
Einum DD,
Huang J,
Possin DE,
Smith AC,
Martinez RA,
Koszdin KL,
Treuting PM,
Ware CB,
Hurley JB,
Ptacek LJ,
Chen S
(2001)
Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of SCA7.
Neuron
31:913-927[Web of Science][Medline].
-
Li M,
Miwa S,
Kobayashi Y,
Merry DE,
Yamamoto M,
Tanaka F,
Doyu M,
Hashizume Y,
Fischbeck KH,
Sobue G
(1998)
Nuclear inclusions of the androgen receptor protein in spinal and bulbar muscular atrophy.
Ann Neurol
44:249-254[Web of Science][Medline].
-
Lin CH,
Tallaksen-Greene S,
Chien WM,
Cearley JA,
Jackson WS,
Crouse AB,
Ren S,
Li XJ,
Albin RL,
Detloff PJ
(2001)
Neurological abnormalities in a knock-in mouse model of Huntington's disease.
Hum Mol Genet
10:137-144[Abstract/Free Full Text].
-
Lindenberg KS,
Yvert G,
Muller K,
Landwehrmeyer GB
(2000)
Expression analysis of ataxin-7 mRNA and protein in human brain: evidence for a widespread distribution and focal protein accumulation.
Brain Pathol
10:385-394[Web of Science][Medline].
-
McCampbell A,
Taylor JP,
Taye AA,
Robitschek J,
Li M,
Walcott J,
Merry D,
Chai Y,
Paulson H,
Sobue G,
Fischbeck KH
(2000)
CREB-binding protein sequestration by expanded polyglutamine.
Hum Mol Genet
9:2197-2202[Abstract/Free Full Text].
-
Nakamura K,
Jeong SY,
Uchihara T,
Anno M,
Nagashima K,
Nagashima T,
Ikeda S,
Tsuji S,
Kanazawa I
(2001)
SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein.
Hum Mol Genet
10:1441-1448[Abstract/Free Full Text].
-
Nawa H,
Takei N
(2001)
BDNF as an anterophin: a novel neurotrophic relationship between brain neurons.
Trends Neurosci
24:683-685[Web of Science][Medline].
-
Orr HT
(2001)
Qs in the nucleus.
Neuron
31:875-876[Web of Science][Medline].
-
Paulson HL
(1999)
Protein fate in neurodegenerative proteinopathies: polyglutamine diseases join the (mis)fold.
Am J Hum Genet
64:339-345[Web of Science][Medline].
-
Paulson HL,
Bonini NM,
Roth KA
(2000)
Polyglutamine disease and neuronal cell death.
Proc Natl Acad Sci USA
97:12957-12958[Free Full Text].
-
Perutz MF,
Windle AH
(2001)
Cause of neural death in neurodegenerative diseases attributable to expansion of glutamine repeats.
Nature
412:143-144[Medline].
-
Ross CA
(1997)
Intranuclear neuronal inclusions: a common pathogenic mechanism for glutamine-repeat neurodegenerative diseases?
Neuron
19:1147-1150[Web of Science][Medline].
-
Ross CA,
Becher MW,
Colomer V,
Engelender S,
Wood JD,
Sharp AH
(1997)
Huntington's disease and dentatorubral-pallidoluysian atrophy: proteins, pathogenesis and pathology.
Brain Pathol
7:1003-1016[Web of Science][Medline].
-
Saudou F,
Finkbeiner S,
Devys D,
Greenberg ME
(1998)
Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions.
Cell
95:55-66[Web of Science][Medline].
-
Schilling G,
Becher MW,
Sharp AH,
Jinnah HA,
Duan K,
Kotzuk JA,
Slunt HH,
Ratovitski T,
Cooper JK,
Jenkins NA,
Copeland NG,
Price DL,
Ross CA,
Borchelt DR
(1999a)
Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin.
Hum Mol Genet
8:397-407[Abstract/Free Full Text].
-
Schilling G,
Wood JD,
Duan K,
Slunt HH,
Gonzales V,
Yamada M,
Cooper JK,
Margolis RL,
Jenkins NA,
Copeland NG,
Takahashi H,
Tsuji S,
Price DL,
Borchelt DR,
Ross CA
(1999b)
Nuclear accumulation of truncated atrophin-1 fragments in a transgenic mouse model of DRPLA.
Neuron
24:275-286[Web of Science][Medline].
-
Sisodia SS
(1998)
Nuclear inclusions in glutamine repeat disorders: are they pernicious, coincidental, or beneficial?
Cell
95:1-4[Web of Science][Medline].
-
Sofroniew MV,
Howe CL,
Mobley WC
(2001)
Nerve growth factor signaling, neuroprotection, and neural repair.
Annu Rev Neurosci
24:1217-1281[Web of Science][Medline].
-
Stennicke HR,
Salvesen GS
(1998)
Properties of the caspases.
Biochim Biophys Acta
1387:17-31[Medline].
-
Trottier Y,
Lutz Y,
Stevanin G,
Imbert G,
Devys D,
Cancel G,
Saudou F,
Weber C,
David G,
Tora L,
Agid Y,
Brice A,
Mandel J-L
(1995)
Polyglutamine expansion as a pathological epitope in Huntington's disease and four dominant cerebellar ataxias.
Nature
378:403-406[Medline].
-
Weiner LP,
Konigsmark BW,
Stoll Jr J,
Magladery JW
(1967)
Hereditary olivopontocerebellar atrophy with retinal degeneration. Report of a family through six generations.
Arch Neurol
16:364-376[Abstract/Free Full Text].
-
Wellington CL,
Ellerby LM,
Hackam AS,
Margolis RL,
Trifiro MA,
Singaraja R,
McCutcheon K,
Salvesen GS,
Propp SS,
Bromm M,
Rowland KJ,
Zhang T,
Rasper D,
Roy S,
Thornberry N,
Pinsky L,
Kakizuka A,
Ross CA,
Nicholson DW,
Bredesen DE,
Hayden MR
(1998)
Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract.
J Biol Chem
273:9158-9167[Abstract/Free Full Text].
-
Yvert G,
Lindenberg KS,
Devys D,
Helmlinger D,
Landwehrmeyer GB,
Mandel JL
(2001)
SCA7 mouse models show selective stabilization of mutant ataxin-7 and similar cellular responses in different neuronal cell types.
Hum Mol Genet
10:1679-1692[Abstract/Free Full Text].
-
Yvert G,
Lindenberg KS,
Picaud S,
Landwehrmeyer GB,
Sahel JA,
Mandel JL
(2000)
Expanded polyglutamines induce neurodegeneration and trans-neuronal alterations in cerebellum and retina of SCA7 transgenic mice.
Hum Mol Genet
9:2491-2506[Abstract/Free Full Text].
-
Zander C,
Takahashi J,
El Hachimi KH,
Fujigasaki H,
Albanese V,
Lebre AS,
Stevanin G,
Duyckaerts C,
Brice A
(2001)
Similarities between spinocerebellar ataxia type 7 (SCA7) cell models and human brain: proteins recruited in inclusions and activation of caspase-3.
Hum Mol Genet
10:2569-2579[Abstract/Free Full Text].
-
Zoghbi HY,
Orr HT
(2000)
Glutamine repeats and neurodegeneration.
Annu Rev Neurosci
23:217-247[Web of Science][Medline].
-
Zuccato C,
Ciammola A,
Rigamonti D,
Leavitt BR,
Goffredo D,
Conti L,
MacDonald ME,
Friedlander RM,
Silani V,
Hayden MR,
Timmusk T,
Sipione S,
Cattaneo E
(2001)
Loss of huntingtin-mediated BDNF gene transcription in Huntington's disease.
Science
293:493-498[Abstract/Free Full Text].
Copyright © 2002 Society for Neuroscience 0270-6474/02/22124897-09$05.00/0
This article has been cited by other articles:
|
|
|
|
 
M. Pennuto, I. Palazzolo, and A. Poletti
Post-translational modifications of expanded polyglutamine proteins: impact on neurotoxicity
Hum. Mol. Genet.,
April 15, 2009;
18(R1):
R40 - R47.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. E. Young, G. A. Garden, R. A. Martinez, F. Tanaka, C. Miguel Sandoval, A. C. Smith, B. L. Sopher, A. Lin, K. H. Fischbeck, L. M. Ellerby, et al.
Polyglutamine-Expanded Androgen Receptor Truncation Fragments Activate a Bax-Dependent Apoptotic Cascade Mediated by DP5/Hrk
J. Neurosci.,
February 18, 2009;
29(7):
1987 - 1997.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. C. Warby, C. N. Doty, R. K. Graham, J. B. Carroll, Y.-Z. Yang, R. R. Singaraja, C. M. Overall, and M. R. Hayden
Activated caspase-6 and caspase-6-cleaved fragments of huntingtin specifically colocalize in the nucleus
Hum. Mol. Genet.,
August 1, 2008;
17(15):
2390 - 2404.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. R. Gatchel, K. Watase, C. Thaller, J. P. Carson, P. Jafar-Nejad, C. Shaw, T. Zu, H. T. Orr, and H. Y. Zoghbi
The insulin-like growth factor pathway is altered in spinocerebellar ataxia type 1 and type 7
PNAS,
January 29, 2008;
105(4):
1291 - 1296.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. E. Young, L. Gouw, S. Propp, B. L. Sopher, J. Taylor, A. Lin, E. Hermel, A. Logvinova, S. F. Chen, S. Chen, et al.
Proteolytic Cleavage of Ataxin-7 by Caspase-7 Modulates Cellular Toxicity and Transcriptional Dysregulation
J. Biol. Chem.,
October 12, 2007;
282(41):
30150 - 30160.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Latouche, C. Lasbleiz, E. Martin, V. Monnier, T. Debeir, A. Mouatt-Prigent, M.-P. Muriel, L. Morel, M. Ruberg, A. Brice, et al.
A Conditional Pan-Neuronal Drosophila Model of Spinocerebellar Ataxia 7 with a Reversible Adult Phenotype Suitable for Identifying Modifier Genes
J. Neurosci.,
March 7, 2007;
27(10):
2483 - 2492.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. S. Thomas Jr, G. S. Fraley, V. Damien, L. B. Woodke, F. Zapata, B. L. Sopher, S. R. Plymate, and A. R. La Spada
Loss of endogenous androgen receptor protein accelerates motor neuron degeneration and accentuates androgen insensitivity in a mouse model of X-linked spinal and bulbar muscular atrophy
Hum. Mol. Genet.,
July 15, 2006;
15(14):
2225 - 2238.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Taylor, S. K. Grote, J. Xia, M. Vandelft, J. Graczyk, L. M. Ellerby, A. R. La Spada, and R. Truant
Ataxin-7 Can Export from the Nucleus via a Conserved Exportin-dependent Signal
J. Biol. Chem.,
February 3, 2006;
281(5):
2730 - 2739.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Adachi, M. Katsuno, M. Minamiyama, M. Waza, C. Sang, Y. Nakagomi, Y. Kobayashi, F. Tanaka, M. Doyu, A. Inukai, et al.
Widespread nuclear and cytoplasmic accumulation of mutant androgen receptor in SBMA patients
Brain,
March 1, 2005;
128(3):
659 - 670.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Helmlinger, J. Bonnet, J.-L. Mandel, Y. Trottier, and D. Devys
Hsp70 and Hsp40 Chaperones Do Not Modulate Retinal Phenotype in SCA7 Mice
J. Biol. Chem.,
December 31, 2004;
279(53):
55969 - 55977.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Goti, S. M. Katzen, J. Mez, N. Kurtis, J. Kiluk, L. Ben-Haiem, N. A. Jenkins, N. G. Copeland, A. Kakizuka, A. H. Sharp, et al.
A Mutant Ataxin-3 Putative-Cleavage Fragment in Brains of Machado-Joseph Disease Patients and Transgenic Mice Is Cytotoxic above a Critical Concentration
J. Neurosci.,
November 10, 2004;
24(45):
10266 - 10279.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Helmlinger, G. Abou-Sleymane, G. Yvert, S. Rousseau, C. Weber, Y. Trottier, J.-L. Mandel, and D. Devys
Disease Progression Despite Early Loss of Polyglutamine Protein Expression in SCA7 Mouse Model
J. Neurosci.,
February 25, 2004;
24(8):
1881 - 1887.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Chen, G.-H. Peng, X. Wang, A. C. Smith, S. K. Grote, B. L. Sopher, and A. R. La Spada
Interference of Crx-dependent transcription by ataxin-7 involves interaction between the glutamine regions and requires the ataxin-7 carboxy-terminal region for nuclear localization
Hum. Mol. Genet.,
January 1, 2004;
13(1):
53 - 67.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Michalik and C. Van Broeckhoven
Pathogenesis of polyglutamine disorders: aggregation revisited
Hum. Mol. Genet.,
October 15, 2003;
12(90002):
R173 - 186.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. P. Huynh, H.-T. Yang, H. Vakharia, D. Nguyen, and S. M. Pulst
Expansion of the polyQ repeat in ataxin-2 alters its Golgi localization, disrupts the Golgi complex and causes cell death
Hum. Mol. Genet.,
July 1, 2003;
12(13):
1485 - 1496.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. T. Libby, D. G. Monckton, Y.-H. Fu, R. A. Martinez, J. P. McAbney, R. Lau, D. D. Einum, K. Nichol, C. B. Ware, L. J. Ptacek, et al.
Genomic context drives SCA7 CAG repeat instability, while expressed SCA7 cDNAs are intergenerationally and somatically stable in transgenic mice
Hum. Mol. Genet.,
January 1, 2003;
12(1):
41 - 50.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Fossale, V. C. Wheeler, V. Vrbanac, L.-A. Lebel, A. Teed, J. S. Mysore, J. F. Gusella, M. E. MacDonald, and F. Persichetti
Identification of a presymptomatic molecular phenotype in Hdh CAG knock-in mice
Hum. Mol. Genet.,
September 15, 2002;
11(19):
2233 - 2241.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
Gene
