 |
 Previous Article | Next Article 
The Journal of Neuroscience, February 1, 1999, 19(3):964-973
Mutant Huntingtin Expression in Clonal Striatal Cells:
Dissociation of Inclusion Formation and Neuronal Survival by Caspase
Inhibition
Manho
Kim1,
H-S
Lee1,
Genevieve
LaForet2,
Charmian
McIntyre1,
Eileen J.
Martin1,
Patrick
Chang1,
Tae Wan
Kim1,
M.
Williams3,
P. H.
Reddy3,
Dan
Tagle3,
Frederick M.
Boyce1,
Lisa
Won4,
Alfred
Heller4,
Neil
Aronin2, and
Marian
DiFiglia1
1 Department of Neurology, Massachusetts General
Hospital, Boston, Massachusetts 02114, 2 Departments of
Medicine and Cell Biology, University of Massachusetts Medical Center,
Worcester, Massachusetts 01655, 3 National Institutes of
Health, Bethesda, Maryland, 20892, and 4 Department of
Pharmacological and Physiological Sciences, University of Chicago,
Chicago, Illinois 60637
 |
ABSTRACT |
Neuronal intranuclear inclusions are found in the brains of
patients with Huntington's disease and form from the
polyglutamine-expanded N-terminal region of mutant huntingtin. To
explore the properties of inclusions and their involvement in cell
death, mouse clonal striatal cells were transiently transfected with
truncated and full-length human wild-type and mutant huntingtin cDNAs.
Both normal and mutant proteins localized in the cytoplasm, and
infrequently, in dispersed and perinuclear vacuoles. Only mutant
huntingtin formed nuclear and cytoplasmic inclusions, which increased
with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features
(membrane blebbing, shrinkage, cellular fragmentation), but those with
mutant huntingtin generated the most cell fragments (apoptotic bodies).
The caspase inhibitor Z-VAD-FMK significantly increased cell survival
but did not diminish nuclear and cytoplasmic inclusions. In contrast,
Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but
did not increase survival. A series of N-terminal products was formed
from truncated normal and mutant proteins and from full-length mutant
huntingtin but not from full-length wild-type huntingtin. One prominent
N-terminal product was blocked by Z-VAD-FMK. In summary, the formation
of inclusions in clonal striatal cells corresponds to that seen in the
HD brain and is separable from events that regulate cell death.
N-terminal cleavage may be linked to mutant huntingtin's role in cell death.
Key words:
NH2-terminal huntingtin fragments; nuclear
inclusions; cytoplasmic inclusions; full-length huntingtin; apoptosis; apoptotic bodies; membrane blebbing; Z-VAD-FMK; Z-DEVD-FMK; striatal
hybrid cells
| |
INTRODUCTION |
The genetic mutation in
Huntington's disease (HD) is a polyglutamine expansion in the
N-terminal region of huntingtin (Huntington's Disease Collaborative
Research Group, 1993 ). The mechanism of HD pathogenesis is unclear.
Recent studies in HD postmortem brain tissue show that the N-terminal
region of mutant huntingtin aggregates in nuclear inclusions (NI) in
cortical and striatal neurons (DiFiglia et al., 1997; Becher et al.,
1998). The appearance of NI in regions most affected in the HD brain
implicates the aggregation of huntingtin in the pathogenesis of HD. In
support of this idea, HD transgenic mice expressing exon 1 with a
highly expanded CAG repeat domain develop NI before the onset of a
clinical phenotype (Davies et al., 1997; Scherzinger et al., 1997) and
before a reduction in mRNAs for neurotransmitter receptors in the
striatum and cortex (Cha et al., 1998). Inclusions also appear in
patients with other expanded CAG repeat disorders including
spinocerebellar ataxia 1 (SCA-1; Skinner et al., 1997), spinocerebellar
ataxia 3 (SCA-3; Machado-Joseph's disease; Paulson et al., 1997), and
dentatopallidoluysian atrophy (DRPLA; Becher et al., 1998), indicating
that a common mechanism may contribute to the formation of these
structures. Despite these interesting observations, the involvement of
inclusions in cell death remains unclear.
Proteins with expanded polyglutamines, when introduced into
non-neuronal cells in vitro, produce minimal or no NI, but
form aggregates in the cytoplasm when expressed from truncated
transcripts (Paulson et al., 1997; Skinner et al., 1997; Igarashi et
al., 1998; Martindale et al., 1998). Nuclear and cytoplasmic aggregates have been observed in non-neuronal cells after the expression of small
N-terminal mutant huntingtin fragments, but not after the expression of
large fragments or full-length huntingtin (Cooper et al., 1998; Hackam
et al., 1998). These results suggest that the intrinsic properties
required to generate N-terminal products from larger proteins may be
cell-specific. Activated caspases cleave normal and mutant huntingtins
near the NH2 terminus (Goldberg et al., 1996; Wellington et
al., 1998), and their presence may be needed to form inclusions from
full-length mutant huntingtin.
Here we report the effects of transient expression in an immortalized
striatal cell line of partial and full-length huntingtin with normal
and expanded polyglutamine regions. Expression of normal or mutant
proteins occurred mainly in the cytoplasm, produced apoptotic effects,
and generated N-terminal huntingtin fragments in Western blots. Mutant
huntingtin formed NI and CI, which were reduced by the caspase
inhibitor Z-DEVD-FMK without altering survival. The inhibitor,
Z-VAD-FMK, had no effect on the formation of inclusions but markedly
increased survival and blocked the cleavage of a prominent N-terminal
product. We speculate that inclusions formed by mutant huntingtin
are independent of events involved with cell death.
| |
MATERIALS AND METHODS |
Construction of expression plasmids. cDNA transcripts
containing the 5 prime one third (3221 bases for wild-type) of
huntingtin or the full-length cDNA (9774 for wild-type) were
constructed with 18, 46, and 100 CAG repeats. The 100 CAG repeat
sequence was interrupted by a CGG between the 28th and 30th CAG
triplets. The truncated transcripts included putative sites of caspase
cleavage. Cloned cDNAs were verified by restriction digestion and
sequencing. The cDNA for the FLAG epitope was placed 5 prime to that of
huntingtin, and the transcripts were cloned into the mammalian
expression vector pcDNA3 (Invitrogen, San Diego, CA) and identified as
FH3221-18, -46, and -100, and FH9774-18, -46, and -100. The pcDNA3 vectors containing truncated huntingtin with
normal and expanded (100) polyglutamines without the FLAG epitope
(H3221-18 and -100) were also made. In another set of
expression plasmids, the shorter huntingtin transcript (3221 bases) was
cloned into pEGFP-N1 (Clontech, Palo Alto, CA) in frame with a 3 prime
transcript for green fluorescent protein (GFP). These expression
plasmids are identified as H3221GFP18, 46, and 100. The
cytomegalovirus promoter drives protein expression in pcDNA3 and
pEGFP-N1. The respective plasmids with FLAG or GFP only were used as
controls in some experiments.
Cell culture. Clonal striatal cells (X57 cell line) derived
by somatic cell fusion of embryonic day 18 mouse striatal neurons and
neuroblastoma cells (N18TG2; Wainwright et al., 1995) were grown on
untreated glass coverslips. A mouse-derived mesenchephalic dopaminergic
cell line (MN9D; Choi et al., 1991) was also used.
In vitro transfection and experimental procedures.
Transfections were performed with Lipofectamine (2.5 µg/35 mm dish;
Life Technologies, Gaithersburg, MD). Mock transfections omitted
the plasmid DNA and were performed as controls. For treatment with caspase inhibitors or transglutaminase inhibitor, cells were grown in
35 mm culture dishes to 60-80% confluence and treated with Lipofectamine and FH3221-18 or -100 (2.5 µg) for 6 hr.
Cells were harvested and plated onto glass coverslips and treated with
the cell-permeable caspase inhibitors Z-VAD-FMK (5-100
µM) or Z-DEVD-FMK (50-200 µM) (obtained
from Enzyme Systems Products, Livermore, CA) or the
transglutaminase inhibitor cystamine (0.2-1 mM)
continuously for 3 d. Fresh inhibitor was replaced with each
feeding every 24 hr. Cells were washed in PBS and fixed and processed
for FLAG immunofluorescence. The concentrations of the caspase
inhibitors Z-VAD-FMK and Z-DEVD-FMK were in the range used by other
investigators to examine effects on survival of neuronal and
non-neuronal cells in culture (Eldadah et al., 1997; MacFarlane et al.,
1997; McCarthy et al., 1997; D'Mello et al., 1998).
Immunohistochemistry. Transfected striatal hybrid cells
grown on glass coverslips were rinsed briefly with PBS and fixed for 20 min with 4% paraformaldehyde in PBS at room temperature. After fixation, cells were rinsed multiple times and stored at 4°C
in PBS. Cells were made permeable by a 60 min incubation in 0.2% Triton X-100 in PBS and were treated with 4% normal goat serum before
incubation in primary antibodies against FLAG (M5 monoclonal antibody;
Eastman Kodak, Rochester, NY), huntingtin (polyclonal Ab 1, Ab
585, Ab 2527; 1-4 µg/ml; DiFiglia et al., 1995; Velier et al.,
1998), or bromodeoxyuridine (BrdU; monoclonal; Boehringer Mannheim,
Indianapolis, IN). For studies of BrdU incorporation, cells were
incubated in 0.2 NHCl at room temperature for 1 hr after fixation. All
single or combined incubations of primary antisera were overnight at
4°C. Secondary antibody was used at a 1:400 dilution and produced no
labeling in the absence of primary antibody. Plasmids containing the
cDNAs for FLAG or GFP only produced no labeling in cells at the DNA
concentrations used for transfection (2.5 µg/35 mm culture dish). For
FLAG immunofluorescence, Bodipy FL anti-mouse IgG was used (Molecular
Probes, Eugene, OR). For double labeling with anti-BrdU, sections were
treated sequentially in Cy3 anti-mouse IgG (Jackson ImmunoResearch,
West Grove, PA) and anti-BrdU-fluorescein (Boehringer Mannheim).
Sections were examined using conventional immunofluorescence microscopy
and a Bio-Rad (Hercules, CA) 1024 laser confocal microscope. Image processing was performed with Adobe Photoshop.
Western blot assays. Total protein extracts from the
transfected cells were detected in Western blot assay with
anti-huntingtin antisera Ab 1 as previously described (DiFiglia et al.,
1995).
| |
RESULTS |
Patterns of huntingtin localization
Wild-type or mutant huntingtin localized diffusely to the
cytoplasm in the majority of cells (X57) studied 1-3 d after
transfection, regardless of polyglutamine length or length of
huntingtin (Fig. 1, Table
1). Among cells expressing mutant
huntingtin in the cytoplasm were some with labeling also in the nucleus
(Figs. 1, 2d). In 1-10% of
neurons, wild-type and mutant huntingtins were heavily concentrated in
irregular or tubular-shaped cytoplasmic vacuoles independent of
polyglutamine length in huntingtin. The vacuoles were distributed
ubiquitously in the cell body (dispersed vacuoles) or as a complex of
interconnected tubules in the perinuclear region (perinuclear vacuoles)
and were more frequent with the truncated than the full-length
proteins. Cells with perinuclear vacuoles were significantly smaller in
cross-sectional area than nontransfected cells in the same culture
dishes (p < 0.05; n = 50 labeled and unlabeled neurons in cultures expressing wild-type or
mutant proteins), suggesting that they were undergoing apoptosis.
View larger version (67K):
[in this window]
[in a new window]
|
Figure 1.
Patterns of localization of FLAG-huntingtin
fusion proteins in clonal striatal neurons 1-3 d after transfection of
FH3221 with 18, 46, or 100 glutamines. Diffuse cytoplasmic
labeling, dispersed vacuoles, and condensed perinuclear vacuoles occur
regardless of polyglutamine length. Neurons with mutant huntingtin show
some diffuse nuclear labeling and nuclear and cytoplasmic
("ring-like") inclusions (arrows).
|
|
View larger version (71K):
[in this window]
[in a new window]
|
Figure 2.
Features of NI and CI in clonal striatal and
midbrain neurons. The inclusions were formed by FH3221-100
(a-c, e) or FH9774-100
(d). Inclusions in striatal cells appear more
consolidated and intensely FLAG-positive (a-d) than in
the midbrain neuron (e). Striatal cell in
a has three small and two large NI. Cells in
b and d have both a nuclear and a
cytoplasmic inclusion. Cell in c shows retraction of
cytoplasm away from the cytoplasmic inclusion. All cells have diffuse
cytoplasmic labeling, and cell in d has diffuse nuclear
labeling.
|
|
Nuclear inclusions (NI; 0.7-10% of neurons) or cytoplasmic inclusions
(CI; 0.9-1.4% of neurons) formed from the shortened (Figs. 1,
2a-c) or full-length huntingtin (Fig.
2d) using FH3221-100 and FH9774-100,
respectively. NI also developed in 0.6-1.4% of neurons using
FH3221-46 (Fig. 1), but they were not found after 3 d
with the full-length cDNA expressed by FH9774-46 (Table 1). Similar to HD brain, the NI were spherical or ovoid and occupied 10-30% of the cross-sectional area of the nucleus. NI developed in
clonal midbrain dopaminergic neurons (Wainwright et al., 1995) with
FH3221-100 (Fig. 2e), but the NI were less
intensely labeled than those found in the striatal cells. Between days
3 and 4, the rate of cell proliferation and the loss of FLAG-positive
cells varied for different transfections and affected the percent of cells with inclusions at these time points. Nevertheless, ~4-6 d
after transfection, the percent of NI and CI induced by
FH3221-100 began to increase, and by day six constituted 43 and 27% of the total labeled cells, respectively (Fig.
3). The rise in cells with NI and CI
overlapped a period of marked loss in the total FLAG-positive neurons.
NI and CI occurred together in only ~10% of labeled cells. Thus, by
day six, 60% of the FLAG- positive neurons that remained in the
cultures had some form of inclusion. CI typically occurred singly in
cells, frequently near blebs in the plasma membrane (Figs.
2c, 5e). CI were spherical and uniformly dense
(Fig. 2b,c) or had clear centers, which gave them
a distinct ring-like appearance (Figs. 1,
4g). CI were distinct from
dispersed and condensed perinuclear vacuoles in a number of ways. CI
occurred singly in neurons and were uniformly spherical, whereas
vacuoles were numerous, interconnected, irregularly shaped, or
tubular.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 3.
Effects of the number of days after transfection
on the formation of NI and CI. Cells were transfected with
FH3221-100. Percent of total FLAG+ neurons with inclusions
progressively increases between 4 and 6 d. Note that the total
number of FLAG+ neurons (indicated over each set of bars) decreases
markedly by 5-6 d after transfection. Results are shown for one
transfection experiment. Results were similar in two other
experiments.
|
|
View larger version (101K):
[in this window]
[in a new window]
|
Figure 4.
Mutant huntingtin with 100 glutamines localized in
clonal striatal cells. a-d show staining with
anti-huntingtin antisera Ab 1 after transfection of
H3221-100 (no epitope tag), and
e-g show GFP after transfection of
H3221GFP100 (tagged at the COOH terminus). Both constructs
produce mainly diffuse labeling in the cytoplasm (a, e),
some cells with perinuclear vacuoles (b, f),
nuclear inclusions (c, e), and cytoplasmic inclusions
(d, g). Note the ring-like structure of the cytoplasmic
inclusion in g. The results indicate that the expression
of mutant huntingtin alone or with a COOH-terminal GFP tag shares the
same subcellular compartments as FLAG-huntingtin shown in Figures 1
and 2.
|
|
The patterns of FLAG staining in striatal cells transfected with
FH3221 constructs were also seen with Ab 1, which
recognizes the NH2-terminus in huntingtin (results not
shown). Ab 1 detected similar patterns of normal and mutant huntingtin
localization after expression of H3221-18 and
H3221-100 (Fig. 4a-d, shown for H3221-100), suggesting that the presence of the FLAG
epitope at the NH2 terminus did not affect protein
targeting. In neurons expressing FH3221,
anti-huntingtin antisera Ab 585, which recognizes an epitope downstream
of the polyglutamine tract in huntingtin, detected mainly diffuse
cytoplasmic labeling in neurons. It also weakly recognized the
cytoplasmic face of dispersed and perinuclear vacuoles that accumulated
normal and mutant proteins. Ab 585 did not label NI or CI. Similarly,
Ab 2527, which recognizes an epitope near the COOH-terminal, detected
mainly diffuse cytoplasmic labeling in neurons and did not recognize
vacuoles or inclusions after expression of FH9774. The
limited recognition of vacuoles and inclusions by Ab 585 and Ab 2527 suggested that these compartments had accumulated mainly cleaved
NH2 huntingtin products.
Neurons transfected with H3221 GFP-100 showed
huntingtin-GFP localization comparable to FLAG-huntingtin 1 d
after transfection (Fig. 4e,g). Among
GFP-positive neurons (n = 7096 cells), 95% had diffuse
cytoplasmic labeling 0.14% had NI, and 1.8% had CI. The presence of
huntingtin-GFP within both nuclear and cytoplasmic inclusions
suggested that the large, uncleaved form of mutant huntingtin was
incorporated into aggregates. CI were identified about as frequently
with the FLAG and GFP-tagged cDNAs. However, NI were labeled more
frequently (5-18×) with the FLAG-tagged construct than the
GFP-tagged cDNA. This suggested that NI incorporated mainly cleaved
N-terminal fragments of mutant huntingtin. In an analysis of
double-labeled cells (n = 161 cells) cotransfected with
FLAG and GFP-tagged cDNAs, the NI (n = 7) detected had
FLAG labeling but not GFP, indicating that in this small sampling of cells only a cleaved N-terminal fragment of mutant huntingtin had
translocated to the nucleus. Vacuoles were also less frequently detected with the GFP-tagged construct (0.23% dispersed vacuoles and
2.4% perinuclear vacuoles) than with the FLAG-tagged cDNA (Table 1;
range, 1.4-8.2%). These results provided additional support that the
vacuoles accumulated primarily NH2 products of the normal
and mutant proteins.
Apoptotic features in neurons expressing wild-type and
mutant huntingtins
Apoptosis occurs in affected neurons of the HD brain
(Portera-Cailliau et al., 1995). Morphological characteristics of
apoptotic cell death including cell shrinkage, membrane blebs, and
cellular fragmentation (Kerr et al., 1972) were evident in cells
expressing partial and full-length wild-type or mutant huntingtins
(Fig. 5). Shrunken cells had a marked
accumulation of wild-type or mutant huntingtin in the cytoplasm (Fig.
5a,h) and were seen under basal conditions and in cultures differentiated with forskolin or sodium butyrate. Severe membrane blebs (blisters) of the plasma membrane, a
feature of early apoptotic cell death, occurred after normal or mutant
protein expression. Membrane blebs were found in cells where mutant
huntingtin appeared diffusely in the cytoplasm (Fig. 5c) or
additionally formed NI and CI (Fig. 5c,d). An
early stage of cellular fragmentation, indicated by protrusions from
the cell surface, was seen with FLAG- (Fig.
5b,g,h) or GFP-tagged
(Fig. 5f) transcripts. Isolated cell fragments that
contained vacuoles appeared when FH3221-18 or 46 were
expressed (Fig. 6a,
left). Cell fragments with ring-like inclusions were
prevalent when FH3221-100 was transfected (Fig.
6a, right) and were increased in proportion to
total cell fragments 1-6 d after transfection (Fig. 6b) in parallel with the rise in CI in neurons (see Fig. 3). The ratio of cell
fragments to neurons was significantly greater in cultures transfected
with FH3221-100 than in cultures treated with
FH3221-18 or -46 (Fig. 6c).
View larger version (121K):
[in this window]
[in a new window]
|
Figure 5.
Features of apoptotic cell death in clonal
striatal cells expressing wild-type (a, b) and mutant
huntingtin (c-h). Expression plasmids were
FH3221-18 in a and b,
FH3221-100 in c-e, H3221GFP-100
in f, and FH9774-100 in g and
h. Shrunken cells (a, h,
large arrows), plasma membrane blebs
(c-e, small arrows), and cellular
fragmentation (b, f-h, arrowheads) were
the most commonly seen apoptotic features. Note membrane blebs along
the plasma membrane appear in neurons that have diffuse cytoplasmic
FLAG labeling or in addition have NI (d) and CI
(e).
|
|
View larger version (24K):
[in this window]
[in a new window]
|
Figure 6.
Analysis of apoptotic cell fragments.
a, Cell fragments with vacuoles (left)
and ring-like inclusions (right). b, Cell
fragments with ring-like inclusions increase in proportion to total
cell fragments 1-6 d after transfection of FH3221-100.
Number of cells is shown at the top of each bar for one
time course experiment. c, Ratio of cell fragments per
neuron is significantly greater in cultures expressing huntingtin with
100 glutamines than with 18 or 46 glutamines
(p < 0.01; n = 6 per
group).
|
|
Effects of caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK
Striatal cells expressing FH3221-18 or 100 and treated
with the caspase inhibitor Z-VAD-FMK showed marked increases in cell number on day 3 (Fig. 7) compared with
neurons without the caspase inhibitor. Increased survival with
Z-VAD-FMK was dose-dependent and maximal at 100 µM (Fig.
7). The proportion of FLAG-huntingtin-negative cells in the cultures
was modestly increased by Z-VAD-FMK (mean increase was 49%) compared
with FLAG-huntingtin-positive cells (240-310%). Moreover, the mean
percent of FLAG+ cells incorporating BrdU, a marker of S-phase
activity, was not significantly changed by Z-VAD-FMK (Student's
t test; p > 0.05) either for the wild-type ( Z-VAD-FMK, 4.8%; +Z-VAD-FMK, 4.3%) or mutant proteins (100 CAGs, Z-VAD-FMK, 25.5%; +Z-VAD-FMK, 16.5%). This showed that the
inhibitor had increased viability and not cell proliferation. Similar
protective effects of Z-VAD-FMK on cell survival were seen when the
full-length wild-type and mutant huntingtins were expressed (mean
increases were 397% for FH9774-18 and 198% for
FH9774-100; n = 6 experiments). In contrast
to its effects on survival, Z-VAD-FMK did not change the proportion of
total neurons that developed NI and CI (Fig. 7).
View larger version (24K):
[in this window]
[in a new window]
|
Figure 7.
Effects of caspase inhibitors Z-VAD-FMK (100 µM) and Z-DEVD-FMK (200 µM) on the number
of FLAG-positive neurons and the proportion of neurons with NI and CI
present 3 d after transfection. Striatal neurons were transfected
with FH3221-18 or -100. Bar graphs show a
significant increase in neuron survival (*p < 0.05; n = 6) after treatment with Z-VAD-FMK and a
significant reduction (*p < 0.05;
n = 6) in the proportion of neurons that develop
nuclear and cytoplasmic inclusions after treatment with Z-DEVD-FMK.
Mean values based on total FLAG-positive neurons counted per coverslip
in six coverslips. Line graphs show effects of
concentration of Z-VAD-FMK on the number of FLAG-positive neurons and
of Z-DEVD-FMK on the percent of FLAG-positive neurons with nuclear
inclusions in cultures treated with FH3221-100. Mean values
are based on analysis of 20 microscopic fields per coverslip in three
coverslips. *Signifies a difference from 0 concentration at
p < 0.05, Student's t test.
|
|
Treatment with the caspase 3 inhibitor, Z-DEVD-FMK, caused a
dose-dependent reduction in the proportion of neurons with NI and CI
formed by FH3221-100 (Fig. 7). This was offset by a
significant rise in the proportion of neurons with diffuse cytoplasmic
labeling (results not shown). Z-DEVD-FMK had no effect on the survival of cells expressing FH3221-18, -46 (data not shown), or
-100 (Fig. 7).
Effects of transglutaminase inhibitors
Transglutaminase is a substrate for polyglutamine-enriched
proteins (Cooper et al., 1997) and causes mutant huntingtin to aggregate (Kahlem et al., 1998). Treatment over 3 d with a 0.2 mM concentration of the transglutaminase inhibitor
cystamine did not reduce NI or CI formed by mutant huntingtin in our
striatal cells, and 0.5 and 1 mM cystamine were toxic to
striatal cells.
Western blot assay of protein extracts from the transfected
striatal cells
Total protein extracts from cells transfected with
FH3221-18, 46, and 100 were examined by Western blot with
anti-huntingtin antisera Ab 1. The fully expressed proteins migrated at
~140 kDa (Fig. 8). A larger fragment of
~175 kDa also appeared when mutant huntingtin contained 100 glutamines. A series of three N-terminal fragments were generated by
the normal and mutant proteins. The size of the products seen with
FH3221-18 was ~60, 70, and 80 kDa. FH3221-46
generated products of ~70, 80, and 90 kDa, and FH3221-100 produced bands of ~80, 90, and 100 kDa. The variation in size of the
N-terminal products of the normal and mutant proteins was consistent
with the variable polyglutamine regions in these proteins. The two
smaller N-terminal products generated by the normal (60 and 70 kDa) and
mutant (70 and 80 kDa for FH3221-46 and 80 and 90 kDa for
FH3221-100) proteins were more prominent than the larger product. In some blots these smaller fragments resolved as doublets, indicating that each arose from cleavage at two nearby sites in huntingtin. These results demonstrated that three to five cleavage products were produced by the large truncated normal and mutant proteins.
View larger version (71K):
[in this window]
[in a new window]
|
Figure 8.
Western blots of huntingtin expression in protein
extracts of transfected striatal cells and the effects of caspase
inhibitors. a, Human huntingtin is seen at the expected
size of ~140 kDa (top arrowhead) in cells exposed to
the FH3221 expression plasmids. A slightly larger band at
~175 kDa is also seen with FH3221-100. N-terminal
products (bottom arrowheads) occur with wild-type (18 CAGs) and mutant (46 and 100 CAGs) huntingtins and show variable size
consistent with polyglutamine expansion. Native huntingtin appears at
the top of first lane. Z-VAD-FMK (100 µM)
markedly inhibits a prominent intermediate N-terminal product in
wild-type huntingtin (70 kDa) and mutant huntingtins (80 kDa for 46 CAGs and 90 kDa for 100 CAGs). b, Expression of
full-length wild-type huntingtin (18 CAGs) and mutant huntingtin (100 CAGs) with FH9774 constructs generates N-terminal products
(arrowheads) only from mutant huntingtin. Lane
c was nontransfected and shows native mouse huntingtin at the
top of the blot. Full-length human mutant huntingtin
migrates above native mouse protein. c,
d, Time course of appearance of N-terminal products
after expression of FH3221-100. Results in c
and d are from different transfections. The blot at the
right in c is a longer exposure of the 7 and 9 hr time points. The 140 kDa band appears at 5 and 6 hr. The 100 kDa product is seen at 9 hr (c, right
blot). All three N-terminal products are present at 15 hr
(d). The 90 kDa band is the last to appear at 15 hr (d) and the most prominent at 45 hr
(c). Native huntingtin appears at the
top of most lanes (c).
e, Effects of Z-VAD-FMK concentration (0-100
µM) on mutant huntingtin expressed from
FH3221-100. The 90 kDa N-terminal product is attenuated at
all concentrations and maximally at 100 µM. Increased
expression of the 140, 80, and 100 kDa proteins are seen and may be
related to increased viability. f, Effects of
concentration of Z-DEVD-FMK (0-200 µM) on mutant
huntingtin expression by FH3221-100. No inhibitory effects
on cleavage are seen at any concentration. N-terminal products are
slightly elevated, but the 140 kDa protein is unchanged in cells
treated with the inhibitor. Protein extracts (20 µg/lane) were taken
24 hr after transfection in a, b,
e, and f. Antibody Ab 1 was used to
detect huntingtin. Molecular mass markers are shown to the
left of each blot.
|
|
The NH2-terminal products produced by
FH3221-100 were also detected after expression of
full-length mutant huntingtin (Fig. 8b). The 90 kDa product
was the most prominent of the three products seen. N-terminal products
were not produced by full-length wild-type huntingtin (Fig.
8b). Cells split from the same transfections were used to
verify by FLAG staining that the transfections were successful for both
the full-length normal and mutant proteins. The results suggested that
polyglutamine expansion in full-length huntingtin facilitated
production of N-terminal fragments.
The generation of N-terminal products was examined 5-45 hr after
transfection of FH3221-100. The 140 kDa protein was
detected at 5-6 hr, was maximal by 24 hr, and was reduced at 45 hr.
The 100 kDa fragment was seen at 9 hr, peaked between 15 and 24 hr, and
was lower by 45 hr. The 90 kDa band was first seen at 15 hr and was
prominent at 24 and 45 hr. The 80 kDa band was low at 9 hr, maximal at
15 hr, reduced by 24 hr, and nearly absent by 45 hr (Fig.
8c,d). After long film exposures of the
chemiluminescence reaction (~1 hr), multiple smaller molecular mass
products (in the range of 20-50 kDa for FH3221-100) were
detected (data not shown). This suggested that the products were being
processed to smaller fragments. The results indicated that the
generation of N-terminal products from FH3221-100 was
time-dependent but coincided with the appearance of inclusions in the
striatal cells. The emergence and duration of the products also
appeared to differ, with the 90 kDa species being the most
long-lived.
Z-VAD-FMK blocked formation of the intermediate 90 kDa fragment seen
with FH3221-100, and the corresponding 70 and 80 kDa products formed from FH3221-18 and FH3221-46,
respectively (Fig. 8a,e). The inhibitory effect
was correlated in a dose-dependent manner (Fig. 8e, shown
for FH3221-100) with increased survival (Fig. 7). Z-VAD-FMK
increased the fully expressed 140 kDa protein and enhanced expression
of the 80 and 100 kDa products generated by FH3221-100
(Fig. 8a,f). The rise in the 140 kDa
protein may have resulted from increased cell survival and/or the
inhibition of the 90 kDa from the 140 kDa protein. Z-VAD-FMK also
blocked the 90 kDa fragment generated by full-length mutant huntingtin with FH9774-100 (results not shown). Z-DEVD-FMK had no
inhibitory effects on N-terminal products of normal or mutant
huntingtin. Treatment with the inhibitor at all doses tested had no
effect on the 140 kDa band but slightly increased the N-terminal
products formed by FH3221-100 (Fig. 8f)
compared with the N-terminal products in untreated cells.
| |
DISCUSSION |
The recent identification in HD brain of nuclear inclusions
derived from N-terminal fragments of mutant huntingtin (DiFiglia et
al., 1997) has heightened interest about their formation and potential
role in the pathogenesis of the disease. We have shown that the
predominant cytoplasmic distribution of huntingtin localization seen in
immortalized mouse striatal cells transfected with human huntingtin
cDNAs fits with that of neurons in normal and HD brain (DiFiglia et
al., 1995, 1997; Sapp et al., 1997). The development and appearance of
NI in the transfected cells also showed a remarkable correspondence
with HD neurons. The formation of NI in culture, as in the HD brain,
was polyglutamine and time-dependent. The CI formed in culture evolved
similarly to the NI and most likely corresponds to the mutant
huntingtin aggregates identified in dystrophic neurites in the HD brain
(DiFiglia et al., 1997), although the ring-like architecture of some CI
has not been seen in HD neurons.
Inclusions may contain huntingtin proteins of various sizes. NI
contained mostly cleaved N-terminal products consistent with other
recent findings (Cooper et al., 1998; Hackam et al., 1998), whereas CI
had a mixture of cleaved and larger complete proteins. Immunohistochemical and biochemical studies in HD brain suggest that
mutant huntingtin may be present in the nucleus as a full-length protein (DiFiglia et al., 1997, their Fig. 3; Sapp et al., 1997). N-terminal products also accumulated in dispersed and perinuclear vacuoles. In contrast to inclusions, which contained only mutant huntingtin, vacuoles incorporated normal or mutant proteins. The subcellular compartment to which vacuoles belong is unclear, but recent
observations suggest that they are part of the endoplasmic reticulum
(K. Kegel and M. DiFiglia, unpublished observations).
Features of apoptosis occurred in striatal neurons expressing normal
and mutant proteins. The characteristics of apoptotic cell death
included membrane blebbing, cellular fragmentation, and shrinkage (Kerr
et al., 1972). The striatal cells most reduced in size were those that
accumulated high levels of N-terminal normal or mutant huntingtin into
dispersed and perinuclear vacuoles, indicating that accumulation into
these compartments could be toxic. The generation of apoptotic
fragments was polyglutamine-dependent and occurred significantly more
frequently in the striatal cells expressing a highly expanded mutant
protein than in cells with the moderately expanded or wild-type
huntingtin. The apoptotic morphology induced by wild-type huntingtin
was somewhat surprising because it has not been reported in studies
with other cell lines. The immortalized striatal cells may be
especially sensitive to huntingtin-induced apoptosis or may be less
able to handle the accumulation of large amounts of huntingtin produced
by transient transfection than other cells. Native huntingtin exists at
low levels in striatal hybrid cells cultured under basal conditions (Kim et al., 1999) and is also low in medium-sized striatal neurons compared with other neurons in mouse and human brain (Bhide et al.,
1996; Sapp et al., 1997).
A pathogenic mechanism for HD has been proposed, whereby polyglutamine
expansion induces the production of an altered protein that causes cell
death possibly through the formation of inclusions (Goldberg et al.,
1996; Davies et al., 1997; Hackam et al., 1998; Martindale et al.,
1998). The caspase inhibitor Z-VAD-FMK increased cell viability without
changing the formation of inclusions. Treatment with the caspase
inhibitors Z-DEVD-FMK (this study) and Z-IETD-FMK (M. Kim and M. DiFiglia, unpublished observations) significantly reduced the
proportion of neurons that formed NI and CI but was not associated with
increased survival. Because caspase inhibitors are not selective and
may affect cell survival and morphological features of apoptosis
through multiple ICE-like proteases and other substrates (Eldadah et
al., 1997; MacFarlane et al., 1997; D'Mello et al., 1998), the
mechanisms by which these inhibitors acted in our study are unclear.
Nevertheless, the results of experiments with Z-VAD-FMK and DEVD-FMK
suggest that the formation of inclusions is not essential to cause cell
death. In accord with this idea, results in transfected 293T cells
exposed to tamoxifen showed that even in the absence of inclusions, the
mutant protein was more toxic than wild-type (Hackam et al., 1998).
During the review of this manuscript, another study reported that the
formation of nuclear inclusions in cultured embryonic rat striatal
neurons by N-terminal fragments of mutant huntingtin did not correlate with the extent of apoptotic cell death (Saudou et al., 1998).
Huntingtin expression in clonal striatal cells produced a series of
three to five N-terminal fragments, consistent with the presence of
endogenous proteolytic activity. These products are in the range of
sizes consistent with cleavage at several putative caspase cleavage
sites near the NH2 terminus of huntingtin (Wellington et
al., 1998). Apoptotic extracts (Goldberg et al., 1996) and purified
caspases 1 and 3 produce N-terminal products (two for each caspase)
from in vitro translated full-length normal and mutant
huntingtin, and deletion mutation has verified at least one caspase
3-sensitive site (Wellington et al., 1998). Thus, some of the
N-terminal products of huntingtin seen in the striatal cells may result
from caspase 1 and/or caspase 3 activity. The marked attenuation by
Z-VAD-FMK of a prominent N-terminal product in huntingtin (90 kDa
product generated from FH3221-100) is compatible with the
interruption of a caspase 1 and/or 3 consensus site in huntingtin. In
the range of concentrations used in our study, Z-VAD-FMK was shown to
inhibit the activity of caspases 1 and 3 in apoptotic cells (Eldadah et
al., 1997; MacFarlane et al., 1997). It is not clear whether the
N-terminal products generated by mutant huntingtin expression in the
striatal cells contributed directly to the formation of nuclear
inclusions or involved further proteolytic digestion as suggested by
biochemical assay in the HD brain (DiFiglia et al., 1997).
Although both normal and mutant proteins expressed from truncated cDNAs
were susceptible to cleavage, only mutant huntingtin was cleaved from
the full-length protein. While our paper was being reviewed, Lunkes and
Mandel (1998) also reported cleavage of full-length mutant but not
wild-type huntingtin using stable and inducible neuroblastoma cell
lines. The findings in both studies support previous evidence that
polyglutamine expansion enhances N-terminal cleavage (Goldberg et al.,
1996). Z-VAD-FMK, which increased survival, blocked a prominent
N-terminal product generated by truncated normal and mutant huntingtin
proteins. The same product was formed by mutant huntingtin but not
wild-type huntingtin when the full-length protein was expressed. These
observations strongly implicate an N-terminal product of mutant
huntingtin in apoptosis but do not prove that this product functions as
an apoptotic initiator or mediator.
Despite evidence that transglutaminase induces polymerization of mutant
huntingtin (Kahlem et al., 1998), the inhibition of transglutaminase
was ineffective in reducing NI and CI or in changing survival. However,
concentrations of the transglutaminase inhibitor cystamine, which were
found effective in reducing cytoplasmic aggregates formed by mutant
DRPLA in COS-7 cells (Igarashi et al., 1998), were toxic to the
striatal cells. Therefore, our negative results do not exclude the
possibility that transglutaminase contributes to the aggregation of
mutant huntingtin in the HD brain.
We used an immortalized cell line derived from a fusion of embryonic
mouse cells of striatal origin and neuroblastoma cells to investigate
huntingtin expression. This cell line shows characteristics of striatal
cells, including an enrichment in dopamine receptors, and forms
neurites when differentiated (Wainright et al., 1995). Also, native
huntingtin in the clonal striatal cells is known to have the same
cytoplasmic localization and vesicle membrane associations found in
mouse striatal cells in vivo (Kim et al., 1999; Bhide et
al., 1996). Because transformed cells have altered growth
characteristics, they may have some limitations for the study of
neuronal function compared with primary cultured neurons. Among the
significant advantages of immortalized neurons, however, is their
potential use for high through-put assays in drug screening.
In summary, our findings show that clonal mouse striatal cells
transiently expressing human huntingtin in vitro exhibit
patterns of mutant huntingtin expression seen in the HD brain,
including the polyglutamine-dependent and time-dependent formation of
NI. Experiments using caspase inhibitors revealed that the formation of
inclusions could be separated from events that control cell survival.
Immortalized striatal cells provide a more accurate model of the HD
cellular phenotype than that achieved with other types of transformed
cells recently studied and should afford a rapid way of dissecting the
signal transduction events involved with HD pathology.
| |
FOOTNOTES |
Received Sept. 21, 1998; revised Nov. 18, 1998; accepted Nov. 20, 1998.
This work was supported by National Institutes of Health Grants
NS16367 to M.D., NS31579 to M.D. and N.A., the Hereditary Disease
Foundation to M.D. and N.A., the Huntington's Disease Society of
America to M.D., the Howard Hughes Medical Institute to G.L., and
MH28942 to A.H. and L.W.
Correspondence should be addressed to Marian DiFiglia, Laboratory of
Cellular Neurobiology, Massachusetts General Hospital, MGH-East, 149 13th Street, Charlestown, MA 02129.
| |
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].
-
Bhide PG,
Day M,
Sapp E,
Schwarz C,
Sheth A,
Kim J,
Young AB,
Penney J,
Golden J,
Aronin N,
DiFiglia M
(1996)
Expression of normal and mutant huntingtin in the developing brain.
J Neurosci
16:5523-5535[Abstract/Free Full Text].
-
Cha JH,
Kosinski CM,
Kerner JA,
Alsdorf SA,
Mangiarini L,
Davies SW,
Penney JB,
Bates GP,
Young AB
(1998)
Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human Huntington's disease gene.
Proc Natl Acad Sci USA
95:6480-6485[Abstract/Free Full Text].
-
Choi HK,
Won LA,
Kontur PJ,
Hammond DN,
Fox AP,
Wainer B,
Hoffman PC,
Heller A
(1991)
Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion.
Brain Res
552:67-76[Web of Science][Medline].
-
Cooper AJ,
Sheu KF,
Burke JR,
Onodera O,
Strittmatter WJ,
Roses AD,
Blass JP
(1997)
Polyglutamine domains are substrates of tissue transglutaminase: does transglutaminase play a role in expanded CAG/poly-Q neurodegenerative diseases?
J Neurochem
69:431-434[Web of Science][Medline].
-
Cooper JK,
Schilling G,
Peters MF,
Herring WJ,
Sharp AH,
Kaminsky Z,
Masone J,
Khan FA,
Borchelt DM,
Dawson VL,
Dawson TM,
Ross CA
(1998)
Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture.
Hum Mol Genet
7:783-790[Abstract/Free Full Text].
-
Davies SW,
Turmain M,
Cozens BA,
DiFiglia M,
Sharp AH,
Ross CA,
Scherzinger E,
Wanker EE,
Mangiarini L,
Bates GP
(1997)
Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation.
Cell
9:537-548.
-
DiFiglia M,
Sapp E,
Chase K,
Schwarz C,
Meloni A,
Young C,
Martin E,
Vonsattel J-P,
Reeves S,
Carraway R,
Boyce FM,
Aronin N
(1995)
Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons.
Neuron
14:1075-1081[Web of Science][Medline].
-
DiFiglia M,
Sapp E,
Chase KO,
Davies SW,
Bates GP,
Vonsattel JP,
Aronin N
(1997)
Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.
Science
277:1990-1993[Abstract/Free Full Text].
-
D'Mello SR,
Aglieco F,
Roberts MR,
Borodezt,
Haycock JW
(1998)
A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K+ deprivation.
J Neurochem
70:1809-1818[Web of Science][Medline].
-
Eldadah BA,
Yakovlev AG,
Faden AI
(1997)
The role of CED-3-related cystein proteases in apoptosis of cerebellar granule cells.
J Neurosci
17:6105-6113[Abstract/Free Full Text].
-
Goldberg YP,
Nicholson DW,
Rasper DM,
Kalchman MA,
Doide HB,
Graham RK,
Bromm M,
Kazemi-Esfarjani P,
Thornberry NA,
Vaillancourt JP,
Hayden MR
(1996)
Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract.
Nat Genet
13:442-449[Web of Science][Medline].
-
Hackam A,
Singaraja T,
Wellington CL,
Metzler M,
McCutcheon K,
Zhang T,
Kalchaman M,
Hayden MR
(1998)
The influence of huntingtin protein size on nuclear localization and cellular toxicity.
J Cell Biol
141:1097-1105[Abstract/Free Full Text].
-
Huntington's Disease Collaborative Research Group
(1993)
A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.
Cell
72:971-983[Web of Science][Medline].
-
Igarashi S,
Koide R,
Shimohata T,
Yamada M,
Hayashi Y,
Takano H,
Data H,
Oyake M,
Sato T,
Sato A,
Egawa S,
Ikeuchi T,
Tanaka H,
Nakano R,
Tanaka K,
Hozumi I,
Inuzuka T,
Takahashi H,
Tsuji S
(1998)
Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch.
Nat Genet
18:111-117[Web of Science][Medline].
-
Kahlem P,
Green H,
Djian P
(1998)
Transglutaminase action imitates Huntington's disease: selective polymerization of huntingtin containing expanded polyglutamine.
Mol Cell
1:595-601[Web of Science][Medline].
-
Kerr JFR,
Wyllie AH,
Currie AR
(1972)
Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.
Br J Cancer
26:239-257[Web of Science][Medline].
-
Kim M, Velier J, Chase K, LaForet G, Kalchman MA, Hayden MR, Won L,
Heller A, Aronin N, DiFiglia M (1999) Forskolin and dopamine
D1 receptor activation increase huntingtin's association with
endosomes in immortalized neuronal cells of striatal origin.
Neuroscience, in press.
-
Lunkes A,
Mandel J-L
(1998)
A cellular model that recapitulates major pathogenic steps of Huntington's disease.
Hum Mol Genet
7:1355-1361[Abstract/Free Full Text].
-
MacFarlane M,
Cain K,
Sun X-M,
Alnemri ES,
Cohen GM
(1997)
Processing/activation of at least four interleukin-1
 converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells.
J Cell Biol
137:469-479[Abstract/Free Full Text]. -
Martindale D,
Hackam A,
Wieczorek A,
Ellerby L,
Wellington C,
McCutcheon K,
Singaraja R,
Kazemi-Esfarjani P,
Devon R,
Kim SU,
Bredesen DE,
Tufaro F,
Hayden MR
(1998)
Length of huntingtin and its polyglutamine tract influences localization and frequency of intracellular aggregates.
Nat Genet
18:150-154[Web of Science][Medline].
-
McCarthy NJ,
Whyte MKB,
Gilbert CS,
Evan GI
(1997)
Inhibition of CED-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak.
J Cell Biol
136:215-227[Abstract/Free Full Text].
-
Paulson HL,
Perez MK,
Trottier Y,
Trojanowski JQ,
Subramony SH,
Das SS,
Vig P,
Mandel JL,
Fischbeck KH,
Pittman RN
(1997)
Intraneuronal nuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3.
Neuron
19:333-344[Web of Science][Medline].
-
Portera-Cailliau C,
Hedreen JC,
Price DL,
Koliatsos VE
(1995)
Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models.
J Neurosci
15:3775-3787[Abstract].
-
Sapp E,
Schwarz C,
Chase K,
Bhide PG,
Young AB,
Penney J,
Vonsattel JP,
Aronin N,
DiFiglia M
(1997)
Huntingtin localization in brains of normal and Huntington's disease patients.
Ann Neurol
42:604-612[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].
-
Scherzinger E,
Lurz R,
Turmaine M,
Mangiarini L,
Hollenbach B,
Hasenbank R,
Bates GP,
Davies SW,
Lehrach H,
Wanker EE
(1997)
Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.
Cell
90:549-558[Web of Science][Medline].
-
Skinner PJ,
Koshy BT,
Cummings CJ,
Klement IA,
Helin K,
Servadio A,
Zoghbi HY,
Orr HT
(1997)
Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures.
Nature
389:971-974[Medline].
-
Velier J,
Kim M,
Schwarz C,
Kim TW,
Sapp E,
Chase K,
Aronin N,
DiFiglia M
(1998)
Wild-type and mutant huntingtins function in vesicle trafficking in the secretory and endocytic pathways.
Exp Neurol
152:34-40[Web of Science][Medline].
-
Wainwright MS,
Perry BD,
Won L,
O'Malley KL,
Wang WY,
Ehrlich ME,
Heller A
(1995)
Immortalized murine striatal neuronal cell lines expressing dopamine receptors and cholinergic properties.
J Neurosci
15:676-688[Abstract].
-
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].
Copyright © 1999 Society for Neuroscience 0270-6474/99/193964-10$05.00/0
This article has been cited by other articles:
|
|
|
|
 
J.-Y. Noh, H. Lee, S. Song, N. S. Kim, W. Im, M. Kim, H. Seo, C.-W. Chung, J.-W. Chang, R. J. Ferrante, et al.
SCAMP5 Links Endoplasmic Reticulum Stress to the Accumulation of Expanded Polyglutamine Protein Aggregates via Endocytosis Inhibition
J. Biol. Chem.,
April 24, 2009;
284(17):
11318 - 11325.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Ratovitski, M. Gucek, H. Jiang, E. Chighladze, E. Waldron, J. D'Ambola, Z. Hou, Y. Liang, M. A. Poirier, R. R. Hirschhorn, et al.
Mutant Huntingtin N-terminal Fragments of Specific Size Mediate Aggregation and Toxicity in Neuronal Cells
J. Biol. Chem.,
April 17, 2009;
284(16):
10855 - 10867.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Takahashi, S. Kikuchi, S. Katada, Y. Nagai, M. Nishizawa, and O. Onodera
Soluble polyglutamine oligomers formed prior to inclusion body formation are cytotoxic
Hum. Mol. Genet.,
February 1, 2008;
17(3):
345 - 356.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. DiFiglia, M. Sena-Esteves, K. Chase, E. Sapp, E. Pfister, M. Sass, J. Yoder, P. Reeves, R. K. Pandey, K. G. Rajeev, et al.
Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits
PNAS,
October 23, 2007;
104(43):
17204 - 17209.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. P. Nguyen, P. Kobbe, H. Rahne, T. Worpel, B. Jager, M. Stephan, R. Pabst, C. Holzmann, O. Riess, H. Korr, et al.
Behavioral abnormalities precede neuropathological markers in rats transgenic for Huntington's disease
Hum. Mol. Genet.,
November 1, 2006;
15(21):
3177 - 3194.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Yamamoto, M. L. Cremona, and J. E. Rothman
Autophagy-mediated clearance of huntingtin aggregates triggered by the insulin-signaling pathway
J. Cell Biol.,
February 27, 2006;
172(5):
719 - 731.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. B.D. Clabough and S. O. Zeitlin
Deletion of the triplet repeat encoding polyglutamine within the mouse Huntington's disease gene results in subtle behavioral/motor phenotypes in vivo and elevated levels of ATP with cellular senescence in vitro
Hum. Mol. Genet.,
February 15, 2006;
15(4):
607 - 623.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. G. Valera, M. Diaz-Hernandez, F. HernANdez, Z. Ortega, and J. J. Lucas
The Ubiquitin-Proteasome System in Huntington's Disease
Neuroscientist,
December 1, 2005;
11(6):
583 - 594.
[Abstract]
[PDF]
|
|
|
|
|
|
|
K. B. Kegel, E. Sapp, J. Yoder, B. Cuiffo, L. Sobin, Y. J. Kim, Z.-H. Qin, M. R. Hayden, N. Aronin, D. L. Scott, et al.
Huntingtin Associates with Acidic Phospholipids at the Plasma Membrane
J. Biol. Chem.,
October 28, 2005;
280(43):
36464 - 36473.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. J. Slow, R. K. Graham, A. P. Osmand, R. S. Devon, G. Lu, Y. Deng, J. Pearson, K. Vaid, N. Bissada, R. Wetzel, et al.
Absence of behavioral abnormalities and neurodegeneration in vivo despite widespread neuronal huntingtin inclusions
PNAS,
August 9, 2005;
102(32):
11402 - 11407.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Poirier, H. Jiang, and C. A. Ross
A structure-based analysis of huntingtin mutant polyglutamine aggregation and toxicity: evidence for a compact beta-sheet structure
Hum. Mol. Genet.,
March 15, 2005;
14(6):
765 - 774.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T.-K. Sang, C. Li, W. Liu, A. Rodriguez, J. M. Abrams, S. L. Zipursky, and G. R. Jackson
Inactivation of Drosophila Apaf-1 related killer suppresses formation of polyglutamine aggregates and blocks polyglutamine pathogenesis
Hum. Mol. Genet.,
February 1, 2005;
14(3):
357 - 372.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Everett and N. W. Wood
Trinucleotide repeats and neurodegenerative disease
Brain,
November 1, 2004;
127(11):
2385 - 2405.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Diaz-Hernandez, F. Moreno-Herrero, P. Gomez-Ramos, M. A. Moran, I. Ferrer, A. M. Baro, J. Avila, F. Hernandez, and J. J. Lucas
Biochemical, Ultrastructural, and Reversibility Studies on Huntingtin Filaments Isolated from Mouse and Human Brain
J. Neurosci.,
October 20, 2004;
24(42):
9361 - 9371.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Tanaka, Y. M. Kim, G. Lee, E. Junn, T. Iwatsubo, and M. M. Mouradian
Aggresomes Formed by {alpha}-Synuclein and Synphilin-1 Are Cytoprotective
J. Biol. Chem.,
February 6, 2004;
279(6):
4625 - 4631.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z.-H. Qin, Y. Wang, E. Sapp, B. Cuiffo, E. Wanker, M. R. Hayden, K. B. Kegel, N. Aronin, and M. DiFiglia
Huntingtin Bodies Sequester Vesicle-Associated Proteins by a Polyproline-Dependent Interaction
J. Neurosci.,
January 7, 2004;
24(1):
269 - 281.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z.-H. Qin, Y. Wang, K. B. Kegel, A. Kazantsev, B. L. Apostol, L. M. Thompson, J. Yoder, N. Aronin, and M. DiFiglia
Autophagy regulates the processing of amino terminal huntingtin fragments
Hum. Mol. Genet.,
December 15, 2003;
12(24):
3231 - 3244.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Busch, S. Engemann, R. Lurz, H. Okazawa, H. Lehrach, and E. E. Wanker
Mutant Huntingtin Promotes the Fibrillogenesis of Wild-type Huntingtin: A POTENTIAL MECHANISM FOR LOSS OF HUNTINGTIN FUNCTION IN HUNTINGTON'S DISEASE
J. Biol. Chem.,
October 17, 2003;
278(42):
41452 - 41461.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D.M. Avila, D.R. Allman, J.-M. Gallo, and M.J. McPhaul
Androgen Receptors Containing Expanded Polyglutamine Tracts Exhibit Progressive Toxicity when Stably Expressed in the Neuroblastoma Cell Line, SH-SY 5Y
Experimental Biology and Medicine,
September 1, 2003;
228(8):
982 - 990.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. J. Slow, J. van Raamsdonk, D. Rogers, S. H. Coleman, R. K. Graham, Y. Deng, R. Oh, N. Bissada, S. M. Hossain, Y.-Z. Yang, et al.
Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease
Hum. Mol. Genet.,
July 1, 2003;
12(13):
1555 - 1567.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U Nagaoka, T Uchihara, K Iwabuchi, H Konno, M Tobita, N Funata, S Yagishita, and T Kato
Attenuated nuclear shrinkage in neurones with nuclear inclusions of SCA1 brains
J. Neurol. Neurosurg. Psychiatry,
May 1, 2003;
74(5):
597 - 601.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Jiang, F. C. Nucifora Jr, C. A. Ross, and D. B. DeFranco
Cell death triggered by polyglutamine-expanded huntingtin in a neuronal cell line is associated with degradation of CREB-binding protein
Hum. Mol. Genet.,
January 1, 2003;
12(1):
1 - 12.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Goffredo, D. Rigamonti, M. Tartari, A. De Micheli, C. Verderio, M. Matteoli, C. Zuccato, and E. Cattaneo
Calcium-dependent Cleavage of Endogenous Wild-type Huntingtin in Primary Cortical Neurons
J. Biol. Chem.,
October 11, 2002;
277(42):
39594 - 39598.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. B. Menalled, J. D. Sison, Y. Wu, M. Olivieri, X.-J. Li, H. Li, S. Zeitlin, and M.-F. Chesselet
Early Motor Dysfunction and Striosomal Distribution of Huntingtin Microaggregates in Huntington's Disease Knock-In Mice
J. Neurosci.,
September 15, 2002;
22(18):
8266 - 8276.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Luthi-Carter, S. A. Hanson, A. D. Strand, D. A. Bergstrom, W. Chun, N. L. Peters, A. M. Woods, E. Y. Chan, C. Kooperberg, D. Krainc, et al.
Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain
Hum. Mol. Genet.,
August 15, 2002;
11(17):
1911 - 1926.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Y.W. Chan, R. Luthi-Carter, A. Strand, S. M. Solano, S. A. Hanson, M. M. DeJohn, C. Kooperberg, K. O. Chase, M. DiFiglia, A. B. Young, et al.
Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease
Hum. Mol. Genet.,
August 15, 2002;
11(17):
1939 - 1951.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. D. Keene, C. M. P. Rodrigues, T. Eich, M. S. Chhabra, C. J. Steer, and W. C. Low
Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of Huntington's disease
PNAS,
August 6, 2002;
99(16):
10671 - 10676.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Kouroku, E. Fujita, A. Jimbo, T. Kikuchi, T. Yamagata, M. Y. Momoi, E. Kominami, K. Kuida, K. Sakamaki, S. Yonehara, et al.
Polyglutamine aggregates stimulate ER stress signals and caspase-12 activation
Hum. Mol. Genet.,
June 15, 2002;
11(13):
1505 - 1515.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Ding, J. J. Lewis, K. M. Strum, E. Dimayuga, A. J. Bruce-Keller, J. C. Dunn, and J. N. Keller
Polyglutamine Expansion, Protein Aggregation, Proteasome Activity, and Neural Survival
J. Biol. Chem.,
April 12, 2002;
277(16):
13935 - 13942.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. B. Kegel, A. R. Meloni, Y. Yi, Y. J. Kim, E. Doyle, B. G. Cuiffo, E. Sapp, Y. Wang, Z.-H. Qin, J. D. Chen, et al.
Huntingtin Is Present in the Nucleus, Interacts with the Transcriptional Corepressor C-terminal Binding Protein, and Represses Transcription
J. Biol. Chem.,
February 22, 2002;
277(9):
7466 - 7476.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. O. S. Mejia and R. M. Friedlander
Caspases in Huntington's Disease
Neuroscientist,
December 1, 2001;
7(6):
480 - 489.
[Abstract]
[PDF]
|
|
|
|
|
|
|
G. A. Laforet, E. Sapp, K. Chase, C. McIntyre, F. M. Boyce, M. Campbell, B. A. Cadigan, L. Warzecki, D. A. Tagle, P. H. Reddy, et al.
Changes in Cortical and Striatal Neurons Predict Behavioral and Electrophysiological Abnormalities in a Transgenic Murine Model of Huntington's Disease
J. Neurosci.,
December 1, 2001;
21(23):
9112 - 9123.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Martin-Aparicio, A. Yamamoto, F. Hernandez, R. Hen, J. Avila, and J. J. Lucas
Proteasomal-Dependent Aggregate Reversal and Absence of Cell Death in a Conditional Mouse Model of Huntington's Disease
J. Neurosci.,
November 15, 2001;
21(22):
8772 - 8781.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. J. Kim, Y. Yi, E. Sapp, Y. Wang, B. Cuiffo, K. B. Kegel, Z.-H. Qin, N. Aronin, and M. DiFiglia
Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis
PNAS,
October 23, 2001;
98(22):
12784 - 12789.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Wyttenbach, J. Swartz, H. Kita, T. Thykjaer, J. Carmichael, J. Bradley, R. Brown, M. Maxwell, A. Schapira, T. F. Orntoft, et al.
Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease
Hum. Mol. Genet.,
August 1, 2001;
10(17):
1829 - 1845.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Petersen, K. E. Larsen, G. G. Behr, N. Romero, S. Przedborski, P. Brundin, and D. Sulzer
Expanded CAG repeats in exon 1 of the Huntington's disease gene stimulate dopamine-mediated striatal neuron autophagy and degeneration
Hum. Mol. Genet.,
June 1, 2001;
10(12):
1243 - 1254.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Chun, M. Lesort, J. Tucholski, C. A. Ross, and G. V.W. Johnson
Tissue Transglutaminase Does Not Contribute to the Formation of Mutant Huntingtin Aggregates
J. Cell Biol.,
April 2, 2001;
153(1):
25 - 34.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. M. Mende-Mueller, T. Toneff, S.-R. Hwang, M.-F. Chesselet, and V. Y. H. Hook
Tissue-Specific Proteolysis of Huntingtin (htt) in Human Brain: Evidence of Enhanced Levels of N- and C-Terminal htt Fragments in Huntington's Disease Striatum
J. Neurosci.,
March 15, 2001;
21(6):
1830 - 1837.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Y. Chang and X. Yang
Proteases for Cell Suicide: Functions and Regulation of Caspases
Microbiol. Mol. Biol. Rev.,
December 1, 2000;
64(4):
821 - 846.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. B. Kegel, M. Kim, E. Sapp, C. McIntyre, J. G. Castano, N. Aronin, and M. DiFiglia
Huntingtin Expression Stimulates Endosomal-Lysosomal Activity, Endosome Tubulation, and Autophagy
J. Neurosci.,
October 1, 2000;
20(19):
7268 - 7278.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Rigamonti, J. H. Bauer, C. De-Fraja, L. Conti, S. Sipione, C. Sciorati, E. Clementi, A. Hackam, M. R. Hayden, Y. Li, et al.
Wild-Type Huntingtin Protects from Apoptosis Upstream of Caspase-3
J. Neurosci.,
May 15, 2000;
20(10):
3705 - 3713.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. I. Diamond, M. R. Robinson, and K. R. Yamamoto
Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor
PNAS,
January 18, 2000;
97(2):
657 - 661.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Chai, S. L. Koppenhafer, N. M. Bonini, and H. L. Paulson
Analysis of the Role of Heat Shock Protein (Hsp) Molecular Chaperones in Polyglutamine Disease
J. Neurosci.,
December 1, 1999;
19(23):
10338 - 10347.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. S. Hackam, A. S. Yassa, R. Singaraja, M. Metzler, C.-A. Gutekunst, L. Gan, S. Warby, C. L. Wellington, J. Vaillancourt, N. Chen, et al.
Huntingtin Interacting Protein 1 Induces Apoptosis via a Novel Caspase-dependent Death Effector Domain
J. Biol. Chem.,
December 22, 2000;
275(52):
41299 - 41308.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
